Natural Products
Throughout history, humans have turned to nature for a variety of resources, including food, fragrances, cosmetics, dyes, and especially medicines The quest for cures to prolong life and alleviate suffering has led to the exploration of natural products, particularly plants and marine sources Cures are often derived from secondary metabolites—compounds that, while not essential for the organism's survival, provide significant advantages, such as defense against predators Unlike primary metabolites, which are crucial for growth and reproduction, secondary metabolites play a key role in the organism's protection These compounds encompass various classes, including alkaloids, peptides, terpenoids, sterols, and polyketides.
Natural products originate from a diverse range of sources, including plants, animals, microorganisms, insects, and the ocean These bioactive compounds possess various therapeutic qualities, exhibiting medicinal characteristics such as anti-tumor, anti-cancer, anti-inflammatory, anti-viral, analgesic, and anti-bacterial properties.
One particularly important example of a natural product with medicinal qualities is quinine (1), an alkaloid isolated from the bark of the South American
The Chinchona tree, identified in 1820 by Pelletier and Caventou, has a rich history of medicinal use, as its bark was utilized by South Americans to combat malaria long before its introduction to Europe around 1640 Quinine, derived from this tree, remains a crucial treatment for malaria, a disease that claims the lives of one to two million people annually, predominantly in impoverished regions Beyond its antimalarial properties, quinine is also recognized for its effectiveness as an antipyretic, analgesic, and anti-inflammatory agent.
The discovery of quinine stemmed from traditional knowledge of a plant's medicinal properties, while taxol was developed through a collaborative effort between botanists and chemists starting in 1960 to find new antitumor compounds Among over 650 extracts tested, the yew tree Taxus brevifolia showed effectiveness against melanoma cell lines, although isolating the active compound proved challenging.
3 compound and low yields, taxol is still used today to combat cancers of the breast, lung, prostate, and ovary 6
Marine Natural Products
Covering about 70% of the Earth's surface, the ocean hosts a vast biodiversity of marine species, influenced by varying factors like light, pressure, and temperature Despite being largely unexplored until the 1940s with the introduction of scuba diving, approximately 300,000 marine species have been identified, with estimates suggesting the total could exceed one million This rich biological diversity leads to a wide range of chemical compounds, presenting significant opportunities for drug discovery As of 2010, over 15,000 natural products have been isolated from marine sources, with more than 28 marine natural products currently in clinical trials for medicinal use.
Recent advancements in marine drug discovery have led to the development of two significant medications: Prialt (ziconotide), a peptide derived from the venom of the Pacific cone snail (Conus magus), and Yondelis, extracted from a tropical sea squirt Approved by the FDA in 2004, Prialt became the first marine natural product used as a drug in the United States, offering a novel treatment for chronic pain Initially isolated in 1979 through bioassay techniques by Olivera’s team at the University of Utah, ω-conotoxin MVIIA is a linear peptide consisting of 25 amino acids linked by three disulfide bridges The complete synthesis of this compound was achieved in 1987, paving the way for clinical trials and regulatory approval from both the FDA and the European Commission.
Yondelis (trabectedin) is the first anticancer drug derived from marine sources, specifically extracted from the tunicate Ecteinascidia turbinate This unique alkaloid features a complex structure characterized by three fused tetrahydroisoquinoline rings connected by a thioether bridge, creating a ten-member lactone ring The complete synthesis of trabectedin was first documented in 1990.
5 following in 1996 In 2007, the European Commission approved Yondelis for the treatment of soft tissue sarcomas and clinical trials indicate the compound also is effective against solid tumors 7
Briareum asbestinum
Genus: Briareum Species: asbestinum Figure 1: Taxonomy of Briareum asbestinum
Coral reefs are renowned for their incredible biodiversity, hosting a vast array of animal life that surpasses even that of tropical rainforests Among the diverse inhabitants of these ecosystems, octocorals are particularly common, contributing to the rich tapestry of life found within coral reef communities.
6 community and are often the majority of the biomass found there Much of the diversity of species on a coral reef is due to octocorals 8
Octocorals, a subclass of the Anthozoa class within the phylum Cnidaria, are colonial marine invertebrates characterized by their eight-fold symmetry This group includes soft corals, blue corals, sea pens, sea whips, and sea fans Unlike stony corals, octocorals lack a stony skeleton; instead, their colonies are formed by polyps embedded in mesogleal tissue, interconnected by tiny channels that facilitate the flow of water and nutrients Each polyp features eight tentacles surrounding a mouth that leads to a pharynx and gastrovascular cavity, supported by eight radial partitions called mesenteries The order Alyconacea encompasses soft corals, which contain microscopic skeletal structures known as sclerites that provide support and deter predation Although soft corals were not historically recognized as reef builders, they contribute to reef foundations by concentrating spicules into spiculite.
