From Relative to Absolute Stereochemistry of Secondary Metabolites: Applications in Plant Chemistry

Structural elucidation of specialized metabolites or natural plant products, especially the stereochemical features of chiral bioactive metabolites, is the most important stage for all investigations in pharmacognosy and phytochemistry. Numerous methods have been established to solve the absolute configuration of natural products, including direct methods such as X-ray diffraction, chiroptical spectroscopy, stereo-controlled partial or total organic synthesis, and chemical correlation by simple organic reactions for derivatization, as well as indirect methods using a standard chiral compound or a derivatizing agent with known absolute configuration, for example, nuclear magnetic resonance by utilization of anisotropic effects of chiral agents. Once the relative configuration of a natural product is resolved, the absolute stereochemistry can be determined with the assistance of chiroptical spectroscopies supported by quantum mechanical-NMR prediction of chemical shifts, scalar coupling constants and simulation of optical rotatory dispersion, in addition to electronic and vibrational circular dichroism spectra. This review summarizes the most practical methods employed in the assignment of absolute stereochemistry of natural plant products with selected examples focused on multichiral center bioactive small molecules from the mega-diverse Brazilian and Mexican flora.


Introduction
Many commercialized drugs (ca. 40%) are directly or indirectly originated from specialized metabolites or natural products (Newman and Cragg 2020); thus, phytochemistry is still one of the key fields of interest of pharmacognosy and medicinal chemistry in the search for pharmacologically active agents (Atanasov et al. 2021). Currently, new multiinformational-based profiling approaches for target-based drug discovery, pharmacological high-throughput screening, and targeted isolation of specialized metabolites, combining taxonomic, genomic, and/or bioactivity data, have been developed. Consequently, pharmacognosy has become a high-tech discipline and the limitations of its traditional approaches have been modernized by application of molecular biology techniques in the -omics era, where molecular (Alamgir 2018), genomic (Yang et al. 2019), and metabolomic pharmacognosy (Allard et al. 2018) have been envisioned as the most advantageous methodologies (Singh et al. 2022).
High-throughput screening of natural product libraries against isolated biological targets has become a routine practice in drug development and involves the correct elucidation of the stereochemical features of hits for their further structural optimization to improve affinity, selectivity to lower the risk of adverse effects, efficacy, potency, metabolic stability to extend the half-life, and oral bioavailability. In these biochemical and pharmaceutical approaches, as well This review was presented in part at the Sociedade Brasileira de Farmacognosia, 2° Simpósio Regional Sudeste de Farmacognosia (27-30 April 2022), Penedo, Rio de Janeiro, Brazil. as in molecular technology, stereochemistry and chirality are of prominent importance in the interaction of drugs and biological targets since all cell receptors are chiral (Jayakumar et al. 2018). Consequently, the structural elucidation of specialized metabolites, including the description of their absolute stereochemistry, is essential for development of pharmaceuticals from natural sources. This stereochemical requirement for the absolute configuration is also crucial for nature-product-inspired drug discovery in the design of synthetically manipulable small molecules that mimic nature's chemistry (Rodrigues et al. 2016).
The spatial configuration of atoms (three-dimensional structure) in all metabolites is intimately related to their biological activity. Thus, it is important to elucidate the chirality of a molecule correctly-the geometric property of an organic compound of not being superimposable with its mirror image or enantiomer (Fig. 1)-since pairs of enantiomeric compounds can present different activities when they act on the same binding site, resulting in inactivity, being metabolized or absorbed by the cell in a discriminated way, interacting differently with the same metabolic pathway, or even being toxic (Jayakumar et al. 2018). Several of the medicines now in use, especially in psychiatry, occur as mixtures of enantiomers, also known as racemic mixtures or racemates. For some therapeutic drugs, singleenantiomer formulations can deliver superior selectivity for their pharmacological targets, enhanced therapeutic indices, and improved pharmacokinetics than racemates (McConathy and Owens 2003). For example, one enantiomer may be responsible for the therapeutic effects of a drug whereas the other is inactive or contributes to undesirable effects. The antidepressant effects of the anesthetic ketamine, an uncompetitive antagonist of the N-methyl-d-aspartate receptor, is the furthermost significant discovery in the prevention and treatment of depression in the last five decades. Ketamine is a racemic mixture composed of equal amounts of (S)ketamine and (R)-ketamine ( Fig. 1). Although (R)-ketamine is a less potent antagonist than (S)-ketamine, preclinical evidence indicated that the (R)-form develops longer-lasting action with fewer side effects (Jelen et al. 2021). In nature, chiral specialized metabolites are biosynthesized in optically pure form (Fig. 1); however, occasionally, both enantiomers could be produced in the same plant (Finefield et al. 2012;Batista et al. 2018).
Undoubtedly, a pair of enantiomers display dissimilar pharmacologic effects and pharmacokinetics. Consequently, the US Food and Drug Administration requests that the pharmacological and toxicological properties of each enantiomer in a ( ±)-racemic drug should be studied independently before the drug be marketed as a racemate (FDA 1992) to avoid toxic calamities such as the thalidomide tragedy (Woolf 2022). In the 1950s, this drug was developed and distributed in Europe and many other countries as a nonaddictive, nonbarbiturate sedative over-the-counter racemic mixture to relieve anxiety and promote sleep. Thalidomide was also a successful anti-emetic to treat morning sickness, a common symptom of pregnancy (Fig. 2). The antiangiogenic effects for the toxic S-( −)-enantiomer were unknown prior to its release for human use. Nevertheless, as the drug can rapidly racemize between both enantiomeric states at physiological pH, it is extremely hard to produce a stable form for the R-( +)-enantiomer that is nonteratogenic (Vargesson 2015). The lack of a premarketing study led to thousands of infants born with severe malformation of the extremities (phocomelia). Failure to determine the correct structure of a bioactive specialized metabolite or a synthetic organic compound can dramatically alter the course of a drug development program.
In the present review, the analytical techniques to establish the absolute configuration of organic compounds are organized into absolute (direct) or relative (indirect) methods in agreement to whether a reference compound with an identified chiral center has been used or not. For -nicotine and its (R)-enantiomer which could be produced under the conditions of cigarette smoking by racemization. (C) Enantioselectivity for celery ketone odor: (R)-enantiomer (threshold 9.1 ng/l air) is reminiscent of celery leaves' scent; the (S)-antipode (threshold 45 ng/l air) is 5 times weaker and reminiscent of licorice and anise example, Mosher's method is a well-known indirect approach for determining the absolute configuration of organic compounds containing hydroxyl, amino, or carboxyl groups (Hoye et al. 2007), while single-crystal X-ray diffraction is a direct method for chiral molecules having anomalous scattering effects (Ameh 2019). Several different methods are used and involved: (i) chemical correlation with compounds of identified absolute configuration by simple derivatization reactions or chemical degradation and comparison of optical rotation by polarimetry (da Silva et al. 2021); (ii) chiroptical spectroscopy methods such as optical rotatory dispersion (ORD), circular dichroism (CD) or exciton chirality methods (Harada 2017;Mándi and Kurtán 2019); and (iii) empirical NMR methods (Bifulco et al. 2007;Costa et al. 2021).
When the relative configuration of a metabolite is confirmed, the absolute stereochemistry can be identified with the support of chiroptical spectroscopy, assisted by quantum mechanical-NMR prediction, and simulation of electronic CD, vibrational CD, and ORD spectra. Therefore, despite the remarkable developments in the methods for determining the absolute configuration, the mischaracterization of specialized metabolites has been a frequent problem in structural elucidation (Chhetri et al. 2018;Menna et al. 2019;Liu et al. 2020;Navarro-Vázquez 2021). Representative examples are illustrated here where distinctive approaches incorporating diverse chemical correlations and spectroscopic and/or computational methods were planned to elucidate the absolute configuration of natural small molecules from the Brazilian and Mexican flora. Accordingly, this review could help readers in the fields of phytochemistry, medicinal chemistry, phytopharmaceutical biotechnology, biochemical pharmacology, and toxicology, among others, for selecting a correct combination of approaches when confronting a specific stereochemical problem in structure elucidation of plant metabolites.

Search Strategy
The keywords used in the Google Scholar search, with absolute configuration being present in all combinations, were NMR, natural product, structure elucidation, structural revision, chirality, Mosher's method, quantum NMR calculations, optical rotatory dispersion, circular dichroism, and X-ray crystallography. Relevant reviews and digital documents (in the English language) were consulted to assemble all available literature to provide an updated survey of the topic absolute configuration or stereochemistry of plant metabolites.

Single-Crystal X-Ray Diffraction
Single-crystal X-ray diffraction is the most powerful analytical technique to establish or confirm the chemical structures of organic compounds, including those derived from natural sources, such as plants and microorganisms of terrestrial and marine origin. The absolute configuration of a great variety of compounds, such as alkaloids (Zhu et al. 2015;Zhang et al. 2021), phenolics (Bueno-Pérez et al. 2020;Huo et al. 2020), polyketides (Li et al. 2019b;Annang et al. 2020), and terpenoids (Le-Huu et al. 2018;Wang et al. 2020), among others, has been derived by using X-ray diffraction analyses.
Single-crystal X-ray diffraction analyses can be classified according to their radiation source (Mo, Cu, and synchrotron), to the methodology followed to solve a chemical structure or determine the absolute configuration, and to the sample preparation (single-crystal and crystalline sponge) (Kunde and Schmidt 2019). However, despite being a powerful analytical tool, this methodology has some limitations associated to the requirements of the sample to be analyzed, such as the amount, its purity, and the crystal size.
The approaches for determination of the absolute configuration through X-ray diffraction can be sub-classified as direct or indirect methods. For the first type, this method relies on the accurate measurement of the small differences in scattering intensities of Friedel opposites due anomalous dispersion effects. In this sense, the magnitude of the anomalous dispersion increases with the increasing of the atomic number. Thereby, the elucidation of the absolute stereochemistry through X-ray diffraction by direct methods can be determined calculating the Flack or Parsons parameters (Le Pevelen 2012; Albright and White 2013). The Flack parameter is the direct method mostly used to establish the absolute stereochemistry and its values ranging Fig. 2 Structure of thalidomide enantiomers. This synthetic drug was distributed as a racemic mixture which can rapidly interconvert (racemize) in body fluids and tissues to form equal concentrations of both enantiomeric forms (R and S) between 0 and 1. A physical translation for this parameter could be that it will indicate the mole fraction of a chiral molecule in a crystal structure versus the mole fraction of its enantiomer. To consider that the absolute configuration has been determined satisfactorily, the following conditions must be met. The Flack parameter is represented by x(u), where x is the numeric value of the parameter, and u, the standard uncertainty. The x value must be near zero, and the u value ranging from 0.04 to 0.1. In addition, the relation |x|/u < 3.0 must hold. Some examples of the use of the Flack parameter have been published (Flack 2008(Flack , 2012Flack and Bernardinelli 2008).
The absolute configuration determination by indirect methods using X-ray crystallography consists in the insertion of an element with recognized absolute configuration or a heavy atom (Z > 8). The introduction of this group can occur by the formation of a salt (counterion) or a covalent bond, or through the co-crystallization or solvate formation (Albright and White 2013). Table 1 provides a comparison of some requirements for structure determination and absolute configuration establishment through single-crystal X-ray diffraction, the crystalline sponge method, and microcrystal electron diffraction. Table 2 describes some crystallization solvent systems to obtain adequate crystals from natural products with the required features to be subjected to X-ray diffraction.

