1 Introduction
Brazil is known for its rich biodiversity, which includes a vast array of plant, animal, and microbial species. Many of these organisms are known to produce a variety of compounds with potent biological activity (Valli et al., 2018). Despite plants being one of the most explored sources of natural products, Brazil’s marine environment is another rich source of secondary metabolites, with a diverse array of organisms that produce bioactive compounds (Berlinck et al., 2004).
For several decades, Brazil has been actively researching marine natural products (MNP) (Berlinck et al., 2004; Ióca et al., 2018). This research field has focused on exploring the diverse marine fauna and flora of Brazil’s extensive coastline, including its coral reefs, mangroves, and seagrass beds, among other habitats. Some of the most promising areas of this research include the discovery of new bioactive compounds with potential pharmaceutical and biomedical applications, as well as the identification of novel structures (Berlinck et al., 2004; Ióca et al., 2018). Up to this point, many bioactive MNPs from both primary and secondary metabolism have been discovered in the marine environment, highlighting the importance of this region for studying natural products and their potential applications in medicine and other fields (Lorente et al., 2014).
Significant taxonomic differences between terrestrial and marine organisms, whose secondary metabolisms are substantially distinct, allowed the isolation of structurally different compounds from those obtained from traditional sources (Stonik, 2009). Another interesting fact about compounds of marine origin is that they are structurally complex, both from an architectural and stereochemical point of view. These data can be observed in a review of the literature on marine compounds, which reveals that the vast majority of the 1,490 substances reported are chiral compounds with complex structures (Carrol et al., 2021). Since the AC of a compound is often closely related to its biological activity, the determination of absolute stereochemistry is an important step in the characterization of bioactive compounds (Batista et al., 2021).
The determination of the AC of secondary metabolites can be achieved using techniques such as X-ray crystallography, chemical synthesis, Nuclear Magnetic Resonance (NMR) spectroscopy, and chiroptical methods (Batista et al., 2018). X-ray crystallography was used in the first AC assignment of a chiral compound (Bijvoet et al., 1951) and is still considered one of the most reliable techniques (Flack and Bernardinelli, 2008; Harada, 2008; Parsons, 2017). Nevertheless, the requirement of a well-defined single crystal can be prohibitive of a direct AC assignment of natural products, which are usually available in non-crystalline form (Mándi and Kurtan, 2019). Another powerful technique for the AC determination is NMR spectroscopy. However, for being intrinsically insensitive to chirality in the normally used isotropic media, this technique requires the use of chiral derivatizing agents or chiral solvating agents for the AC determination of a target compound (Wenzel and Chisholm, 2011; Seco et al., 2012; Zanardi et al., 2018). Chemical synthesis is also widely used to determine the AC of natural products. By synthesizing the natural product or related compounds using chiral starting materials or chiral catalysts, the AC can be assigned by comparing the physical and spectroscopic properties of the synthesized compounds with those of the natural product. This approach is challenging, since it is necessary to identify viable synthetic routes to obtain the molecules of interest (Sun and Sahinidis, 2022). Furthermore, stereocontrolled organic synthesis is highly dependent on the unequivocal AC of starting materials and products, in addition to being usually laborious, time-consuming, and expensive (Sadlej et al., 2010). Chiroptical methods rely on the differential interaction of a non-racemic chiral sample with left and right circularly polarized radiation and represent an important tool for AC determination of natural products (Grauso et al., 2019). These techniques, which include optical rotation (OR), optical rotational dispersion (ORD), electronic circular dichroism (ECD), vibrational circular dichroism (VCD) and Raman optical activity (ROA), are non-destructive and their measurements are obtained directly in solution (Batista et al., 2018). Despite these well-established techniques, it is worth noting that determining the AC of secondary metabolites can be challenging and often requires the use of associated techniques for confirmation (Batista et al., 2021). Difficulties associated with AC determination are probably because there is no universal technique since each of these methods has advantages and some limitations for complex molecules such as natural products.
3 Absolute configuration
This section presents a list of natural compounds isolated from the Brazilian coast that had their AC mentioned. The method used in the assignment will also be specified.
After a careful evaluation of about 250 articles that deal with marine secondary metabolites, it was found that more than half of them describe the isolation of chiral compounds. The rest of the articles mainly leads with identifying compounds in the extract, isolation of non-chiral compounds, or just the biological activity of previously isolated metabolites. Worth mentioning that about 70% of these articles that describe the isolation of chiral compounds do not name any data about their AC and that many works discriminate the AC of only some of the reported metabolites. In addition, several works only mention the AC of the isolated compound but do not describe the method used for this assignment. It is noteworthy that all structures were drawn and the ACs were specified as in the original publication.