Another common feature of the Alyconacea is the symbiotic relationship between soft corals and zooxanthellae, dinoflagellates of the genus
Symbiodinium The relationship is mutualistic with the coral providing the
Zooxanthellae play a crucial role in the survival of coral by providing essential carbon dioxide for photosynthesis, as well as shelter and protection In exchange, these symbiotic algae supply corals with vital products of photosynthesis, including glucose, glycerol, and amino acids Remarkably, zooxanthellae can fulfill up to 100% of a coral's energy needs, highlighting their importance in coral ecosystems.
B asbestinum, commonly referred to as Corky Sea Fingers or Deadman’s Fingers, exists in two growth forms: stalk and encrusting This soft coral thrives in tropical and subtropical marine environments, found at depths ranging from 1 to 750 meters, primarily in the western Atlantic Ocean, including the Caribbean, Florida, and the Gulf of Mexico Its ability to be easily propagated in captivity has made it a favored choice for aquarium enthusiasts.
The Chemistry of Briareum asbestinum
The wealth of secondary metabolites contained in the tissues of octocorals has generated excitement among chemists 13 Such abundance is unseen among
Soft corals differ from hard corals primarily due to their vulnerability to predation, as they lack physical defenses and regenerate slowly Despite being largely undefended, soft corals are rarely consumed, highlighting the necessity for chemical defenses The complexity and concentration of secondary metabolites produced by soft corals suggest that their synthesis is metabolically costly, underscoring their significance While these metabolites may serve various roles, including competitive and reproductive functions, their primary purpose appears to be chemical defense against predators.
In Octocorallia, terpenoid chemistry plays a significant role, with B asbestinum being a notable example The secondary metabolites from B asbestinum are primarily cembranes, which are cyclic diterpenes made up of four isoprene units The biosynthesis of these diterpenes occurs through geranylgeranyl diphosphate, which is synthesized from isopentyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) There are two established pathways for the production of IPP and DMAPP, one of which is the mevalonate pathway.
Acetyl-CoA is generated from D-glucose through a series of reactions Two acetyl-CoA molecules undergo a Claisen-like condensation to form acetoacetyl-CoA, which then reacts with another acetyl-CoA in an aldol reaction to produce 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) This compound is subsequently reduced by nicotinamide adenine dinucleotide phosphate (NADPH) to form mevalonic acid The mevalonic acid is then phosphorylated using adenosine triphosphate (ATP) to create mevalonic acid diphosphate Finally, this molecule undergoes decarboxylation and dehydration to yield isopentyl diphosphate, which isomerizes to dimethylallyl diphosphate.
Alternatively, the deoxyxylulose phosphate pathway also affords both isopentyl diphosphate and dimethylallyl diphosphate from D-glucose (Figure 4)
In the initial stages of the metabolic pathway, D-glucose is transformed into pyruvate Pyruvate then interacts with thiamine diphosphate to produce hydroxyethylthiamine diphosphate, which participates in a condensation reaction with D-glyceraldehyde-3-phosphate This reaction leads to the formation of 1-deoxy-D-xylulose-5-phosphate (DXP) through an intermediate rearrangement that alleviates charge strain Subsequently, DXP reductoisomerase catalyzes an intramolecular rearrangement and reduction, resulting in the creation of 2-C-methyl-D-erythritol-4-phosphate (MEP) This process involves multiple enzymatic steps and transformations.
11 convert MEP to 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (19) Through mechanisms not fully understood this compound is transformed to isopentyl diphosphate(IPP) (11) and dimethylallyl diphosphate(DMAPP) (12) 3
Finally, IPP and DMAPP undergo a series of reactions to form geranylgeranyl diphosphate, which upon dephosphorylation cyclizes to afford a cembrane (Figure 5)
Cembranes are 14-membered cyclic compounds featuring three double bonds, methyl substituents, and an isopropyl group Their reactive double bonds make cembranes prone to further cyclizations and oxidations, as observed in the secondary metabolites of B asbestinum.