Current Trends
The structure determination of tricolorin A was establish based in high-resolution MS (FAB) and NMR analyzes, and constitutes the first example of a fully characterized resin glycoside by X-ray crystallography obtained from a Mexican species of the morning glory family, Ipomoea tricolor Cav., Convolvulaceae (Rencurosi et al. 2004). This tetrasaccharide of 11S-hydroxyhexadecanoic acid inhibited seed germination and radicular growth, acting as a pre-and post-emergence plant growth inhibitor (Lotina-Hennsen et al. 2013). Several pharmacological properties, such as antibacterial, antifungal, and cytotoxic activities, as well as its potential as a multidrug-resistance modulator in bacterial and cancer cells, have been described (Pereda-Miranda et al. 2006;Castañeda-Gómez et al. 2019;Lira-Ricárdez and Pereda-Miranda, 2020). To determine the crystal structure of this polar and high-weight (1022 Da) natural product, a great effort was made, first to isolate and purify enough quantities of tricolorin A through recycling reversed-phase HPLC, and then to assay several conditions for the crystallization of proteins with the application of the reserve vapor diffusion by the hanging drop method (Hou et al. 2019).
Mineral oil was used as a barrier to vapor diffusion between the reservoir and the drop, both containing the   Bautista et al. 2013bBautista et al. , 2015Ortega et al. 2017 Triterpenoids Acetone/hexanes, CH 2 Cl 2 /MeOH (3:1), Wang et al. 2020 sample. In this way, the amount of water in the drop was lowered. The drops contained a higher reagent concentration than the reservoir. Water vapor left the reservoir and traveled into the drops promoting the crystallization of the glycolipid in the interface of the mineral oil and water (Fig. 3). Crystals of tricolorin A (0.5 × 0.01 × 0.01 mm 3 ) were analyzed with application of the synchrotron radiation due to the size of the crystal cell unit (Rencurosi et al. 2004). Diffraction data were collected at The European Synchrotron Radiation Facility (Grenoble, France) on the beam line ID29 (l = 0.81570 Å). The software SIR2002 was used to solve the 3D structure. Refinement was performed using the Shelx-97 program and applying a few restraints on C-C distances (R 0.0998, wR 0.2283). Tricolorin A packing shows an alternance of hydrophilic and hydrophobic interfaces. Eighteen water molecules mediate the contacts between the two glycolipid bilayers in the asymmetric unit where four independent tricolorin A molecules with slightly different conformations were observed (Fig. 4).
To avoid the inconveniences of repetitive laboratory work associated to single-crystal X-ray diffraction determinations, which imply the purification of enough amounts of a natural product (target compound) and, subsequently, the optimization of crystal growth with enough size and quality to be analyzed by this technique, Fujita developed the crystalline sponge method (Inokuma et al. 2013). This method uses trace amounts of the target compound (order μg-ng) and does not require an initial crystal growth process of the analyte. For this purpose, crystalline sponges constituted by networked complexes are used. [[(ZnI2)3(tpt)2]•x(solvent) ]n complex (tpt = tris(4-pyridyl)-1,3,5-triazine) was originally proposed as a crystalline sponge and has been greatly employed. Figure 5 illustrates this method, which makes use of single-crystalline porous coordination networks (crystalline sponges) that absorb small analyte molecules within the pores and make the absorbed guest compound observable by conventional single-crystal diffraction (Zigon et al. 2021).
The crystal structure of the known sesquiterpene guaiazulene (1) was determined with only 80 ng of sample. Nevertheless, the standardization of the methodology established the use of 5 μg of the target compound, which still is a very small quantity of sample to carry out the X-ray diffraction analysis. Even better, the crystalline sponge method also allows the absolute configuration determination with the same amount of sample necessary for the crystallographic analysis and does not require the introduction of heavy atoms through derivatization because the guest framework contains heavy atoms (Zn and I), which display anomalous scattering effects. Thus, the absolute configuration of santonin (2) constitutes the first example of this type of determination using the crystalline sponge method.
This method was also applied to characterize the regiostereochemistry when a chiral oxidant agent was used for all the sub-products of oxidation of α-humulene (3) with samples of 5-50 μg (Zigon et al. 2015), during the structural revision of natural products (Lee et al. 2017;Chhetri et al. 2018), and in the chemo-typing of crude extracts (order mg) for natural product discovery (Wada et al. 2018). The conjunction of the crystalline sponge method and synchrotron radiation was used to establish the absolute configuration of the lignan asarinin (4) (Li et al. 2019a).
The microcrystal electron diffraction (microED), also called electron crystallography, is an analytical tool that allows determining the molecular structure of very small crystals of synthetic and natural compounds. This methodology is based on the use of electrons as radiation source Images provided by Prof. Anne Imberty, Centre de Recherches sur les Macromolécules Végétales, Centre National de la Recherche Scientifique (affiliated with University Joseph Fourier), Grenoble, France instead of X-rays, in which the diffraction patterns obtained reach atomic resolution (Gruene et al. 2018). In comparison with single-crystal X-ray diffraction, the microED uses microcrystals a small as 1 × 2 μm 2 , while a crystallographic determination using synchrotron radiation requires crystals with lengths ranging from 5 to 10 μm and a laboratory diffractometer needs crystals of 50 μm length. The analysis time of microED (30 min-4 h) also is less than the required by single-crystal X-ray diffraction determinations (4-24 h) (Gruene et al. 2018;Kunde and Schmidt 2019). The number of molecular structures determined so far by the application of electron diffraction is around 425, against ~ 78,500 structures determined by single-crystal X-ray diffraction. This novel methodology has been applied for the structure determination of various bioactive natural products: biotin (5), brucine (6), cinchonine (7), and progesterone (8) Danelius et al. 2021).

Chiroptical Spectroscopy
Specific rotation, a physical constant distinguishing chiral or optically active compounds, was formerly discovered by Arago and Biot in the early nineteenth century. Then, a diversity of methods based on the interactions of asymmetric molecules with left-and right-circularly polarized light have been developed, e.g., optical rotatory dispersion (ORD), electronic circular dichroism (ECD), and vibrational circular dichroism (VCD). Nevertheless, they involve different characteristics (Table 1): OR and ORD are based on circular birefringence or the difference in velocity of circularly polarized lights through the medium (Δn), while circular dichroism (CD), including ECD and VCD, depends on the difference in absorption (ΔA or Δε). In addition, ECD is concerned with the absorption of UV-Vis light (electronic Fig. 4 Tricolorin A structure. Chemical structure determined by NMR and MS analyses (A); ORTEP drawing of tricolorin A determined by application of synchrotron radiation (B); crystal packing where molecules of water are represented by red spheres Step i: guest soaking into the crystalline sponge; step ii: conventional single-crystal X-ray diffraction study. Reproduced from Zigon et al. 2021 transition), while VCD involves the absorption at the mid-IR region (vibrational transition). Typical sample amounts for measurements depend on the molecular weight and physicochemical properties of the analyte along with the limitations for the three methods (Table 3). For a recent review on the principles and applications of chiroptical spectroscopy aided by quantum-chemical calculations for conformational and configuration analysis of natural products, see Mándi and Kurtán (2019).

Specific Optical Rotation
Optical rotation or specific optical rotation, also known as polarization rotation or circular birefringence, is the difference in refractive indices of left-and right-circularly polarized light and is normally recorded at 589 nm (Na D-line). The dependence of specific rotation on wavelength is called optical rotatory dispersion, and it could be measured across diverse scanning wavelengths to generate the so-called optical rotatory dispersion (ORD) spectrum, especially suitable for compounds without chromophores in the UV-Vis region. For that, the specific optical rotation at other wavelengths such as 633, 578, 546, 436, 405, and 365 nm can be measured by using mercury or tungsten-halogen lamps.
The specific optical rotation varies with the wavelength, temperature, solvent, and sample concentration of the chiral molecule (1-10 mg of sample); consequently, these parameters should be indicated for all the reported [α] λ T values in the literature. For example, argentilactone displays a 5,6-dihydro-2H-pyran-2-one moiety with a negative specific optical rotation for the natural ( −)-form (levorotatory enantiomer) due to its single chiral center at C-6 ( Fig. 6). This biodynamic 5,6-dihydropyran-2-one has been isolated from Aristolochia argentina Griseb., Aristolochiaceae, Ceiba crispiflora (Kunth) Ravenna, Malvaceae, and Annona haematantha Miq., Annonaceae. This natural 5,6-dihydropyran-2-one has displayed in vitro antiprotozoal activity against Plasmodium falciparum (ED 50 0.5 μM), Leishmania panamensis (ED 50 51.5 μM), and Leishmania amazonensis (ED 50 51.5 μM), as well as cytotoxic activity against leukemia (P-388: IC 50 21.4 μM), lung non-small cells (NCI 460: IC 50 14.3 μM), and breast (MCF-7: IC 50 14.7μM) cells. Its synthetic dextrorotatory ( +)-enantiomer also displayed antiproliferative activity against MCF-7 (IC 50 > 13.9 μM) and NCI 460 (IC 50 > 17.9 μM) cell lines. However, the natural levorotatory ( −)-enantiomer proved to be more potent for multidrug-resistant breast cancer cells (NCI-ADR, levorotatory ( −)-form: IC 50 >13.9 μM; dextrorotatory ( +)-form:  The chirality is associated to the 5,6-dihydro-2H-pyran-2-one moiety with its asymmetric center at C-6 IC 50 >100 μM), whereas melanoma cells (UACC62) were more sensitive to the synthetic dextrorotatory ( +)-enantiomer, i.e., levorotatory ( −)-form: IC 50 >55.0 μM; dextrorotatory ( +)-form: IC 50 >17.8 μM (de Fatima et al. 2004). Every optical active natural product has its own specific rotation which is determined by its intrinsic molecular structure. Chiral plant products are mainly synthesized in optically pure form as part of a family or class of secondary metabolites with a common origin (Finefield et al. 2012). Therefore, a simple specific rotation value is frequently used to assign the absolute stereochemistry of a new isolated product by comparison of the [α] D values of structurally related compounds from the same structural group. This practice is usually performed for natural products with different substitution patterns to conclude on the absolute configuration of novel isolated compounds from the same plant source. However, it must be considered with precaution since it could lead to erroneous conclusions. Even minor structural variations in the substitution pattern of the basic skeleton can invert or modify the sign and magnitude of the specific optical rotation. The case for the chiral furanones sotolon, found in aged Japanese sake, and the maple furanone exemplifies the chances for absolute configuration misinterpretations from the assignment only based on the comparisons of optical rotation values for a particular family of specialized plant metabolites. These chiral 5-substituted-2(5H)-furanones are naturally occurring with an enantiomeric excess, and each of the enantiomers have dissimilar organoleptic properties (Nakahashi et al. 2011). Sotolon is produced by the thermal degradation of 4-hydroxy-l-isoleucine. Instead, maple furanone is formed spontaneously from l-threonine. Their chemical structures diverge from each other by the presence of a single methylene unit, and despite having identical absolute configuration, they have opposite optical rotation signs (Fig. 7). For these products, geometry optimizations were carried out using DFT calculations at the B3PW91/6-31G(d,p) level with the Gaussian 03 program. These spectra were averaged with the Boltzmann-weighted populations for (R)-sotolon. The calculated IR and VCD spectra of the (R)isomer exhibited an excellent agreement with the experimental spectra in the fingerprint regions from 1500 to 1000 cm −1 (Fig. 4). Consequently, the absolute stereochemistry for both enantiomers was undoubtedly concluded to be ( −)-(R)-and ( +)-(S)-sotolon. The observed IR absorptions for the maple furanone are like those of sotolon. However, the observed VCD spectrum of ( +)-isomer diverged particularly around 1254 cm −1 . The calculated VCD spectrum of (R)-maple furanone showed good agreement with the experimental spectrum ( Fig. 7), the absolute stereochemistry of the enantiomers of maple furanone were determined as the ( +)-(R)-and ( −)-(S)-enantiomers (Nakahashi et al. 2011).
For most of the members of a particular class of secondary metabolites, slightly structural modifications such as changes in the substitution pattern, for example, the substitution of a hydroxyl group with an acetoxy group, cannot invert the sign of the optical rotation; only changes in the order of magnitude The optical rotations for both natural products are opposite, but their absolute configurations are identical by application of VCD. Analytical condition: [α] D c 0.30, CHCl 3 ; VCD spectra were recorded on a FT-VCD spectrometer (BioTools, Inc.) with a resolution of 8 cm. −1 for 3 h. Samples were dissolved in CCl 4 (0.15 M) and placed in a 72-mm CaF 2 cell. Adapted from Nakahashi et al. (2011) for the [α] value could be detected. For example, the same dextrorotatory optical rotations for natural boronolide and its derivatives, 1,2-dideacetylboronolide and deacetylboronolide isolated for species of Tetradenia, Lamiaceae, were recorded (Davies-Coleman and Rivett 1987;Chandrasekhar et al. 2003;Prasad and Anbarasan 2006). The inversion for the sign only depended on the absolute configuration for the chiral center C-6 of the 5,6-dihydro-2H-pyran-2-one stereoclusters in the synthetic enantiomers since both series share the same absolute configuration for the side chain (Fig. 8).
In contrast, during the total synthesis of spicigerolide and three of its stereoisomers, which shared the same absolute configuration for the chiral centers at the side chain, which are equivalent to those of the commercially available l-rhamnose (Fig. 9), it was observed that the specific rotation of this natural 5,6-dihydro-2H-pyran-2-one was of the same sign but with a different order of magnitude as those for its two synthetic 6-epi-steroisomers (Falomir et al. 2003). This situation did not directly indicate an antipodal relationship for the two epimers, i.e., 6(R)-( −)-and 6(S)-( +)-spicigerolide, as described above for 6(R)-( −)and 6(S)-( +)-argentilactone ( Fig. 6) and 6(R)-( +)-and 6(S)-( −)-boronolide (Fig. 8). However, comparison of 6(R)-( +)-1E-spicigerolide with epimers at the chiral center C-6, e.g., 6(S)-( −)-1E-spicigerolide ( Fig. 9), proved that all these related compounds possessed a negative sign for their optical rotation, as it should be expected for natural ( −)-sicigerolide with a divergent optical rotation, but with the same absolute configuration as the 6(R)-epimer (López-Vallejo et al. 2011). The deviation in optical rotation could be traced to a conformational asymmetry effect to obtain coplanar and fully extended zigzag conformations for the multichiral center side chain, but with exclusion of 1,3-parallel non-bonded oxygen-oxygen interactions, mainly between the ether oxygen of the lactone unit and the acetyl group at C-3′, which represent the main contribution to their conformational equilibrium as demonstrated by DFT calculations (López-Vallejo et al. 2011). All the low-energy conformers gave negative specific optical rotation at the DFT B3LYP/ DGDZVP level. The Boltzmann-averaged [α] D was − 25.9 which corroborated the experimental [α] D − 23.0 (c 2.26, CHCl 3 ). Consequently, DFT-optical rotation calculations can afford a useful verification for the absolute configuration of flexible natural products if low-energy conformers have the same optical rotation signs even in cases when some conformers provide opposite signs (Grauso et al. 2019;Mándi and Kurtán 2019). Thus, it is recommended that if optical rotation responses for different conformers of a chiral molecule have opposite signs, the application of other chiroptical techniques like VCD and/or ECD is encouragingly suggested.
ORD curves are obtained by plotting specific rotation vs. wavelength. Near the UV-maximum absorption of a substance, the ORD curve has an S-shaped appearance with a positive peak and a negative trough. This change is called the Cotton effect or anomalous dispersion. Anomalous curves, depending on the number of absorbing substituents, show various intense peaks and troughs which contain an asymmetric carbon as part of a chromophore (Fig. 10). However, plain optical rotatory dispersion curves without any peak, with no inflection point, i.e., the curve do not cross the zero-rotation line, could be also obtained for compounds that do not have absorption in the wavelength region where optical activity is examined (Fig. 11). Elucidation of the absolute stereochemistry for a novel natural product is generally based on comparison of the ORD curves of the examined molecule with the ORD spectra of closely related compounds of known configurations. For example, the optical rotatory dispersion curves for natural ( −)-spicigerolide and related synthetic derivatives are shown in Fig. 12. These ORD experimental data suggest the presence of the same Fig. 8 Specific optical rotation data for natural boronolide and synthetic 6-epi-boronolide, as well as for its derivatives, 1,2-dideacetylboronolide and deacetylboronolide, having the chiral α,β-unsaturated δ-lactone functionality