3.1 Terpenoids
The class of secondary metabolites with more reports of stereochemical description is terpenes. Chamigrane is an abundant subclass of sesquiterpenes isolated from a variety of sources from terrestrial and marine environments. These compounds have displayed a diverse array of biological activities (White et al., 2010) and most of them are generally produced by the red alga of the genus Laurencia (Family Rhodomelaceae) and endophytic fungi (Zhao et al., 2021). Eleven chamigranes, of which four were reported for the first time, have been isolated from a Brazilian specimen of the red alga Laurencia scoparia (Davyt et al., 2001). The chamigranes (−)-isorigidol (1) and ma’ilione (2) were submitted to X-ray diffraction analysis, which allowed to determine the AC of 1 and 2 as 3R,6S,9S,10S and 6S,9R,10S, respectively (Davyt et al., 2001). The (−)-dendroidiol (3), another chamigrane sesquiterpene, was isolated from the red alga Laurencia dendroidea collected from the southeastern Brazilian coast. The AC of 3 was also assigned by X-ray analysis as 3R,4S,6S,9R,10S (Machado et al., 2014). The phytochemical study of a Brazilian specimen of Ophionereis reticulate, a brittle starfish widespread along South America´s northeastern coast, led to the isolation of the chamigrene sesquiterpenes (−)-isoobtusadiene (4), (−)-acetyl isoobtusadiene (5), and (+)-elatol (6) (Nuzzo et al., 2017). The AC assignment of the isoobtusadiene skeleton was determined as 6S,9R,10S based on modified Mosher’s method analysis of 4. The comparison of the experimental and calculated ECD spectra of 5 has supported this assignment. The full characterization of elatol (6) was based on NMR data and the comparison of the measured OR value with those previously reported (Nuzzo et al., 2017). Elatol is a typical chamigrene sesquiterpene normally isolated from algae (Nuzzo et al., 2017). A handful of articles surveyed during this bibliographical research reported the occurrence of elatol in organisms collected from the Brazilian coast but did not mention any data related to its stereochemistry.
Algae of the genus Laurencia is known to produce halogenated sesquiterpenes in a wide and variety of skeleton types (Cikos et al., 2021). In addition to the chamigrane, the bisabolane skeleton is also reported in this kind of organism. A halogenated bisabolene sesquiterpene (7) was isolated from Laurencia scoparia, a red alga collected at the coast of Ubatuba (SP, Brazil). The AC of (+)-7 was determined by X-ray crystallography as 2S,3S,6R,9S (Davyt et al., 2006).
Four nitrogen-containing sesquiterpenes (8-11) isolated from the Brazilian endemic sponge Dysidea robusta had their AC determined (Williams et al., 2009). The compounds (+)-isopyrodysinoic acid (8) and (+)-13-hydroxyisopyrodysinoic acid (9) had their AC assigned as 4S,6S,11R and 4R,6S,11R, respectively, by comparison of the sign of the OR with related compounds. The AC of (+)-pyrodisinoic acid (10) has been suggested as 6S,11R,13S based on the common biogenetic origin for the skeletons of pyrodisinoic and isopyrodisinoic acids. Furthermore, the NMR analysis of (+)-pyrodysinoic acid B (11) established that this compound is an epimer of 10.
Chemical investigation of the endophytic fungus Nemania bipapillata (AT-05) isolated from the Brazilian alga Asparagopsis taxiformis led to the isolation of the botryane sesquiterpenes (+)-(2R,4S,5R,8S)-4-deacetyl-5-hydroxy-botryenalol (12), (+)-(2R,4R,5R,8S)-4-deacetyl-5-hydroxy-botryenalol (13), (+)-(2R,4S,5R,8R)-4-deacetyl-botryenalol (14), (+)-(2R,4R,8R)-nemenonediol A (15), and (+)-(2R,4S,8S)-nemenonediol B (16). The ACs of 13, 14, and 15 were assigned by ECD, and the ACs of 12 and 16 by VCD, both associated with quantum chemical calculations (Medina et al., 2019).