Secondary metabolites derived from B asbestinum are noteworthy for their unique chemical diversity and significant biological activities Research has demonstrated that these compounds exhibit cytotoxic and anti-inflammatory effects, highlighting their potential therapeutic applications.
Specimens of B asbestinum produce secondary metabolites with four different carbon diterpenoid skeletons; briaranes (26), an unusual briarane diterpenoid containing a C-19 methyl ester (briareolate ester) (27), asbestinins
(28), and briarellins (29) All are comprised of fused six-member and ten-member rings The briaranes have a ɣ-lactone ring while asbestinins and briarellins possess an additional 7-member heterocylic ring
Briareolate esters, resembling briaranes, are unique compounds produced exclusively by the asbestinum species Unlike the typical ɣ-lactone ring, these molecules feature an unusual C-19 methyl ester and have demonstrated biological activity Previously isolated only from specimens off the coast of Tobago, briareolate esters have recently been discovered in samples collected by our group off southeast Florida.
The briaranes are the result of a 3,8-cyclization of a cembrane (25) followed by oxidation (Figure 6) 19 We believe that briareolate esters (26) could be a precursor to briaranes (27).
Eunicellins (30) are derived from the cyclization of a cembrane, beginning with a 2,11-cyclization that produces a bicyclic precursor This process is followed by additional cyclization and oxidation, resulting in the formation of the asbestinin (28) skeleton Briarellins (29) emerge from further oxidation and a 1,2 methyl shift of the methyl substituent on the six-member ring.
Figure 7: Asbestinin and Briarellin Formation
Research Objectives
This research aimed to create a qualitative analytical method for distinguishing the diterpenoid carbon skeletons, or chemotypes, present in B asbestinum Additionally, it sought to identify the presence of unique diterpenoids within the species.
C-19 methyl ester diterpenoids, briareolate esters, in B asbestinum from the southeast coast of Florida
In developing a method to differentiate chemotypes of Briareum sp., an extensive literature review was performed to catalog the various compounds isolated from this genus A comprehensive database was created, detailing each molecule's structure, molecular weight, formula, and collection location This analysis led to the classification of three chemotype categories: briareolate esters, briaranes, and eunicellins (including asbestinins and briarellins) Among these, the briarane chemotype is the most prevalent, with 292 reported compounds, followed by eunicellins with 60, while briareolate esters are the least common, with only 14 compounds identified.
Sclerite Analysis
Sclerites, composed of calcium carbonate, provide structural support to soft corals and help deter predation First noted in octocorals in 1755 and utilized for identification in 1865, sclerite morphology is considered species-dependent Since sclerite analysis serves as a taxonomic indicator, it was a logical focus for studying B asbestinum chemotypes However, findings revealed that sclerites do not vary among individuals of the same species.
Thin Layer Chromatography
To differentiate between chemotypes, thin layer chromatography (TLC) was initially conducted on crude extracts, but results were inconsistent due to the extract's complexity To improve accuracy, a solid phase extraction (SPE) technique was employed, fractionating the extract on HP20 resin into three distinct fractions: 40% acetone/water, 75% acetone/water, and 100% acetone The 75% and 100% fractions were selected to enhance chromatographic consistency Ultimately, a 10% MeOH/CH2Cl2 system provided the most effective separation of these fractions.
The TLC chromatograms for each chemotype displayed striking similarities, making differentiation challenging The Rf values were nearly identical, suggesting that the compounds in the mixtures possess similar polarity Consequently, this separation method may not be effective for distinguishing between the chemotypes.
Briareloate Ester Asbestinin Briarellin Briarane 100% 75% 100% 75% 100% 75% 100% 75%
18 was unsuccessful for identifying the chemotypes, a difference may be seen in a more sophisticated separation.
High Performance Liquid Chromatography
Reverse phase high performance liquid chromatography (HPLC) is an effective technique for separating complex mixtures of small molecules This method utilizes a non-polar stationary phase paired with a polar mobile phase, allowing polar compounds to elute first, which contrasts with normal phase chromatography A diverse range of columns and solvents can be employed to separate various mixtures, including peptides, proteins, steroids, and other biological molecules Enhanced separation capabilities may reveal differences in chemotypes within a chromatogram.
The crude extract's complexity necessitated an initial solid-phase extraction, which divided it into polar, medium polar, and non-polar fractions Subsequently, high-performance liquid chromatography (HPLC) was conducted on each of these fractions to analyze the distinct chemotypes.