Circular Dichroism
Circular dichroism (CD) or electronic circular dichroism (ECD) is the differential absorption of left-and right-handed circularly polarized light. In CD, the circular polarized light is used and is converted to elliptical light in contrast to ORD where this conversion does not occur. CD spectra are absorptive while ORD spectra are dispersive. CD curves are obtained by plotting molar ellipticity vs wavelength. Generally, the CD effect is related to the ORD anomaly, since both reproduce the interaction of the polarized light with the same chiroptical chromophore. The CD curve maximum corresponds with the wavelength of an anomalous ORD crossover, and the signs of both the Cotton effect in ORD and CD spectra match (Fig. 13).
The main restriction of the ECD is that it needs the occurrence of at least one chromophore for absorption of visible/ UV radiation, which needs to be in the proximity of the stereogenic elements. This limitation is partly corrected by derivatization reactions created to introduce chromophores. Among the chiroptical methods, ECD is mostly used for the establishment of the absolute configuration of natural products. This can be credited to the low amount of sample needed, i.e., few micrograms of sample (Table 1). When molecules exist in more than one conformation in solution, each conformer will have its owned CD curve, in a similar way to ORD. A review on the general features of ECD was published by Vázquez (2017). Different approaches have been established for CD interpretation (Mándi and Kurtán 2019) which are summarized below.

ECD Spectral Comparison
The empirical correlation of experimental CD spectra of structural related compounds is the most frequently used for the absolute configuration assignment of a new natural product, which can be effortlessly deduced by a simple comparison of its measured CD spectrum with the CD data of previously related compounds for which their absolute stereochemistry has been established. For example, chiroptical measurements were undertaken to explore the ECD spectra of the brevipolide series from Hyptis brevipes Poit., Lamiaceae (Suárez-Ortiz et al. 2013). A positive n → π* Cotton effect for the α,β-unsaturated δ-lactone was observed for these compounds centered at λ max 235-264 nm (Fig. 14), confirming the same stereogenic center at C-6-(R), which was supported by the relative configuration defined from X-ray diffraction analysis (Suárez-Ortiz et al. 2013). ECD curves for the chiral trans-cinnamoyl derivatives exhibited a positive Cotton effect centered around 300 nm, in contrast with the cis-compounds, which showed a negative Cotton effect at 319 nm (Δε = − 1.3).
It must be underlined that in this situation, an undisputable attribution of the absolute stereochemistry was achievable because the reference has an identical chromophore and Fig. 10 Comparison of optical rotatory dispersion curve spectra: plain curves (black) and anomalous curves with positive (red) and negative (blue) Cotton effects, and its relationship with near-UV maximum absorption (λ max ). Adapted from Maksimenka 2010

Fig. 11
Experimental optical rotatory dispersion curves of (R)-and (S)-limonene measured from the visible to the near-infrared by the application of broadband optical activity spectroscopy with interferometric Fourier-transform balanced detection. Reproduced from Ghosh et al. (2021). https:// creat iveco mmons. org/ licen ses/ by/4. 0/ diverging only in the substitution pattern of the molecule not disturbing the α,β-unsaturated γ-lactone chromophore, since small alterations in the chromophore or minor conformational changes may cause significant fluctuations in the CD spectrum, as illustrated by the opposite Cotton effects observed for the geometric isomer of the cinnamoyl residues at λ max 300-325 nm (Fig. 14).
Another instructive example is provided by two neoclerodane-type diterpenoids from Salvia, Lamiaceae, salvimicrophyllin C and linearolactone, which effectively demonstrate the influence resulting in the Cotton effect by a change in the dissymmetric region of the molecule in the vicinity of the chromophore, i.e., the n → π* transition for the γ-lactone conjugated to the diene system in ring A with an UV absorption band near λ max 275 nm (Bautista et al. 2014a). Both diterpenes displayed an opposite CD spectrum with near-identical structures and the same molecular formula but diverging in the relative configuration for the fusion of rings A-C. This difference is the result of distinctive conformational distributions as demonstrated by NOESY experiments for both compounds (Fig. 15).
The relative configuration for salvimicrophyllin C was demonstrated by the values for the H-12 coupling constants (J 11α-12 = 12.0 and J 11β-12 = 3.5 Hz), which established an α-orientation for the furan ring. The NOESY spectrum showed a correlation between H-12 and H-8, thus establishing the β-orientation for these protons as well as the presence of a trans-fused δ-lactone. The cross-peaks of H-8 with H-10 and H-19 pro-R , as well as the correlation between these two protons suggested a cis fusion for the decalin. In contrast, the NOESY spectrum of linearolactone showed cross-peaks of H 3 -20 with H-8 and H-19 pro-S and a correlation of H-19 pro-S with H-10. Finally, the absolute configuration of both compounds was also supported by  comparing their ECD curves as illustrated in Fig. 15(C). A mirror-like CD behavior with opposite Cotton effects at 260-262 and 302-304 nm were observed with a positive curve for salvimicrophyllin C and a negative one for linearolactone (Bautista et al. 2014a).

Semi-empirical Sector Rules
Several rules have been proposed to elucidate the absolute configuration of chiral natural products having an intrinsic achiral chromophore, such as alkene, carbonyl, and benzene to correlate the ECD transitions of the chromophore with the chiral environment in its vicinity. The Cotton effect sign coupled to a specific transition is extremely sensitive to contributions from all substituents in a chromophore surrounding which can be evaluated by splitting the 3D space around the achiral chromophore into sectors with different signs of contribution. The octant rule for saturated alkyl ketones is the most extensively accepted sector rule, which correlates the n →π* transition of the carbonyl chromophore with the absolute configuration by using eight sectors surrounding the ketone carbonyl group (McNaught and Peckham 2012). Many effective empirical rules have been recommended by researchers such as Djerassi, Crabeé, and Snatzke (Legrand and Rougier 1977). Examples for the application of the semiempiral Snatzke's rule for the C-6 chiral α,β-unsaturated δ-lactone chromophore are illustrated in the present review (see Case Study Examples) with 5,6-dihydro-2H-piran-2-ones from the mint family (Lamiaceae). Finally, for compounds possessing inherently chiral chromophores, such as distorted β,γ-unsaturated ketones, dienes, and disulfides, numerous helicity rules have been proposed, which are based on the determination of the direction of a helical path corresponding to the specific electronic transition. The reader interested in the application of these sector rules may consult the comprehensive review by Legrand and Rougier (1977).

Exciton Chirality Method
The CD exciton chirality is a non-empirical method that does not impose the use of a reference, based on non-empirical coupled oscillator and on group polarizability theories. This technique has played a significant role for the establishment of the absolute configuration of many complex aromatic natural products, such as stereoidal bisbenzoates and biaryls (Pescitelli 2022). For this application, it is necessary that the molecule under study has two or more strongly absorbing chromophores (non-conjugated), that is, groups with a high molar extinction coefficient, where the chromophores with intense π-π* absorptions are located close in space and have a chiral mutual alignment that they cannot be excited independently; the interactions between their transition dipoles account for substantial rotational strengths, i.e., interactions through a symmetric and antisymmetric dipole-dipole coupling manner, often surpassing those connected with the perturbations on each chromophore produced by the chiral non-chromophoric skeleton.
These excited states will delocalize over all chromophores within the system and become an exciton coupling. This produces a split of the excited state energy level (∆λ Davydov splitting) for this transition with an enlargement of the corresponding UV absorption band while the CD band becomes bisignate (Fig. 16). The intensity of such a CD split is inversely proportional to the square of the inter-chromophoric distance. The exciton couplings interact with each other, thus generating a characteristic pair of intense ECD bands with opposite signs and comparable band areas at shifted wavelengths, called an ECD exciton couplet (Pescitelli et al. 2011). Initially, these coupled chromophores can be present in the molecule or otherwise be introduced by simple organic reactions to suitable functional groups, such as hydroxyl, amino, or carboxyl substituents (Mándi and Kurtán 2019).
The ECD exciton couplet displays a sign which correlates with the absolute angle of the twist defined by the two electric transition dipole moments (TDM) in a fashion recapitulated by the exciton chirality rule (Fig. 16). A positive couplet signifies that the two TDM outline a positive chirality, namely, when viewed along the line connecting the dipoles, one would need a clockwise rotation to move from the dipole in the front onto that in the back. When viewing the CD spectrum from the longer to the shorter wavelengths, the first band of the CD couplet shows a positive Cotton effect followed by a negative one; the CD is said to represent a positive chirality. In this case, a mutual orientation of the two chromophores corresponds to a clockwise twist between the respective transition moments (Fig. 16). And, vice versa, the CD spectrum with the first negative CE indicates a negative chirality, and hence an anticlockwise twist for the two chromophores. Therefore, the chirality defined is an outcome of both the conformation and the absolute configuration of the analyzed molecule. If a good molecular model is available, looking at the ECD spectrum will provide an immediate absolute configuration assignment (Pescitelli 2022).
The following conditions are necessary to consider for the correct use of the CD exciton chirality method (Pescitelli 2022): the existence of two identical or structurally and electronically comparable chromophores, the CD couplet should be isolated from additional strong CD bands, the knowledge of the molecular conformation, together with the certitude that the CD exciton bands dictate the ECD spectrum, namely other optical activity mechanisms of intrinsic chirality could be ignored such as chromophore distortion or conjugation extension. Quasi-degenerate exciton coupling in polyenone derivatives has been described; as well, the nondegenerate exciton coupling may also occur between any two different chromophores with suitable electronic-dipole properly arranged in space to allow transition dipoles, as enones and more extended conjugated systems, such as dienones, trienones, and so on (Pescitelli 2022). An example of the use of this CD exciton chirality method was the determination of the absolute configuration of a phenyl-substituted chiral dihydrofuroangelicin, 4-methyl-8-(2-E-phenylethenyl)-8,9-dihydro-2H-furo[2,3-h]-1-benzopyran-2-one, by taking advantage of the existence of the two aromatic chrormophores in the structure, the coumarin and the styrene, which produce strong UV absoprtions, as illustrated in Fig. 17(A). The ECD spectrum showed a positive couplet between 230 and 350 nm with an amplitude A = + 15.7 ∆ε for the distance between the CD curve peak and trough; the absolute configuration of the depicted enantiomer was determined to be C-8(S) (Pescitelli et al. 2003). Another example is gymnocin-B , a polyether marine toxin illustrated in Fig. 17(B), and by making use of the presence of two hydroxyl groups at positions C-10 and C-37, two chromophore groups were introduced, p-meso-triphenylporphyrin-cinnamate. By applying the exciton chirality method, a spectrum with a positive Cotton effect was obtained, which led to the assigning of the configuration absolute as (S) in both position C-10 and C-37 (Tanaka et al. 2005).