In 1988, Kelecom and Texeira described the isolation of two new dolastane diterpenes (17 and 18) from the brown alga Dictyota ceroicornishave (Kelecom and Teixeira, 1988). The species in question was collected in Angra dos Reis, State of Rio de Janeiro, Brazil. In fact, most of the Dictyotacean diterpenes are dolastanes and secodolastanes types in the Tropical Atlantic American coastal region (Valim et al., 2005). The AC of (−)-17 and (−)-18 were established from spectral data and chemical correlation with known compounds as 4R,9R,l4S, and 4R,8S,9R,l4S, respectively (Kelecom and Teixeira, 1988). An epimer of 17 (19) was isolated from the brown alga Padina sanctae-crucis collected in the coastal region of João Pessoa, Paraiba, Brazil (Nogueira et al., 2017). The work suggests that the AC of 19 is 4R,9S,14S however there is no mention of how this configuration was assigned. Moreover, in the original publication there is a discrepancy between the AC reported and one represented in the drawn structure. Herein, the stereochemical descriptors in the text were adjusted to ensure consistency with the drawn structure. The dolastanes 20 and 21 were isolated from the Brazilian brown alga Canistrocarpus cervicornis (Bianco et al., 2009; Santos et al., 2011; Figueiredo et al., 2019; Cirne-Santos et al., 2020). The AC of 20 and 21 were mentioned as 4R,9S,14S and 4R,7R,14S, respectively. Again, there is no report on how this assignment was accomplished. A new dolastane diterpene 22 and four known seco-dolastane diterpenes were isolated from the same Brazilian brown alga Canistrocarpus cervicornis (Bianco et al., 2015). X-ray analysis of 22 determined its AC as 5R,8R,9S,10R,12S,14S. The dolastane diterpenes (4R,7R,14S)-23, (4R,7R,14S)-24, and (4R,14S)-25, also isolated from Canistrocarpus cervicorni collected in Brazil, had their AC discriminated but apparently, no method was used for this purpose (Figueiredo et al., 2019). The AC of the dolabellane diterpene (−)-10,18-diacetoxy-8-hydroxy-2,6-dolabelladiene (26), isolated from the marine brown alga Dictyota pfaffii, collected in Atol das Rocas, Brazil, was assigned by X-ray analysis as 1R,2E,4R,6E,8S,10S,11S,12R (Pardo-Vargas et al., 2014).
The unusual triterpenoid raspacionin (27) was isolated from the red sponge Raspaciona aculeate and had its AC determined by the Mosher method leading to the assignment of the stereochemistry at C-4 as S (Cimino et al., 1993). Finally, as steroids are chemically related to terpenes, the two examples involving this class of compounds will be included in this section. The steroidal glycoside (−)-riisein A (28) and (−)-riisein B (29) were isolated from the Brazilian octocoral Carijoa (Telesto) riisei. The arabinose sugar in these compounds was determined as D series by chiral GC analysis and OR comparisons (Maia et al., 2000).
3.2 Alkaloids
The second major class of secondary metabolites with reported absolute stereochemistry are alkaloids, including bromotyrosine derivatives. Bromotyrosines metabolites are normally considered chemical markers of sponges from the Verongiida order (Moriou et al., 2021). The dibromotyrosine derivative (+)-fistularin-3 (30), isolated from the Brazilian sponges Aplysina cauliformis and A. caissara, had its AC assigned as 1R,1′R,6S,6′S,11S by a combination of Marfey’s analysis for C-11 and ECD for the spiroxazolidine moieties (Rogers et al., 2005; Lira T. O. et al., 2006).
There are reports that fistularin-3 presents stereoisomers with different configurations in the two secondary carbinols C-11 and C-17 and the AC assignment of these stereogenic centers has been challenging (Ji et al., 2021). The relative and absolute configurations of the spiroxazolidine moieties presented in fistularin-3 established by analysis of 1H NMR and ECD contributed to the AC assignment of others dibromotyrosine derivatives. Based on the these analyses, the same AC of the spiroxazolidine moieties was assigned for all the following metabolites: (+)-caissarine C (31) and (+)-11-hydroxyaerothionin (32) (Lira T. O. et al., 2006); 11-oxoaerothionin (33), aerothionin (34), 11-keto-12-hydroxyaerothionin (35), 11-ketofistularin-3 (36), aeroplysinin-2 (37), verongidoic acid (38) and its methyl ester (39) (Gandolfi et al., 2010; Medeiros et al., 2012), (+)-40 (Kossuga et al., 2007), and (+)-aplysinafulvin (41) (Nuñez et al., 2008). All these compounds were isolated from the Brazilian species of Aplysina. The dibromotyrosine derivatives (+)-30 and (+)-11-deoxyfistularin-3 (42) were isolated from Aplysina cauliformis and in spite of the mention in the work that the ECD spectra obtained were essentially the same as described in the literature, the authors assigned the ACs of these compounds as 1S,1′S,6R,6′R,11R (Oliveira et al., 2006). Despite bromotyrosines metabolites being considered chemical markers of sponges from the Verongiida order, the compounds 30, 32 e 38 were isolated from cultures of the bacterium Pseudovibrio denitrificans Ab134, isolated from the marine sponge Arenosclera brasiliensis collected in Búzios, Brazil (Nicacio et al., 2017). The ACs of the spiroxazolidine moieties were assigned by ECD analysis as 1R,1′R,6S,6′S for (+)-30 and (+)-32, and 1R,6S for (+)-38.