To achieve effective separation of small molecules, an appropriate HPLC column is essential Initially, a semi-preparative C18 column was utilized, known for its hydrophobic interactions between the stationary phase (octadecylsilane) and the analyte However, this column failed to deliver sufficient separation, resulting in minimal differentiation between the chemotypes observed in the results.
The next column utilized was a semi-preparative PRP-1, in which the stationary phase consists of poly(styrene divinylbenzene) While this column did
19 provide a better separation, it could be improved upon Furthermore, the chromatograms showed little difference between the chemotypes
The third stationary phase utilized to differentiate between chemotypes was a semi-preparative pentafluorophenyl (PFP) column, which significantly enhanced separation and was ultimately chosen to finalize the HPLC analysis.
The solvent gradient was optimized to achieve optimal separation, starting with a MeCN/H2O gradient at 20% for the initial phase The gradient increased to 80% between 5 and 35 minutes, followed by a rise to 100% from 35 to 40 minutes, remaining isocratic until 50 minutes Subsequently, the gradient decreased back to 20% from 50 to 60 minutes However, this method did not yield ideal separation results, as it was primarily designed for basic separation of all three fractions, including the polar 40% acetone/water fraction, and could be further adapted for medium polarity and non-polar fractions.
To enhance compound separation, a new method was tested, starting at 50% MeCN/H2O and increasing to 100% over 15 minutes, followed by a 45-minute isocratic phase However, this approach was found to be unsatisfactory due to the rapid transition to 100% within just 15 minutes Extending the time taken to reach 100% could lead to improved separation results.
In a subsequent attempt, a method was used that began at 60% MeCN/H2O then increased to 80% over 10 minutes The gradient was increased
The method demonstrated a separation efficiency that increased from 20% to 95% within the first 30 minutes, reaching 100% by the 50-minute mark However, from 50 to 60 minutes, the efficiency decreased to 60% Despite achieving the best separation results among all trials, there is potential for simplification of this method.
The optimal separation method commenced with a 60% MeCN/H2O mixture, gradually increasing to 90% over 30 minutes, then reaching 100% at the 40-minute mark, and maintaining isocratic conditions until 50 minutes Subsequently, the gradient was reduced back to 60% from 50 to 60 minutes This method was utilized for analyzing the chemotypes using a PFP column.
Despite enhanced separation, the chromatograms exhibited significant similarity, hindering the identification of chemotypes The congruent polarity of the mixtures led to chromatograms that closely resembled each other, with comparable retention times across different chemotypes.
- 13) Therefore, another method was required
Figure 12: Briareolate Ester HPLC Chromatogram
Differentiation of B asbestinum Chemotypes using NMR spectroscopy
The soft coral B asbestinum has been found to contain four chemotypes
The variation of specific compounds in organisms is significantly influenced by geographic location, though the causes—whether natural, environmental, genetic, or related to extraction and preservation techniques—remain unclear To explore the ecological relationships of this biochemical phenomenon, a sensitive and reproducible qualitative assay for different chemotypes is essential Notably, the distinct 1 H NMR signals observed in each compound suggest that 1 H NMR analysis could effectively differentiate and analyze these compounds within a mixture.
23 advantage of quantification by NMR spectroscopy is that one does not necessarily need a pure, accurately weighted sample of the compound to be identified.
Analytical Method
For effective analysis using 1 H NMR, it is essential that the sample is soluble in the NMR solvent and that the compounds are present at detectable concentrations Due to the high concentrations and complex nature of B asbestinum extracts, a pre-concentration and purification method is necessary The concentration and purification steps, detailed in Scheme 1, were derived from a scaled-down version of the large-scale purification process used for isolating briareolate diterpenoids.
To ensure accurate mass measurements of wet weight soft coral samples, specimens were lyophilized and ground into a homogenous powder A 0.5 g sample was found to be sufficient for quantitative analysis using 1 H and COSY NMR The powdered sample was then mixed with 6.0 mL of methanol and sonicated for ten minutes The resulting extract was filtered and treated with HP20 polymeric resin, concentrated to dryness, and transferred to a solid phase extraction column The column was washed with water and sequentially eluted with 15 mL aliquots of 40%, 75%, and 100% acetone Finally, the 75% acetone fraction was dried in a vacuum centrifuge and transferred into an NMR tube for further analysis.