Vibrational Circular Dichroism
In addition to the traditional chiroptical methods described in the above sections, vibrational optical activity (VOA), represented by vibrational circular dichroism (VCD) and Raman optical activity (ROA), has demonstrated a gradually important protagonism in modern stereochemical analysis. VCD and ROA are associated to the vibrational transitions within the same electronic ground state of a molecule while ORD and CD are associated with transitions between the electronic states of a molecule. VCD deals with differential absorptions of left-and right-circularly polarized infrared radiation, by analogy with ECD, whereas ROA is expressed as a differential scattering for left-and right-circularly polarized components of visible light. These methods similarly permit determining the absolute configuration of organic chiral molecules due to the probability of complementary analysis with high-level quantum calculations. Additionally, in comparison with ECD curves, vibrational optical activity spectra contain much more transitions, through which the absolute stereochemistry can be determined. Furthermore, from a theoretical point of view, VCD spectra are easier to calculate than ECD curves, since only ground electronic state properties are considered (Maksimenka 2010). For a recent analysis of the use of the vibrational chiroptical spectroscopic methods for the stereochemical elucidation of natural products, the reader may refer to the comprehensive reviews by Batista Jr. et al. (2015) and Joseph-Nathan and Gordillo-Román (2015).
Vibrational circular dichroism (VCD) is an extension of ECD from the UV to the IR regions. The great advantage of this change is that in the IR region, the vibration modes of atoms are observed, and therefore, in principle, the method is quite general for organic molecules. In most cases, the VCD methodology does not use model compounds since the absolute configuration follows from comparison of the experimental spectrum and a spectrum generated from quantum mechanical (QM) calculations (Batista et al. 2021). VCD can be used with almost any chiral molecule from natural origin (Batista Jr and da Silva Bolzani 2014), and there is no need for the presence of a chromophoric unit, derivatization, and/or simulation of excited states. Furthermore, VCD spectra show fully detailed bands, even at the fingerprint region (Fig. 7), which can be simulated in DFT calculations; even saturated optically active molecules can be analyzed, which is not possible with ECD.
This chiroptical method has some drawbacks as compared to the advantages offered by the VCD (Table 3). The intensity of the VCD bands, in comparison to the corresponding IR transitions, is more than 10 4 times weaker, and consequently, VCD measurements require a comparatively higher sample concentration ( ∼ 0.01-0.1 M), and longer acquisition time ( ∼ 1-12 h). An expensive and unusual instrumention is also required, and any simple method exists for interpretation of VDC results since comparison with QM predicted VCD spectra is always required. A higher sample concentration can induce aggregation or other unfavorable intermolecular interactions in the presence of unprotected carboxyl and hydroxyl groups, which are difficult to predict by computation. A strongly coordinating solvent such as DMSO can alter the Boltzmann distribution remarkably and change the conformation of a molecule, which is why ECD and VCD calculations are difficult to interpret in this type of solvent. Deuterated analogs of the common IR solvents, such as CDCl 3 and CD 2 Cl 2 , can be used to study the VCD regions, where regular non-deuterated solvents have interfering absorption bands. Despite the potential advantages, the use of VCD for elucidation of absolute configuration of natural products is still limited Mándi and Kurtán 2019).

Nuclear Magnetic Resonance
Nuclear magnetic resonance (NMR) is a potent and attractive device, since it is a non-destructive methodology, for analyzing the structure and dynamics of molecules in solution, but it is not uncommon for some cases that the structural assignment is incomplete or incorrect. The structural elucidation of natural products presents several challenges because of their inherent architectural complexity and often with unprecedented structures, high C/H ratio, and high structural flexibility with numerous conformations. In addition, temperature and solvent effects on 1 H-NMR chemical shifts are observed and the differences may be detected depending on the solvation state of the analyzed compounds, especially for hydroxylated compounds (Mari et al. 2019). Further additional challenges must also be confronted when the information collected from the NMR spectra is interpreted into a 3D structure, such as molecules with separated stereoclusters (Tarazona et al. 2018), further complicated by sample availability, signal overlapping, and impurities. Substantial NMR improvements in mass sensitivity of natural products at the nanomole scale (submilligram samples) have been achieved through low-volume tubes and capillary probes, coupled with cryogenic cooled radiofrequency probes of high-field instruments (300-1200 MHz) (Molinski 2010;Krunić and Orjala 2015).
Several NMR parameters such as chemical shifts, the nuclear Overhauser effects (NOEs), spin-spin couplings (J-based analysis), and anisotropic NMR parameters (residual dipolar couplings, RDCs) have been employed to establish empirical correlation with structural data for natural products. NMR parameters such as two-and three-bond proton-proton ( 3 J HH ) and carbon-proton ( 2,3 J CH ) coupling constants, NOEs, and, to a lesser extent, RDCs (Liu et al. 2017;2019) provide the most valuable information for conformational and/or configurational assignments of rigid and cyclic structures with an expected major conformation. However, the assignment of the absolute configuration of flexible multichiral acyclic systems is not an easy task since it is necessary to assume a thermal equilibrium where the estimation of a properly conformational distribution of flexible systems in solution must recognize the contribution of each conformer by the corresponding Boltzmann weighting factor w i (Zanardi et al. 2020).
The J-based analysis allows determination of the relative configuration in acyclic systems (Barone et al. 2002;Bagno et al. 2006;Menna et al. 2019). 3 J HH coupling constants offer valuable information in conformational and stereochemical studies as they provide relevant geometric information. The 3 J HH coupling constants are directly related to their dihedral angles through the Karplus equation and depend on molecular parameters such as the type of substitution, bond angles and lengths, electronegativity, and the relative position of the substituents attached to the H-C-C-H fragment; also, the heteronuclear coupling constants ( 2,3 J CH ) follow a relationship similar to that of Karplus and can therefore be used to discriminate additional angular constraints (Palermo et al. 2010). The observed coupling constant is used to differentiate between the lowest-energy rotamers around the bond, thus defining at the same time both the preferred conformation and the relative conformation.

NMR Quantum Calculations
Quantum mechanical-NMR calculations of chemical shifts and scalar coupling constants appeared as ideal complements to simplify the elucidation process when experimental NMR data is inconclusive. The power of NMR spectroscopy has been greatly improved by the application of theoretical quantum mechanics parameters calculated mainly with density functional theory (DFT) and Hartree-Fock (HF) protocols (Lodewyk et al. 2012;Sarotti 2016, Semenov andKrivdin 2019). In most cases, the calculated DFT coupling constant ( 3 J HH ) and GIAO (gauge including atomic orbitals) calculation of 13 C chemical shifts are compared with the experimental values by means of basic statistical analysis (Chini et al. 2008;López-Vallejo et al. 2011;Juárez-González et al. 2015), allowing the determination of the correct configuration among several structural hypotheses (Grimblat et al. 2019;. The reader interested in J-based configurational analysis may consult the authoritative review by Bifulco and co-workers (2007).
Numerous significant reports have demonstrated that quantum mechanical-NMR approaches provide NMR accurate predictions to differentiate among closely related isomers of natural products (Bagno et al. 2006;Bifulco et al. 2007;Costa et al. 2021). The major advantage of this computational NMR approach is the opportunity of completing the structural elucidation of natural products without requiring authentic samples. Currently, most DFT protocols for the prediction and simulation of NMR chemical shift calculations have been mainly devoted to exploring the absolute configuration of rigid molecules with few conformations. A quick geometry optimization for the analyzed molecule via molecular mechanics (MM) is initially executed with a suitable molecular mechanics force field. The use of MM3 force field and derivatives such as MMFF94 is especially popular since they are well parameterized for a large collection of chemical structures. Solvation effects unquestionably influence the experimental chemical shifts in molecules with polar character. The simplest way to consider solvation in quantum calculations of chemical shifts is to use implicit solvation models as the polarized continuum (PCM) family than the corresponding gas phase calculations. Recently, the comparison of the differences between the calculated/experimental chemical shifts instead of the shifts themselves led to the advantage of avoiding errors arising from calibration procedures, reducing systematic errors and highlighting the most diagnostic differences . Through DFT-NMR calculation, in conjunction with chiroptical spectroscopy, and X-ray crystallography, the 3-D structure of various natural products with complex structures has been established and revised (Chhetri et al. 2018;Menna et al. 2019;Costa et al. 2021). The achievement of a structural accuracy only depends on the comprehensiveness of the correct analysis of all experimental evidence (Pauli et al. 2016;Marcarino et al. 2020b).
A well-established computer-assisted structure elucidation (CASE) methodology has also been applied to solve the structure elucidation problems by comparing experimental results with the quantum-chemical NMR calculations (Elyashberg et al. 2010). These algorithms can derive all plausible structures from a set of structural constraints, defined by the available spectroscopic data and associated chemical structure information. Currently, a wide variety of powerful methods, the so-called expert systems (ESs), intended for the purpose of molecular structure elucidation from spectral data have been developed and are available for molecules with large conformational freedom (Grimblat and Sarotti 2016), including Artificial Neural Networks-Pattern Recognition Analysis (ANN-PRA) (Zanardi and Sarotti 2015), Case 3D (Navarro-Vázquez et al 2018; Koos et al. 2020), and DU8 + (Kutateladze et al. 2019). Another type of popular toolboxes to correlate one set of experimental data to two or more sets of calculated NMR data is based on Bayesian statistics, represented by DP4 (Kutateladze et al. 2019), and its multiple updates, such as J-DP4 (Grimblat et al. 2019;Marcarino et al. 2020a).
Outline for NMR Calculation Workflows for the stereochemical elucidation of natural products with multiple asymmetric centers by NMR quantum calculations, the so-called DFT-NMR methodology, have been previously described (Di Micco et al. 2010;Petrovic et al. 2010;Bally and Rablen 2011;Willoughby et al. 2014;Kim et al. 2020;Costa et al. 2021;Semenov and Krivdin 2021). In brief, the first step is the establishment of the initial relative stereochemical structure of a natural product based on a detailed 1 H and 13 C NMR spectral analysis with the assistance of a variety of 2D-correlation spectroscopy methods ( 1 H − 1 H, COSY, TOCSY, NOESY/ROESY, HSQC, and HMBC) supported by the calculation of the molecular weight of the analyte which is inferred by high-resolution mass spectrometry (Jarmusch and Cooks 2014 This approach provides an unambiguous solution for each set of calculated NMR data for all possible diastereoisomers of a candidate molecule. However, since the DFT 3 J HH values mainly depend on the H-C-C-H dihedral angles, they have been primarily used by our group to validate the conformational analysis performed by quatum-NMR calculations in highly flexible multichiralcenter small molecules, since they improved the accuracy for stereochemical predictions over the 13 C chemical shifts (López-Vallejo et al. 2011;Suárez-Ortiz et al. 2013;Juárez-González et al. 2015;Martínez-Fructuoso et al. 2019. A crucial issue that needs to be addressed is the accuracy of the calculated chemical displacements and coupling constants. It is always necessary to evaluate a statistic term that expresses the intrinsic precision for the calculation model (Navarro-Vázquez 2021). There are several classical statistical methods, e.g., absolute difference, medium absolute error (MAE), conditional-mean-adjusted estimator (CMAE), root mean square deviation (RMSD), r-squared (R 2 ), or chi-square statistics (X 2 /n), and recently developed probabilistic scores (e.g., CP3, DP4, DP4+, and J-DP4) to evaluate computation results and identify the correct isomer (Kim et al. 2020;Marcarino et al. 2020b). Frequently, a graph of calculated vs experimental data is presented, and the accuracy is judged according to (a) the linear correlation between the two; (b) a statistical parameter such as the correlation coefficient which must be sufficiently high; (c) the average absolute error that must be minimal; and (d) the slope that must be near the unit and the intersection close to zero for R 2 . Convergence between DFT-calculated and experimental 1 H-1 H NMR coupling constant values and chemical shifts occurs from the exact calculation for each conformer's geometry; the precise estimation of the free energies and Boltzmann population for each analyzed stereoisomer; and the appropriate selection of the coupling constant calculation method, as B3LYP/DGDZVP for all the discussed examples in the present review.