Two new alkaloids citrinalins A (43) and B (44) were isolated from the marine Penicillium citrinum, isolated from a seaweed collected in Brazil. The X-ray analysis of 43 enabled the assignment of its AC as 1S,14R,16S,22S. The AC of 44 was suggested as 1S,14R,16R,22S since both alkaloids presented similar OR values with the same signal (Pimenta et al., 2010). The guanidine alkaloids (5aR,7S,8R)-mirabilin B (45), (5aS,7S,8bS)-8bβ-hydroxyptilocaulin (46), (5aS,7S)-ptilocaulin (47), (5aR,7S,8S)-8β-hydroxymirabilin B (48), (5aR,7S,8R)-8α-hydroxymirabilin B (49), isolated from the sponge Monanchora arbuscula, had their AC indicated however there is no mention of how these configuration were assigned (Ferreira et al., 2011).
3.3 Polyketides
Peroxide and peroxide-derived polyketide are metabolites commonly isolated from marine sponges (Norris and Perkins, 2016). Three peroxide-derived polyketides, the plakilactones 6-desmethyl-6-ethyl-9,10-dihydrospongosoritin A (50), spongosoritin A (51), and 9,10-dihydrospongosoritin A (52), were isolated from the Brazilian sponge Plakortis angulospiculatus. The AC of these compounds was deduced based on their negative OR values and comparison with literature data (Epifanio et al., 2005; Santos et al., 2015). The ACs of the plakortides (+)-7,8-dihydroplakortide E (53), (−)-54, (−)-55, (−)-56, (−)-57, and (−)-plakortide P (58), and of the plakilactone (−)-6-desmethyl-6-ethyl-spongosoritin A (59), isolated from the same species, were also assigned based on similarities between OR values with those of structurally related compounds (Santos et al., 2015). The ACs of spongosoritin A (51) and 9,10-dihydrospongosoritin A (52) were confirmed to be (−)-(6R,8R) and (−)-(6R,8S), respectively, using VCD, ECD, NMR and OR, all associated with quantum chemical calculations (Batista et al., 2019).
The dichlorinated polyketide roussoellatide (60), isolated from the marine-derived fungus Roussoella sp. DLM33, had its AC established by X-ray diffraction analyses as 2S,3S,7R,8S,9R,12S (Ferreira et al., 2015). Finally, the structures and ACs of six macrocyclic curvularin-related polyketides, isolated from a marine-derived Penicillium sp. DRF2, were established by spectroscopic data and X-ray diffraction analysis as (15S)-10,11-dehydrocurvularin (61), (15S)-12-keto-10,11-dehydrocurvularin (62), (15S)-cis-10,11-epoxycurvularin (63), (10R,11R,15S,18S)-cyclothiocurvularin A (64), (10S,11S,15S,18R)-cyclothiocurvularin B (65), and (10S,11S,15S,18R)-cyclothiocurvularin B methyl ester (66) (Castro et al., 2016).
3.4 Peptides
Nonribosomal peptides are secondary metabolites with a vast array of biological activities with medical and industrial relevance (Alonzo and Schmeing, 2020). Chemical studies of a Brazilian ascidian of the family Didemnidae led to the isolation of the depsipeptides tamandarins A (67) and B (68). The ACs of the tamandarins were assigned by Marfey’s method and Mosher´s analysis (Vervoort et al., 2000). Two new cyclodepsipeptides, [β-MePro] destruxin E chlorohydrin (69) and pseudodestruxin C (70), isolated from the marine-derived fungus Beauveria feline, had their ACs established by Marfey’s method and derivatization with R-MPA-Cl followed by NMR analysis (Lira S. et al., 2006). The modified diketopiperazine rodriguesines A (71) and B (72) and N-acetyl-rodriguesine A (73) and B (74) were isolated from two Brazilian ascidians of the genus Didemnum and their ACs were assigned by Marfey’s analysis and comparison of OR data with reported literature data for related compounds as 3S,19R (Kossuga et al., 2009). Chemical investigation of the Brazilian endemic marine sponge Clathria (Clathria) nicoleae led to the isolation of a new hexapeptide, clathriamide (75), whose AC was determined by advanced Marfey’s analysis (Freire et al., 2019).