The analysis of CD3OD using 1H and COSY NMR techniques enabled the differentiation of various chemotypes, as illustrated in Scheme 1 This approach facilitated the effective enrichment and purification of analytes to a concentration level sufficient for qualitative analysis Notably, the similarities observed in the 1H NMR spectra of the asbestinin and briarellin chemotypes led to their classification under a single eunicellin chemotype.
MeOH (6mL), sonication (10 mins) EXTRACTION
Scheme 1: Concentration and purification steps for the diterpenoids from B asbestinum
Analysis of the Briareolate Ester Containing Chemotype
To validate the analytical method and establish reproducibility, a sample of
B asbestinum, identified as containing the briareolate ester chemotype, was lyophilized and processed into a homogeneous powder Approximately 0.5 g samples were analyzed following a specific analytical protocol The 1H NMR spectrum of the 75% acetone/water fraction from the briareolate ester chemotype is illustrated in Figure 14, highlighting the presence of briareolate esters through distinct double bond resonances at δH 7.68, 6.62, 6.21, and 6.07, which do not overlap with other chemotypes' proton signals These resonances correspond to H-6 and H-7 in briareolate ester G and L, as shown in Figures 15 and 16, with the variance in chemical shifts attributed to changes in double bond geometry.
26 Figure 14: Typical 1 H NMR spectrum of the 75% acetone/water fraction of the briareolate ester containing B asbestinum chemotype (400 MHz, CD3OD)
27 Figure 15: 1 H NMR spectrum of briareolate ester G (31) (400 MHz, CD3OD)
Figure 16: 1 H NMR spectrum of briareolate ester L (32) (400 MHz, CD3OD)
Comparison of the spectra for briareolate esters G and L with the mixture confirms the presence of these protons in the 75% acetone/water fraction (Figure
Figure 17: 1 H NMR Spectrum Overlay, Briareolate Esters (400 MHz,
The identification of the briareolate ester chemotype can be enhanced by utilizing both 1 H NMR and 1 H-1 H COSY techniques Notably, the correlation detected between H-6 and H-7 in the briareolate esters provides significant insights into their structural characteristics.
30 with the long range correlations observed from both H-6 and H-7 to the olefinic methyl signal H3-16 at δH 1.73 in briareolate ester L
Figure 18: Typical 1 H- 1 H COSY NMR spectrum of the 75% acetone/water fraction of the briareolate ester containing B asbestinum chemotype indicating cross peaks characteristic for the briareolate ester chemotype (400 MHz, CD3OD)
Analysis of the Briarane/Eunicellin Containing Chemotype
Diterpenoids from the briarane chemotype exhibit complex 1H NMR spectra due to their high oxygenation levels The 1H NMR spectrum of the 75% acetone/water fraction from the briarane and eunicellin-containing B asbestinum chemotype indicates that 1H-1H COSY may be more effective for distinguishing this chemotype Notable compounds isolated from this chemotype include the briarane diterpenoid briarein G (33) and the eunicellin 11-acetoxy-4-deoxyasbestinin B (34) To identify diagnostic signals and correlations for these compounds, 1H and 1H-1H COSY analyses of briarein G (33) and 11-acetoxy-4-deoxyasbestinin B (34) were conducted for comparison.
The 1 H NMR spectrum of briarein G (33) reveals a notable presence of deshielded proton signals in the range of 5.0 to 6.0 ppm Many of these signals exhibit correlations either with one another or with the aliphatic region of the spectrum.
1H- 1 H COSY spectrum (Figure 22) The proton signals are from the large number
Briarane diterpenoids are characterized by 32 acetylated oxygenated methines or olefinic bonds, with a notable diagnostic feature being the signal from H-7, which is a consistent element of the briarane structure Additionally, C-6 is either connected to a heteroatom or has a double bond with C-5, leading to the presence of the methine proton, H-6 The resonance of H-7 is observed at δH.
5.00 and occurs as a doublet, being split by one proton at position 6
An overlay of the 1 H- 1 H COSY spectrum of the 75% acetone/water fraction of the briarane and eunicellin containing B asbestinum chemotype and briarein
The study of G (33) has revealed crucial diagnostic correlations for the briarane carbon skeleton, particularly highlighted by the unique spectral features between 4.5 and 6.0 ppm, which are absent in other chemotypes Notably, a significant correlation at δH 5.30 and 3.75 indicates a coupling between H-2 and H-3 Additionally, the numerous correlations observed between the aliphatic region and the acetylated or olefinic region further underscore the diagnostic importance of this analysis.