H NMR Diamagnetic Anisotropy
In the internal reference method, it is essential to correlate the relative configuration against the internal reference of a derivatizing agent with a known absolute configuration. Therefore, it is an indirect NMR-based method for determining the configuration of an unknown stereogenic center, predominantly secondary alcohol R 1 R 2 CHOH (R 1 ≠ R 2 ) or the analogous amines (where OH is replaced by NH 2 ). The most commonly used derivatizing agents for alcohols, amines, thiols, β-chiral primary alcohols, and cyanohydrins are α-methoxy-α-trifluoromethyl phenylacetic acid (MTPA), α-methoxyphenylacetic acid (MPA), 1-naphthylα-phenylacetic acid (1-NPA), 2-naphthyl-α-tert-butoxyacetic acid (2-NTBA), and 9-anthrylmethoxyacetic acid (9-AMA), while the hydroxyl group of ethyl 9-anthryl-2-hydroxyacetic acid ethyl ester (9-AHA) allows the assignment of chiral methine bearing a carboxylic group (Seco et al. 2004(Seco et al. , 2012(Seco et al. , 2015. The most popular NMR-based Mosher ester analysis, originally reported by Dale and Mosher (1973), relies on the fact that the protons in diastereomeric α-methoxy-αtrifluoromethylphenylacetic acid (MTPA) esters, i.e., those derived from combination of the carbinol under examination with (R)-and (S)-MTPA chlorides, display different arrays of chemical shifts in their 1 H NMR spectra. The ΔδRS value is calculated for the protons on both substituents ΔδRSR 1 and ΔδRSR 2 , where ΔδRS is defined as the difference between the proton chemical shift in the (S)-ester derivative (δS) and in the (R)-MPA derivative (δR), i.e., (∆δ H S-MTPA-R-MTPA) (Fig. 18). When a chiral compound (A*) of unknown absolute configuration is derivatized with both (S) and (R)-MTPA, the favored conformation for both diastereoisomer A*-(S)-MTPA and A*-(R)-MTPA esters is determined by the syn-coplanarity (0 • dihedral angle) for the carbonyl group plane with both the proton on the stereocenter and the CF3 moiety (Fig. 18). In this highly favored conformation that dominates the NMR spectra, the R 1 substituent in A*-(S)-MTPA faces the benzene ring which induces an up-field shielding of the R 1 protons. Instead, the methoxy group does not exert any shielding effect on the R 2 substituent. The opposite effects are evident in the A*-(R)-MTPA derivative where R 2 faces the phenyl, and its signals are consequently up-field shifted (Cimmino et al. 2017). Consequently, with compound A* (Fig. 18), protons in the right side of the MTPA plane must present positive values (∆δ H S-MTPA-R-MTPA > 0) and protons on the left side of the MTPA plane must display negative values (∆δ H S-MTPA-R-MTPA < 0). Accordingly, by recognizing the shifting effect sign for a given substituent in the two diastereomeric MTPA derivatives, it is possible to derive its position at the stereogenic center by applying the conformation models described by Ohtani et al. (1991), as illustrated in Fig. 19.

X-Ray Crystallography of Selected Terpenoids
Terpenoids are the largest class of natural products and represent a rich reservoir of candidate compounds for drug discovery. Numerous bioactive terpenoids have been isolated from plant, including monoterpenoids, sesquiterpene lactones, diterpenoids, triterpenoids, and tetraterpenoids, many are relevant natural products because of the ethnopharmacological importance of their plant sources, where sesquiterpene lactones and triterpenoids are most extensively studied especially in anti-cancer research. They are biosynthesized by plants, animals, and fungi via the HMG-CoA reductase or the deoxy-xylulose phosphate pathways with isopentenyl diphosphate and dimethylallyl diphosphate as the building blocks.
The best example for the isolation of a bioactive terpenoid based on traditional therapeutic knowledge is Artemisia annua L., Asteraceae, or sweet wormwood. The use of this herb for the treatment of malaria comes from traditional Chinese medicine as first described in "A Handbook of Formulas for Emergencies" by Ge Hong (283-363 CE). The active compound artemisinin has been a crucial molecule in the treatment of malaria and the first antimalarial drug of the endoperoxide class, owing to the presence of a peroxide containing a 1,2,4-trioxane ring presumed to act as the pharmacophore group of the molecule. Figure 20 illustrates the artemisinin structure for which the location of the peroxyl linkage and the relative structure of artemisinin were determined by spectroscopic analysis (NMR) and chemical reactions (Guo 2016). The absolute configuration was determined by ORD for the n → π* Cotton Fig. 19 1 H NMR spectra of S-MTPA (cyan blue) and R-MTPA (red) ester derivatives for the trans-diaxial 1,2-diol, ( +)-1S, 2S, 4R limonene-1,2-diol, in CDCl 3 (300 MHz). The preparation of these derivatives was performed in NMR tubes, and 1 H spectra were recorded directly without purification of reaction mixtures. The Ohtani conformational models are depicted; ∆δ H SR chemical shifts (in ppm) are also inserted in the 2D conformation for the MTPA ester of the trans-diaxial 1,2-diol effect of the δ-lactone moiety in conjunction with X-ray diffraction analysis of crystals obtained from 50% H 2 O/ EtOH with an orthorhombic P2 1 2 1 2 1 unit cell at the Institute of Biophysics, Chinese Academy of Sciences. Recrystallization from cyclohexane provided a polymorphic crystal belonging to the triclinic space group P1. The conformations of both polymorphs of artemisinin in solid state are quite similar but with small differences in bond angles and torsion angles between the two which accounted for the higher solubility and a faster dissolution rate of the triclinic crystals. The triclinic crystals produced a higher maximum aqueous concentration of 48 μg/ml after 4 h at 37 °C, whereas the concentration of orthorhombic crystals was 20 μg/ml after 18 h at the same temperature (Chan et al. 1997).
Nevertheless, issues with solubility and extensive first-pass metabolism made artemisinin somewhat unsuitable as a therapeutic medicine. The first generation of semisynthetic derivatives, dihydroartemisinin (DHA), artemether, artether, and artesunate, was designed with clinical use (Woodley et al. 2021). However, poor solubility of the artemisinin-ethers necessitates administration by intramuscular injection or oral routes which is not ideal for the rapid systemic exposure required for clearing parasite burden in severe malaria. Artesunate is the succinic ester of DHA displaying greatly improved aqueous solubility which enables administration by intravenous (IV) infusion allowing for effective treatment of severe malaria. The terpenoids exemplified in this section were chosen to illustrate bioactive natural products from the Mexican flora through the resolution of their relative and/or absolute configuration by X-ray diffraction and, as instances, for demonstrating their chemical diversity and disclosing the structural isomerism among plant terpenoids such as epimers, diastereoisomers, and regiosiomers, as discussed below.

Sesquiterpene Lactones
Sesquiterpene lactones represent a large group of secondary metabolites that are widely distributed in several angiosperm plant families and a few bryophytes, including liverworts. These metabolites are particularly diversified in the family Asteraceae, in which more than 5000 compounds have been reported so far (Adekenov 2017), which display a wide range of protective activities in plants, such as anti-herbivory and antimicrobial substances or inhibiting the growth of competing plants (Padilla- Gonzalez et al. 2016). Most of them exhibit antimicrobial, antifungal, antifeedant, insecticidal, antioxidant, anti-inflammatory, antitrypanosomal, antimalarial, neuroprotective, hepatoprotective, immune-stimulant, cytotoxic, and antitumor properties (Moujir et al. 2020). These biological activities are essentially associated to their characteristic α-methylene-γ-butyrolactone functional group, the structural requirement for their pharmacological activities, which participate in Michael-type additions with biological nucleophiles of proteins, especially sulfhydryl groups. Therefore, sesquiterpene lactones are alkylating agents that form covalent adducts in vivo and inhibit enzymes and key proteins. They are also potent apoptotic inducers in several cancer cells (Quintana and Estévez 2018). This type of cell death is recognized as a property that is useful for identifying anti-cancer drugs.
Investigations on the long-known antitumoral potential of sesquiterpene lactones have recently received new impetus by the finding that certain compounds of this class possess a hitherto unknown mechanism of action that may make them interesting leads, or even therapeutic agents, against certain types of leukemia and some other tumors known to be characterized by an excessive activity of the transcription factor c-Myb with important roles in cell proliferation and differentiation by controling gene expression in a wide variety of cell types. Sesquiterpene lactones were discovered as the first type of low-molecular-weight inhibitors of c-Myb and identified as an attractive drug target (Schmidt 2018). Recently, a review of the most relevant techniques that have been used for the determination of the absolute configuration of sesquiterpenes has been published (Pardo-Novoa and Cerda-García-Rojas 2021).
Incomptines A and B (9 and 10) are two heliangolide-type sesquiterpene lactones derived from Decachaeta incompta (DC) R.M.King & H.Rob., Asteraceae. The structure of 10 was first directly established by NMR analysis and X-ray diffraction, while for 9, its structure was determined by the observed differences in the 1 H and 13 C NMR spectra and EIMS spectrum with 10 (Bautista et al. 2012a). Recently, incomptines A (9) and B (10) have been identified as interesting leads due to their biological properties as antibacterial, antiprotozoal (Calzada et al. 2009), anti-diarrheal (Calzada et al. 2020), phytotoxic (Rial et al. 2016), cytotoxic, and antitumoral agents Pina-Jimenez et al. 2021). Both compounds are easily distinguished by 1 H NMR; for compound 10, the H-8 signal appears at δ H 4.18 (broad singlet), while for compound 9, this signal is downshifted at δ H 5.20 (ddd, J = 4.5, 3.0, 0.5 Hz), and the methyl of the C-8 OAc group has a chemical shift of δ H 2.05. Incomptine B (9) is the main constituent in the leaves of the plant and is obtained in high yield (1-1.5% of dry weight), which has allowed the preparation of 10 by acetylation, and for the semi-synthesis of 8-O-acyl and 8-O-alkyl derivatives (Bautista et al. 2014b).