3.5 Others compounds
The halogenated metabolite (+)-5-acetoxycaespitol (76) was isolated from the Brazilian red alga Laurencia catarinensis and its AC was assigned using the modified Mosher’s method as depicted (Lhullier et al., 2010). The structure and AC of 8-methoxy-3,5-dimethylisochroman-6-ol (77), isolated from Penicillium steckii obtained from a Brazilian alga of the genus Sargassum, was established by NMR analysis and by comparison of its OR value with those reported in the literature as S (Kossuga et al., 2012). Four compounds (78-81) were isolated from marine-derived fungal strains obtained from Brazilian marine invertebrates and had their ACs established by comparison of their OR values with literature data (Ióca et al., 2016). Chemical investigation of the deep-sea fungus Penicillium coralligerum YK-247 led to the isolation of the new compound (−)-cladomarine (82) and the known (−)-cladosporin (83). The ACs of these compounds were determined to be 3R,10R, 14S by comparison of the OR values and ECD analysis (Takahashi et al., 2017). The compounds (3R)-scytalon (84), (3R,4R)-4-hydroxy-scytalone (85), and (3R,4R)-3,4,5-trihydroxy-1-tetralone (86) were isolated from endophytic fungi isolated from red alga Asparagopsis taxiformis and had their ACs established by ECD spectroscopy (Medina et al., 2018). The amphidinolides named amphidinolide T1 (87), C4 (88), PX1 (89), PX2 (90), PX3 (91), and stragulin A (92) were isolated from the Brazilian octocoral Stragulum bicolor (Nuzzo et al., 2016; Nuzzo et al., 2019). The AC of 87 was assigned by comparison of OR value with those reported in the literature for the same compound while the ACs of 88 and 92 were suggested based on similarities with analog compounds and modified Mosher’s analysis (Nuzzo et al., 2016; Nuzzo et al., 2019). Since compounds 89-91 share the same carbon skeleton of the stragulin A (92), the same AC of 92 was suggested for these related compounds (Nuzzo et al., 2019).
5 Conclusion
This review covers the state of the art of MNP research in Brazil over the last decades, focusing on the ACs of the isolated secondary metabolites. The AC determination, especially in the case of bioactive compounds, is extremely important. However, from the data presented herein, it was evident that this last step of the structural characterization of chiral compounds is still under-explored by the Brazilian community of natural products. As already mentioned, several methods can be used for the determination of the AC of a given compound. However, a recent review carried out by our research group demonstrated that several misassignments can occur when an inappropriate method is used (Batista et al., 2021). In this context, some highlights regarding the main methods used in the AC assignment of Brazilian MNPs can be made. However, the purpose of the present review is not to question the reliability of the AC assignments reported in the literature but to raise awareness for a more critical analysis when choosing an assignment method.
As can be seen in the data presented herein, a series of compounds were subjected to X-ray analysis. This methodology is very reliable, nevertheless, obtaining crystals is not a trivial task in the case of natural products, which limits its use. The methods that require derivatizations, such as Mosher´s method, were also used but they have limitations since their use depends on the presence of specific structural motifs in the compounds under consideration. All described peptides had their ACs established by Marfey’s method. This technique has proved to be suitable and the most frequently described for the determination of the AC of amino acid residues of marine peptides (Phyo et al., 2018). The main attention has to be paid to assignments made by comparison of OR or ECD data with analogous compounds as well as by chemical correlations. Despite the widespread use, AC assignments based on these empirical correlations are not recommended. Although incorrect relative configurations are the main cause for misassignments of the AC of natural compounds in the first place, the practice of determining the AC based on comparisons of experimental OR and/or ECD data with those described for analogous molecules or spectral analyses based on empirical rules do not always generate reliable results (Batista et al., 2021). This is because similar molecules, even from a stereochemical point of view, can present OR values with opposite signs (Freedman et al., 2003; Nakahashi et al., 2011). Furthermore, empirical rules can present exceptions (Batista et al., 2010; Santos et al., 2018). On the other hand, chiroptical methods associated with quantum chemical calculations have proven to be reliable and, especially in the case of vibrational methods (VCD and ROA), can be applied to virtually all types of secondary metabolites (Batista et al., 2015; Mándi and Kurtan, 2019; Polavarapu and Santoro, 2020; Zhu et al., 2023).
Given the fact that Brazil is home to one of the richest biodiversity in the world, it is expected that this crucial step in the structural characterization of MNPs will be more widely considered. This field of research is highly promising and, if well explored, will greatly contribute to the knowledge of the diverse marine natural product chemical space in Brazil.