Diterpenoids
Diterpenes are a class of structurally diverse C 20 compounds widely distributed in nature and composed of four isoprene units. In nature, they are commonly found in a polyoxygenated form with keto and hydroxyl substituents, which can be esterified by small-size aliphatic or aromatic acids.
Although thousands of diterpenes have been isolated from plants, only few of them became clinically valuable (Lanzotti 2013). Mainly, the drug taxol from the bark of the Pacific yew, Taxus brevifolia Nutt., Taxaceae, used in therapy against ovarian, breast, and lung cancer, with its synthetic water-soluble analog taxotere (docetaxel), is an example of unusual structure discovered from nature and used as medicine. Taxol was the first microtubule-targeting agent described in the literature, with its main mechanism of action consisting of the disruption of microtubule dynamics, thus inducing mitotic arrest and cell death, making it one of the most widely employed antitumoral drugs (Gallego-Jara et al. 2020).
Salvinorin A (13) is perhaps the most representative example of a bioactive neo-clerodane diterpenoid. It was isolated as the major constituent from the leaves of Salvia divinorum Epling & Jativa, Lamiaceae (Ortega et al. 1982), a medicinal psychotropic plant used by the Mazatec ethnicity in most of the northern part of the state of Oaxaca, Mexico, since pre-Hispanic times (Wasson 1962). Modern ethnobotanical use of this plant in its endemic region includes small doses to cure dysfunctions such as diarrhea, swollen belly, headaches, and rheumatism (Casselman et al. 2014). Today, this sage is also used for divination and spiritual healing in shamanic ceremonies and religious rituals product of the contemporary syncretism in Christianity among the Mazatec community. A water potion of crushed leaves produced short-lasting light-headedness, dysphoria, tactile and proprioceptive sensations, a sense of depersonalization, amplified sound perception, and an increased visual and auditory imagery with concomitant disorientation, but not actual hallucinations (Díaz 2013).
Two decades after reporting the chemical structure of salvinorin A (13) by X-ray diffraction (Fig. 22), this natural diterpenoid was confirmed as responsible for the hallucinogen effect of the plant and its molecular mechanism of action was elucidated as the first nonalkaloidal kappa-opioid-mediated psychotropic molecule (Roth et al. 2002). The absence of a protonated amino group, common to all previously naturally occurring hallucinogens, such as N,N-dimethyltryptamine, psilocybin, and mescaline (alkaloidal hallucinogens which interact with specific serotonin receptor subtypes), results in a fast metabolism with its parallel elimination and simultaneous loss of activity. Salvinorin A (13), as a selective kappa-opioid-receptor (KOP) agonist, does not elicit an addictive effect and, therefore, is considered the most potent known psychotropic agent of natural origin (Yan and Roth 2004). Hence, S. divinorum has gained increased popularity as a recreational drug, used as an alternative to marijuana, which accounts for its misuse and categorization as a drug of abuse now banned in many countries (Zawilska and Wojcieszak 2013).
The establishment of the salvinorin A (13) psychotropic effects allowed the development of semisynthetic derivatives and their application to treat NCS disorders such as depression, as well as for the treatment of drug addiction (Prisinzano and Rothman 2008;Hernández-Alvarado et al. 2020). Several synthetic efforts have been focused on the improvement of physicochemical and biological properties of salvinorin A (13): from total synthesis to hundreds of analogs. The pharmacology of analogs modified at C-2 dominates the literature when compared to the ones from other positions, such as modification at C-1, C-4, C-12, and C-17, which have been mostly associated with a reduction in KOP binding affinity (Zjawiony et al. 2019). The distinctive highest KOP binding affinity of these C-2 analogs seems to correlate with their chemical structure and in vivo antinociceptive effects, such as those described for 2-O-cinnamoylsalvinorin B with moderate dual agonism to the subtypes of opioid receptors MOP/KOP to retain analgesic effect without addiction (Roach and Shenvi 2018;Zjawiony et al. 2019). In addition to salvinorin A (13), the structural determination through single-crystal X-ray diffraction analyses of natural products derived from the Mexican flora has been extensively applied to the study of neo-clerodane and rearranged neo-clerodane diterpenoids, which are the most common types of diterpenoids isolated from Salvia species endemic to Mexico. Chemical structures of tehuanins A (14) and E (15) obtained from S. herbacea Benth. (Bautista et al. 2012b) and sepulturins A-D (16-19) isolated from S. shannonii Donn.Sm. (Bautista et al. 2013a) have been confirmed by X-ray diffraction analyses.
The establishment of the absolute configuration of terpenoids based only on X-ray diffraction analyses have been determined by anomalous dispersion using heavy atoms and by the measurement of the Flack parameter. Thus, the absolute configurations of infuscatin (20) Likewise, crystal X-ray diffraction analyses are an excellent analytical tool for the structural elucidation of natural products containing new carbonated frameworks and the establishment of their absolute configurations, as occurred with microphyllandiolide (28, Fig. 24), a diterpenoid with microphyllane backbone derived from S. microphylla Kunth aerial parts (Bautista et al. 2013b). Compound 28 displayed 1 H and 13 C diagnostic signals of a neo-clerodane diterpenoids that include a furan ring (δ H 7.41, m, H-16; 7.37, t, J = 1.5 Hz, H-15; 6.36, dd, J = 1.5, 1.0 Hz; δ C 124.4, C, , and a δ-lactone (δ H 5.25, dd, J = 12.5, 3.5 Hz, H-12; δ C 173.3, C, C-17; 69.8, CH 2 , C-12). However, the presence of an olefinic quarternary carbon at δ C 160.6 (C, C5) indicated a 5,10-seco-neo-clerodane (Ortega et al. 2006;Bautista et al. 2014a, b). The heteronuclear HMBC correlations of H-1 (δ H 5.99, dd, J = 11.0, 6.5 Hz) with C-8 (δ C 32.8, C) indicated the presence of a cyclopropane moiety and suggest a new carbonated framework, named microphyllane, additionally confirmed by X-ray diffraction (Fig. 24) (Bautista et al. 2013b). The biosynthesis of rearranged neoclerodanes, such as the microphyllane diterpenoids represented by microphyllandiollide (28), has been proposed since specific neo-clerodanes, also present in the same plant, could play a role as precursors though pericyclic reactions that produce unexpected and uncommon new frameworks (Gonzalez et al. 1988). Thus, the biosynthesis of 28 could be initiated with salvimicrophyllin C through an electrocyclic opening of the decalin A and B rings to give rise to the triene (a), as a hypothetic intermediate, followed by the C-8 epimerization to produce salvimicrophyllin A. This natural compound can also be the object of a cyclopropanation to generate the intermediate (b), which by an allylic oxidation would generate microphyllandiollide (28) (Fig. 24) (Bautista et al. 2014a(Bautista et al. , 2013bPosada-Salgado et al. 2015).
Teotihuacanin (29) is another example of a carbonated backbone derived from the neo-clerodane class. It was isolated from  Ortega (Bautista et al. 2015), and as demonstrated for microphylladiolide (28), it displayed the characteristic functional moieties of neo-clerodanes and the 13 C NMR signals that indicated a 5,10-seco function (δ C 157.6 and 133.9 for C-5 and C-10). The 2D NMR correlations displayed in the HMBC spectrum of 29 between H-10 (δ H 5.52, d, J = 17.2 Hz) and C-20 (δ C 65.0, CH), and from H-20 (δ H 4.43, s) with C-13 (δ C 123.7, C) and C-16 (δ C 150.9), evidenced the existence of a C-C bond between C-20 and C-16, and in consequence the presence of spiro-cyclohexene moiety in the new framework, named amarisane. The structure of 29 and its absolute configuration was established by single-crystal X-ray diffraction using Cu radiation (Fig. 25)  . As occurred with microphyllandiollide (28), the biosynthesis proposed for teotihuacanin (29) could proceed via the electrocyclic opening of the decalin ring in the intermediate (a) to produce the triene (b), followed of the addition of the furan ring to the carbonyl at C-20 to give 29 (Fig. 25). Alternatively, in intermediate (a), the addition of the C 12 -OH to the carbonyl at C 20 also could occur to generate amarissinin B (Fig. 25)  .
In addition to amarissinin B and teotihuacanin (29), other neo-clerodane diterpenoids derived from S. amarissima, can be biogenetically correlated through the hypothetic intermediate (c). This intermediate could be subjected to a lactonization to produce amarissinin C which, in turn, would be oxidized to generate the reaction intermediate (d) and, after a dehydratation, produced amarissinin D. Then, the H-8 acid proton abstraction would generate a 9,10-seco-neo-clerodane intermediate (e) to produce amarissinin A (23). In a second and independent sequence of reactions, the intermediate (c) could be oxidized to give the amarissinin E analog (f) and be subjected to an O-glucosylation and further acylation to generate amarisolide F (Fig. 26).
Single-crystal X-ray diffraction application, alone or in combination with NMR and HRMS analyses, could be complemented with the use of adequate chromatographic methods to complete the resolution of natural products contained in complex mixtures of epimers, diastereoisomers, and regioisomers. Some examples were selected to illustrate the resolution of isomeric mixtures by chromatographic methods and subsequently analyzed by NMR, HRMS, and X-ray diffraction. One example where it was not possible to accomplish the chromatographic separation of isomers is also presented. These examples include the establishment of the absolute configurations by mainly using the Flack parameter.
Salvinicins A (36) and B (37) are two diastereomer analogs of salvinorin A (13) derived from S. divinorum (Harding et al. 2005). Each constituent was successfully isolated by chromatographic methods, and their structural differences were determined by NMR analysis. Compounds 36 and 37 differed by the orientation of their substituents at C-15 and C-16 of the neo-clerodane framework (36: δ H 4.93, d, J = 3.5 Hz, 4.70,s,δ C 110.6,107.9,37: δ H 4.92,d,J = 3.4 Hz,4.94,s,δ C 111.3,108.4,. In addition, the structure of 36 was successfully confirmed by X-ray diffraction. A second and rare case of diastereoisomerism is represent by hirsutolides A-D (38-41), which were isolated as a solid mixture containing four microphyllane diterpenoids derived from S. hirsuta Jacq (Toscano et al. 2020). The structures of these compounds were deduced by the observation of just one ammonium adduct at m/z 406.1493 [M + NH 4 ] + , consistent with the molecular formula C 20 H 24 NO 8 . Its 13 C NMR spectrum displayed similar chemical shifts for the signals at C-13, C14, C-15, and C-16 of the four diastereomers, as well as a pair of signals for those assigned to C-1, C-2, and C-3. This isomerism was clearly demonstrated by subsequent analysis through single-crystal X-ray diffraction (Fig. 28).

5,6-Dihydro-2H-pyran-2-ones
Members of the genus Hyptis from the mint family (Lamiaceae) contain a group of chemical markers with a 6-heptyl-5,6-dihydro-2H-pyran-2-one skeleton, which is responsible for their antimicrobial (Pereda-Miranda et al. 1993;Fragoso-Serrano et al. 2005) and cytotoxic properties (Falomir et al. 2003). The pharmacophore for this class of active plant principles corresponds to the α,β-unsaturated δ-lactone system, a well-known Michael acceptor, which is complemented with the lipophilicity dispensed by the Scheme 2. Examples of 5,6-dihydro-2H-piran-2-ones from the mint family with their revised structures by DFT-NMR though calculation of the Boltzmann-averaged 3 J HH values 6-alkyl substituent to enable interactions with cell membrane (Bjerketorp et al. 2017).
An exhaustive conformational search was conducted using molecular mechanics (MMFF94), followed by a geometric optimization using DFT B3LYP and the DGZVP bases, to obtain the vibrational frequencies and thermodynamic parameters at 298 K and 1 atm by the GIAO method (Gauge Independent Atomic Orbital). The Boltzmann-averaged 3 J HH Scheme 3. Four candidate stereoisomers at the side chain (SA-1-SA-4) for synargentolide A with the correct biogenetic (S)-configuration at C-6′. Structure for synthetic stereoisomer SA-5 were established to achieve their correct structural assignment and absolute configuration (Scheme 2). For the geometry optimization of this class of flexible multichiral center compounds with the application of ab initio methods, the C(5)-C(6)-C(1′) = C(2′) and C(1′) = C(2′)-C(3′)-C(4′) dihedral angles were rotated in steps of 180° and the remaining dihedral angles of the heptenyl moiety were rotated in steps of 120°, starting at 60° for each bond. The acetyloxy H-C sp3 -O-C sp and C sp3 -O-C = O dihedral angles were explored within the range from + 60° to − 60° to find the more favored alignment, and the conformation for the acetyloxy moieties was adjusted to the most favorable anticlinal geometry prior to the minimization procedure but was left without any geometry restriction during the calculations. Conformers with at least one forbidden gauche P ± gauche M sequence which resulted in O//O 1,3 interactions were excluded (Pereda-Miranda et al. 2001). DFT single-point calculations were achieved at the B3LYP/6-31G(d) level theory for each minimum-energy structure falling between E MMFF = 0-5 kcal/ mol and geometry optimization at the B3LYP/DGDZVP level were carried out for all the structures within an E DFT range of 0-3 kcal/mol. This procedure yielded a selected pool of conformers; their Gibbs free energy values (ΔG) and conformational populations (P), together with NMR chemical shifts and coupling constants, were calculated from the minimum-energy structures with the gauge-invariant atomic orbital (GIAO) method.
The first synthesis of synagentolide A (Scheme 3, SA1), a natural product from Syncolostemon argenteus N.E.Br., Lamiaceae, a South African plant (Collett et al. 1998), recognized that the absolute configuration did not correspond to the originally described as the 6R,4′R,5′R,6′S-stereoisomer (García-Fortanet et al. 2005). All synthetic approaches have also overlooked the biogenetic (S)-configuration for C-6′ present in all natural 6-heptenyl-5,6-dihydro-2H-pyran-2-ones from the mint family (Sabitha et al. 2009(Sabitha et al. , 2011Yadav et al. 2012). Additionally, detailed 1 H NMR chemical shifts and 3 J HH values were not accurately examined to the point that visual comparison of 1 H NMR spectroscopic features was used to describe the signals as multiples for both the natural product SA1 and its synthetic steroisomer SA5 (Sabitha et al. 2009). A detailed analysis of both chemical shifts and coupling constants by nonlinear fitting of the spectra parameters based on the original 1 H NMR plots for the natural product and the synthetic diastereoisomer SA-5 highlighted important differences (Fig. 30) that supported the need for a revision of the original proposed synthetic structure (Juárez-González et al. 2015). Circular dichroism established the 6R-configuration for the lactone chiral center. Therefore, this result permitted the possibility of only four stereoisomers at the side chain (SA-1-SA-4), all depicted in Scheme 3 with the correct biogenetic (S)-configuration at C-6′.  Figure 30 shows the discrepancies established in the 1 H NMR spectra for both H-5′ and H-6′ that are upfield shifted (Δδ 0.04 ppm) for SA-5 in relation to SA-1. Major variations in the 3 J HH values were also registered for proton H-6 and H-4′. There was also an inversion in the value between J 5proR,6 = 5.0 and J 5proS,6 = 9.8 Hz in natural compound SA-1 in relation with the values found in the synthetic isomer SA-5 (J 5proR,6 = 9.7, J 5proS,6 = 5.4 Hz), which modified the shape of these signals in both compounds as expected from the configurational inversion of all chiral centers in the side chain. RMSD statistic comparisons for the DFT computation simulations of four possible diastereoisomers are shown in Fig. 31. Among the four stereoisomeric possibilities, the minimum value was found for the 6R,4′S,5′S,6′S-stereoisomer (Scheme 3, SA-1) because of the match for theoretical and experimental 3 J HH with a RMSD value of 0.66 Hz (Juárez-González et al. 2015). In another case of a flexible 6-heptenyl-5,6-dihydro-2H-pyran-2-one, after the exploration of the full conformational space for eight possible diastereoisomers of spicigerolide (SP-1 to SP-8, Scheme 4), the averaged 3 J HH at the B3LYP/DGDZVP calculation level and the GIAO method with the spin-spin command were used (López-Vallejo et al. 2011). A micro-scale preparation of diastereoisomers SP-2 to SP-4 by epimerization and acetylation of synrotolide, 6R-[3′S,6′-diacetoxy-4′S,5′Sdihydroxy-1′Z-heptenyl]-5,6-dihydro-2H-pyran-2-ones, isolated from a member of the mint family only found in Pondoland, South Africa (Syncolostemon rotundifolius E.Mey. ex Benth.) was used to obtain stereoisomers SP-2 to SP-4. Experimental 3 J HH values for the natural product SP-1 and the semisynthetic compounds SP-2-SP-4 (Scheme 3) were compared to determine the accuracy of the calculations. This comparison was only concentrated on the coupling constants arising from the C(3′)-C(4′)-C(5′)-C(6′) fragment, i.e., J 3-4 , J 4-5 , and J 5-6 , where the stereochemical differences take place along this series of 6-heptenyl-5,6-dihydro-2H-pyran-2-ones, the RMSD values for diasteroisomers SP-1-SP-4 were 0.10, 4.94, 2.47, and 1.77, respectively (Fig. 32). Three scaling factors were found for values of the neighborhood coupling constants 1 H-C-C-1 H that depend on the hybridization of the carbon atom ƒ(sp3)-(sp3) = 0.910, ƒ(sp3)-(sp2) = 0.929, ƒ(sp2)-(sp2) = 0.977, which were used to minimize the mean quadratic error of the calculated values (López-Vallejo et al. 2011). Thus, these results demonstrated an excellent correlation with the correct absolute configuration of natural spicigerolide as diasteroisomer SP-1 (Scheme 4). For the most relevant conformer of spicigerolide, which corresponded to P = 82.3% of the Boltzmann population, a coplanar and fully extended zigzag conformation for the side chain, but with exclusion of 1,3-parallel non-bonded oxygen-oxygen interactions for the acetyloxy substituents at C 3′ -C 6′ , was DFT simulated (Fig. 32).
For the structural elucidation, the 6-heptyl-5,6-dihydro-2H-pyran-2-one skeleton was divided into two stereoclusters, the α,β-unsaturated δ-lactone group and the side chain. The structural elucidation for the cyclic cluster was easily established by the interpretation of the 1 H NMR (relative configuration) and ECD (absolute configuration) spectroscopic data. The pseudo-equatorial side chain at C-6 and the axially oriented substituent at C-5 of the 5,6-dihydro-2H-pyran-2-one were established by the H-5/H-6 coupling constant value (J 5-6 = 2.5-3.5 Hz). This 1 H NMR parameter was correlated with the positive Cotton effect in the CD curve (Δε 260-290 nm) for the assignment of the C-6(S) absolute configuration, as described for the Snatzke's rule relating the sign of the α,β-unsaturated δ-lactone n →π* Cotton effect (Fig. 33) for all 6-substituted 5,6-dihydro-α-pyrones (Davies-Coleman and Rivett 1989). With this semi-empirical rule for the coplanar enelactone system (Snatzke 1968), in which the C-6 atom is displaced by 0.5 Å from the plane containing C-2, C-3, C-4, Fig. 33 A positive Cotton effect in the circular dichroism curve (∆ε 260-290 nm) was correlated with the C-6(S) configuration for pectinolides A-C by application of the semiempiral Snatzke's rule for the coplanar enelactone system in which the side chain must be equatorial Fig. 34 Coplanarity for the -O-C(O)-CH = CH-system as corroborated by X-ray, including the C-6(S) and C-6′(S) configurations found for all 6-substituted 5,6-dihydro-α-pyrones from Hyptis and C-5, when the chiral center C-5 and C-6 are asymmetrically substituted and the relative axial or equatorial orientations of the substituents are known (Fig. 34), the absolute configuration at these centers could be established (Beecham 1972).
The first chemical correlation to complete the elucidation of the absolute configuration for compounds 44 − 47 (Scheme 5) was performed by the ozonolysis of pectinolides A (44) and B (45) to yield ( +)-2-acetyloxy-hexanoic acid (Pereda-Miranda et al. 1993), which was saponified to afford ( +)-2-(S)-hydroxyhexanoic acid ([α] D + 14.8) with a positive Cotton effect (Δε 209 = + 1.58) and a weak negative long-wave length CD max (Δε 244 = − 0.01). This hydroxylated carboxylic acid was also produced by ozonolysis of pectinolide B (45). However, to avoid the possibility of an error in the absolute configuration assignment of these products as a consequence of a probable racemization of the asymmetric center alpha to the carboxyl group, it was decided to confirm the C-3′(S) configuration through derivatization of pectinolide B (45) with (S)-or (R)α-methoxy-α-(trifluoromethyl)phenylacetyl chloride [(S)-or (R)-MTPA-Cl] (Fragoso-Serrano et al. 2005). 1 H NMR chemical shifts data for the diagnostic signals of the (S)-and (R)-MTPA ester derivatives of compound 45, after considering the configurational models described for this methodology (Δδ = δ S-MTPA − δ R-MTPA ) by Ohtani et al. (1991), are Scheme 5. Chemical correlations to confirm the absolute configuration of chiral center C-3′ at the side chain of pectinolides A-D  Fig. 35. It was observed that the cyclic cluster H-3 to H-6 signals, including the H-1′ and H-2′ vinylic signals of the side chain, displayed a positive ∆δ SR value, due to the diamagnetic effect of the Mosher ester aromatic ring. For the rest of the side chain signals (H-4′ to H-7′), negative ∆δ SR values were calculated. Accordingly, the C-3′(S) configuration for the pectinolides A-D (44-47) was finally corroborated by the application of this internal reference method to pectinolide B (45) (Fragoso-Serrano et al. 2005). Finally, on acetylation, pectinolides B (45), C (46), and I (47) afforded the same acetylated derivative identical by GC-MS, TLC, and NMR to pectinolide A (1). Scheme 5 summarizes all the chemical correlations for the elucidation of the absolute configuration of the pectinolide series.
Pectinolide J (48) was found to have the molecular formula C 14 H 20 O 5 , as determined by HRESIMS for the cationized molecule (m/z 291.12061 [M + Na] + ). The 1 H NMR spectrum somewhat differed in the chemical shifts and multiplicities for H-1′, H-2′, and H-3′ signals from those reported for pectinolide C (46) (Fig. 36). The pseudo-equatorial side chain at C-6 and the axially oriented hydroxy group at C-5 were also established by the J 5,6 value of 2.9 Hz. In addition, the C-6(S) absolute configuration agreed with the circular dichroism (Δε 278 + 0.57 DFT chemical shifts and 3 J HH calculations in the gas phase and CHCl 3 solution for the conformational and configurational analysis of compounds 46 and 48 were performed by taking advantage of the fact that the absolute configuration for pectinolide C (46) was unambiguously confirmed. The 1 H NMR chemical shifts and 3 J HH values were sufficiently different between this product and its counterpart with an inverse stereochemistry at C-3′, pectinolide J (48), to permit the appropriate epimer differentiation after comparison with the DFT-calculated values. In contrast, experimental 13 C NMR data was not useful to discriminate between these stereoisomers. This situation was previously reported for pairs of stereoisomers when two chiral stereoclusters are separated by a flexible connective chain (Juárez-González et al. 2015), thus producing identical 13 C NMR spectra but distinguishable 1 H NMR spectra, as observed for 46 and 49. The conformational distributions of 46 and 48 were obtained by application of the previous outline for DFT-NMR calculations. For full details on theoretical chemical shifts calculations for each contributing conformer via geometry optimization with the use of ab initio methods, refer to Martínez-Fructuoso et al. (2019). This procedure yielded 14 conformers for 46 and 20 for 48; their Gibbs free energy values (ΔG) and conformational populations (P), together with NMR chemical shifts and coupling constants, were calculated from the minimum-energy structures with the gauge-invariant atomic orbital (GIAO) method. Subsequently, the minimumenergy structures, chemical shifts, and coupling constants were refined and recalculated with the polarizable continuum model (PCM) using CHCl 3 as the solvent. The major differences were detected in the chemical shift for the C-1′ = C-2′-C-3′-C-4′ fragment of the side chain. Mainly, the C-4′ proR and proS protons (∆δ H-4′proR -H-4′proS = 0.1 ppm) were conclusively distinguished by the DFT chemical shift predictions. The Boltzmannaveraged 3 J HH values for these protons in pectinolide J (48) in CHCl 3 solution were J 3′,4′proR = 5.12 and J 3′,4′proS = 7.26 Hz after considering 20 conformers at the equilibrium, which agreed with the experimental values (J 3′,4′proR = 5.90 and J 3′,4′proS = 7.38 Hz). A low RMSD was obtained when the precise combination of theoretical and experimental 3 J HH values were compared, i.e., 46-theor/46-exp and 48-theor/48-exp in contrast to 46-theor/48-exp and 48-theor/46-exp (Fig. 37), which was used to confirm the biosynthetic enantiodivergence at the chiral center C-3′ of the pectinolides' side chain.
The computational calculations in the aprotic solvent (CHCl 3 ) accounted for the stabilizing interaction resulting from an intramolecular hydrogen bond formed between the hydroxyl hydrogen at C-5 in the lactone ring and the carbonyl group of the acetyloxy substituent at C-3′ in pectinolide C (46), while for pectinolide J (48), a higher conformational dispersion was found. This approach also allowed the understanding of the conformational behavior of these flexible natural products to gain insight into the role of their interactions with molecular receptors (Martínez-Fructuoso et al. 2019), as demonstrated by the high affinity for the pironetin-binding site of α-tubulin of these 6-heptyl-5,6-dihydro-2H-pyran-2-ones (Fig. 38). Pironetin, isolated from cultures of Streptomyces sp., is the representative example of this type of cytotoxic compounds, which is the only natural compound currently known to bind to α-tubulin subunit through a Michael-type addition of Lys352 to the β-carbon atom of the α,β-unsaturated δ-lactone (Bañuelos-Hernandez et al. 2014).
Compounds 52-55 afforded the same peracetylated derivative 50, identical to the natural product (Scheme 6). A correlation of pectinolide A (44) with monticolides A (50) and B (51) was visualized through the oxidation of the doble bound at the size chain. This derivatization was achieved by treatment with m-chloroperbenzoic acid (mCPBA/H 2 O 2 ), followed by acetylation. The natural compound 51 was identical to the derivative obtained from the acid-catalyzed hydrolysis of the epoxide intermediate, which would be formed by the epoxidation of the less hindered re-si face of the side chain double bond to form the C-1′(S), C-2′(S) epoxide, followed by ring opening due to a nucleophilic attack of water at the C-2′ position. The most relevant DFT-conformer of pectinolide A (44) is shown in Fig. 39, where it is evident that the syn-stereoselectivity for the oxidation of the re-si face depended on the sterically hindered si-re face of the side double bond (C 1′ = C 2′ ), which is mainly imposed by the axially oriented substituent at C-5 of the lactone ring located in an anti-parallel alignment to the C-3′ acetyloxy group to avoid non-bonded oxygen-oxygen interactions. The absolute configuration for the threo-diol 51 was validated as C-1′(S) and C-2′(R) from the diagnostic ∆δ H SR signs for the syn-1,2 bis-MTPA-diesters (Martínez-Fructuoso et al. 2019).
Monticofuranolide A (56), a butenolide or 2(5H)furanone, was also isolated by recycling normal-phase HPLC from the CH 2 Cl 2 -soluble fraction from H. monticola ). Comparison of the 3 J HH values for its acetylated derivative (57) with those of peracetyl-5′-epi-synrotolide (SP-4, Scheme 4) allowed simulating the 1 H NMR spectrum for compound 56. The appropriate diastereoisomer (5S,1′R, 2′S, 3′R, 4′S) for 56 was among the stereochemical alternatives, and it was established on the following results: (1) the equivalence between experimental and DFT-ECD spectra at the B3LYP/DGDZVP level confirmed the chiral center 5S-configuration by the observed negative Cotton effect at 275 nm for the n → π* and a positive broad shoulder (240 nm); (2) Mosher ester analysis accredited the C-2′ (S) absolute configuration; (3) the described absolute stereochemistry for the side chain of its hypothetical 5,6-dihydro-α-pyrone precursor monticolide A (50), isolated from the same plant material (Martínez-Fructuoso et al. 2019), and finally, (4) the calculated DFT-NMR chemical shifts and 3 J HH values for 56 and 57. The differences were also quantified for monticofuranolide A (56) by application of the root mean square deviation, resulting in a satisfactory correlation with values ≤ 0.45 ppm for 1 H-NMR and < 9.4 ppm for 13 C-NMR chemical shifts with inclusion of PCM solvation in CHCl 3 (Martínez-Fructuoso et al. 2020).

Brevipolide Series
Ten cytotoxic compounds, brevipolides A − J (58 − 67), with a 6-heptyl-5,6-dihydro-2H-pyran-2-one core bearing a cyclopropane moiety, were isolated from Hyptis brevipes Poit. (Deng et al. 2009;Suárez-Ortiz et al. 2013). These products were structurally related to the secondary active metabolites 66-67 from Lippia alba (Mill.) N.E.Br. ex Fig. 38 Structure of the most frequent and stable pectinolide K (49)-tubulin complex. Tubulin α-subunit (cyan); β-subunit (blue). The distance between the Lys352 amino group (red) and the β carbon atom of the α,βunsaturated lactone was 5.30 Å, close enough to favor a Michael addition. Docking energy (E f kcal/mol) and hydrogen bond distances are indicated. Reproduced from Martínez-Fructuoso et al. 2019 Britton & P.Wilson,Verbenaceae, which were recognized as inhibitors of the chemokine receptor CCR5, the principal human immunodeficiency virus type 1 co-receptor. These compounds were also active in an enzyme-based ELISA NF-κB assay (Deng et al. 2009). However, the C-2′ and C-6′ absolute configurations were not confirmed for any of these 6-heptyl-5,6-dihydro-2H-pyran-2-ones in these preliminary studies (Deng et al. 2009). To elucidate the unresolved stereochemistry for this brevipolide series, the following approach was used (Fig. 40): a catalytic hydrogenation using Pd/C to reduce the double bonds of compounds 65 and 66, yielding the same tetrahydro derivative (68) with identical physical data and NMR spectra, followed by application of the Mosher's ester protocol involving its free C-1′ hydroxy group which indicated the absolute configuration to be S. The relative stereochemistry for all the stereogenic centers was elucidated from the X-ray diffraction analysis of brevipolide F (63) which confirmed, in combination with the Mosher analysis, the 6R, 1′S, 2′S, 4′S, and 6′S absolute configuration (Suárez-Ortiz et al. 2013).
A bent U-shaped geometry is adopted by this brevipolide core which favors an intermolecular hydrogen bond (2.06 Å) in the crystal packing between the oxygen of the lactone group and the hydroxy moiety at C-1′. A hypothetical 3D representation of the (S)-and (R)-MTPA esters for brevipolide F (63) helped in understanding the unexpected value for the ∆δ H S-MTPA-R-MTPA > 0 ppm for protons CH 3 -7′ (+ 0.07), H-8″ (+ 0.02), and H-7″ (+ 0.01) (Suárez-Ortiz et al. 2013). These positive values were similar in sign to those observed for the lactone sterocluster ( Fig. 41) which, according to the conformation models described by Ohtani et al. (1991), should be placed opposite to the side chain. In the angular geometry demonstrated by X-rays, the terminal methyl group (C-7′) and H-6′ are directly oriented at the edge of the benzene ring aligned to the deshielding ( −) portion of the cone produced for the diamagnetic anisotropy of this substituent of the MTPA residue and, therefore, did not exert any shielding effect on CH 3 -7′ and H-6′ (∆δ H ≅ 0) . The nonlinear U-geometry of this side chain was imposed by the cyclopropane moiety and the C-5′ ketone (Fig. 42), as opposed to the commonly observed linear, coplanar, and fully extended zigzag conformations for the side chain of most of the 6-heptyl-5,6-dihydro-2H-pyran-2-ones.

Fig. 40
Methodological approach to solve the absolute configuration for the brevipolide series containing a cyclopropane ring. Hydrogenation (Pd/C) to reduce the double bonds to yield a convergent tet-rahydro derivative (68) from the cis-(63) and trans-cinnamoyl (65) residues followed by application of the Mosher's ester method. An ORTEP drawing of brevipolide I (63) is shown A second series of cytotoxic 6-heptyl-5,6-dihydro-2H-pyran-2-ones, brevipolides K-O (69-73), was also isolated by recycling reversed-phase HPLC from H. brevipes (Suárez-Ortiz et al. 2017). Their structures, containing a distinctive tetrahydrofuran ring, were established by quantum mechanical calculations and spectroscopic analysis of their NMR and CD data. From the positive Cotton effect at 260 nm in the CD spectra, the C-6 (R) configuration for this lactone chiral center was confirmed. The n → π* transition for the cinnamoyl geometric isomers also established the S configuration for the C-6′ at the side chain.  (68) of brevipolides with a cyclopropane ring at the side chain. The preparation of these derivatives was performed in NMR tubes, and 1 H spectra were recorded directly without purification of reaction mixtures to verify the C-1′S configuration. Note the unexpected ∆δ H SR chemical shifts (in ppm) > 0 for Me-7′, H-7″, and H-8″ resulting from the nonlinear U-geometry of the side chain Fig. 42 Conformational models for the (S) and (R)-MTPA esters for the brevipolide framework with diamagnetic shifts (upfield) induced by the benzene ring indicated with the arrows. The terminal methyl group (C-7′) and H-6′ of the side chain are directly oriented at the edge of the benzene ring aligned to the deshielding ( −) portion of the diamagnetic anisotropy cone of this substituent and, therefore, did not exert any shielding effect on the CH 3 -7′ (+0.07) and H-6′ (∆δ H ≅ 0) By application of the Mosher's ester methodology in the NMR tube, the absolute configuration for C-1′, adjacent to the tetrahydrofuran ring, was determined for brevipolide M (71). The chemical shift differences for the S-and R-Mosher esters indicated that the absolute configuration for the chiral center C-1′ was (S). Quite unexpectedly, the chemical shift difference for the H-2′ proton does not reproduce in a qualitative way their vicinity to the ester moiety since there was a positive low magnitude value (δ + 0.08) for this α-proton instead of the high diamagnetic values usually produced (Fig. 43). Previous studies involving acetogenins have also shown this abnormal behavior of the α-protons (Δδ H ≅ 0) when the hydroxyl group and the adjacent C-O bond of the tetrahydrofuran have a threo relationship (Rieser et al. 1992). Therefore, the absolute configuration for the C-2′ was assigned as (R), because of the threo relationship between the chiral centers C-1′(S)/C-2′(R). Consequently, in accordance with the cis configuration for the tetrahydrofuran ring, the absolute configuration for the C-5′ center as (S) was supported by the observed nuclear Overhauser effect (Suárez-Ortiz et al. 2017).
DFT molecular models for the minimum-energy conformers of the esters at the B3LYP/DGDZVP level were used to understand this effect: first, the Mosher ester moiety is located at the opposite side to H-2′, causing an irrelevant anisotropic effect upon this atom (Fig. 43); second, the threo configuration prevents H-2′ from the strong shielding effect of the aromatic moiety in the S-esters; note the contrasting strong protection for the methylene at C-4′ (Fig. 44); and third, there is a deshielding effect caused by the lactone oxygen atom parallel to proton-2′ in the S-b MTPA conformer (Fig. 44). In the (R)-Mosher ester, conformers R-a and R-b are largely stabilized by π − π interaction between aromatic ring (MTPA) and the α,β-unsaturated lactone ring which accounted for the 68% of the Boltzmann populations for the full conformational equilibrium.

Perspectives and Future Directions
Discovery and supply of bioactive phytochemicals depend on the availability of preparative-scale analytical methods with the capability of resolving complex secondary metabolomic mixtures that are usually isolated from plant sources. The purification process must definitively provide a single chemical entity suitable for structure elucidation and identification of their relative and absolute configuration for further biological evaluations. Accordingly, modern high-end detectors for high-resolution separation techniques-state-ofthe-art preparative chromatography applications in natural product, such as refractive index (RI) (Al-Sanea and Gamal 2022) and evaporative light scattering detection (ELSD) (Ali et al. 2021), are highly recommended for compounds with little or no UV chromophore. These hyphenations have allowed the isolation of individual constituents contained in complex mixtures of natural products that in the past were considered unmanageable of separation. However, coupling HPLC-RI with UV, MS, or both is essential for detection of all chromophoric and non-chromophoric compounds, especially by coupling HPLC-ELSD with ESI-MS (LC-ESI-QqTof-MS) for the identification and quantification of active known secondary metabolites (Ali et al. 2021).
Several types of mass spectrometers can be combined with HPLC, for example, tandem mass spectrometers (QqQ), ion trap mass spectrometrs (IT-MS), hybrid MS systems such as time-of-flight (QqTOF) instruments with quadrupole mass analyses (Q) followed by collison cell and TOF, or Fourier-transform mass spectrometers as LC/Q-Orbitrap HPLC is limited by the on-line detector system, and structural elucidation of novel metabolites is always performed off-line following chromatographic purification. Accordingly, deconvolution of NMR spectral data through 2D experiments is not always the first choice, due to the long acquisition time required. This challenge has been properly addressed through the development of hybrid platforms combining direct-infusion mass spectrometry with 1 H NMR spectroscopy, enabling the fast chemical profiling of large sample sizes, without the need of a preceding chromatographic step, thus considerably reducing the acquisition time per sample for the prioritization of active plant metabolites (Gomes et al. 2018).
To overcome the difficulties associated with the availability of enough amount of sample for active natural products from plant sources, in addition to the complications for their crystallization, the crystalline sponge method has emerged as an innovative methodology which has immensely facilitated X-ray analysis of natural products to solve unequivocally their chemical structures and to fully determine their absolute configurations. Similarly, the microcrystal electron diffraction crystallography is a technique that allows establishing and confirming chemical structures. However, this technique is not accessible as a routine laboratory analysis and the data processing is still complicated to be widely applied in natural product chemistry.
Finally, it is important to mention that there is a natural predisposition during the resolution of complex mixtures of secondary metabolites derived from plants to obtain mixtures of enantiomers, epimers, diastereoisomers, and regioisomers as those described in this review, as well as mixtures of monomers, dimers, and oligomers, which might require supplementary efforts for their purification process to yield single chemical entities to unequivocally accomplish their structural elucidation analysis and absolute configuration determination.

Conclusion
Total synthesis has been an effective and broadly practiced approach for structure validation (or revision) of complex natural products; however, it appears that computational methods for structure elucidation are gradually becoming a better alternative, being faster and more reliable, as found in the case studies presented here. The broad diversity of quantum-chemical methods available now permits the study of virtually any molecule by selecting the most appropriate method, regarding the desired accuracy, time consumption, and required computer resources. In conclusion, the use of quantum calculations together with NMR anisotropic experiments presents advantages for integration of the observed NMR chemical shifts, vicinal coupling constant values, residual dipolar couplings, the nuclear Overhauser effects, and circular dichroism data for the conformational and configurational assignment of highly flexible small natural products. This approach has validated its utility for the stereochemical elucidation of natural products with multiple chiral centers, isolated in very small quantities and, particularly, where crystallographic procedures were impossible to apply.