33 Figure 19: Typical 1 H NMR spectrum of the 75% acetone/water fraction of the briarane and asbestinin containing B asbestinum chemotype (400 MHz, CD3OD)
Figure 20: Typical 1 H- 1 H COSY NMR spectrum of the 75% acetone/water fraction of the briarane and eunicellin containing B asbestinum chemotype (400
35 Figure 21: 1 H NMR spectrum of briarein G (33) (400 MHz, CDCl3)
36 Figure 22: 1 H- 1 H COSY NMR spectrum of briarein G (33) (400 MHz, CD3OD)
The overlay of the 1 H-1 H COSY NMR spectra highlights the distinctive features of the briarane and eunicellin chemotype found in B asbestinum, specifically the 75% acetone/water fraction (black) compared to briarein G (red) This analysis reveals critical regions that are indicative of the briarane carbon skeleton, recorded at 400 MHz in CD3OD.
The 1 H NMR analysis of the compound 11-acetoxy-4-deoxyasbestinin B (34) reveals a lower number of deshielded protons due to the less oxygenated and olefinic characteristics of eunicellins Notably, several signals observed between 3.5 and 4.0 ppm indicate the presence of two ether functionalities, specifically attributed to the methine hydrogens H-2 and H-9, as well as the methylene protons H-16α and H-16β.
The overlay of the 1 H-1 H COSY spectrum for the 75% acetone/water fraction of the B asbestinum chemotype, which contains eunicellin and briarane, along with 11-acetoxy-4-deoxyasbestinin B (34), reveals crucial correlations that help identify the eunicellin carbon skeleton Notably, a significant correlation is observed between H-6 and H-7 at δH 2.25 and 5.25, as well as between H-16α and H-16β at δH 3.60 and 4.25 The correlation patterns in the oxymethine region further characterize this chemotype.
39 Figure 24: 1 H NMR spectrum of 11-acetoxy-4-deoxyasbestinin B (34) (400 MHz,
40 Figure 25: 1 H- 1 H COSY NMR spectrum of 11-acetoxy-4-deoxyasbestinin B (34)
The overlay of the 1H-1H COSY NMR spectra reveals key diagnostic regions for the eunicellin carbon skeleton in the 75% acetone/water fraction of the briarane and eunicellin-containing B asbestinum chemotype (black) compared to 11-acetoxy-4-deoxyasbestinin B (red), analyzed at 400 MHz in CD3OD.
Analysis of the Briarane-rich Chemotype
The final chemotype isolated from B asbestinum exclusively features the briarane skeleton The 1H NMR spectrum of the 75% acetone/water fraction for this chemotype, depicted in Figure 27, exhibits a complex structure characterized by numerous downfield signals resulting from oxygenated compounds and double bonds.
11-hydroxybrianthein U (35) is another notable briarane, analyzed using 1H and 1H-1H COSY NMR spectra for comparison with the briarane-rich chemotype's 75% acetone/water fraction Similar to briarein G (33), this compound exhibits numerous olefinic and acetylated bonds in the downfield region of the 1H NMR spectrum, along with multiple correlations between 5.50 and 6.50 ppm in the 1H-1H COSY Notably, H-7 resonates at approximately δH 5.00 as a doublet.
An overlay of the 1 H-1 H COSY spectrum of the 75% acetone/water fraction from the briarane-rich B asbestinum chemotype and 11-hydroxybrianthein U (35) revealed critical correlations essential for identifying the briarane carbon skeleton This analysis, similar to previous studies on briaranes, demonstrated numerous significant correlations.
The diagnostic analysis of this chemotype reveals significant couplings in the downfield region, particularly between δH 2.75 (H-10) and δH 5.80 (H-9) Additionally, a notable coupling is observed between δH 3.60 (H-13) and δH 4.60 (H-12), further supporting the identification of this chemotype.
In summary, the briareolate ester chemotype can be readily identified through 1 H NMR analysis, which reveals four distinct resonances linked to the conjugated diene in either (E,Z) or (Z,Z) configurations Additionally, the presence of a briareolate ester is further validated by the correlations between these protons and the methyl substituent at C-16.
The briarane/eunicellin chemotype exhibits intricate 1H NMR spectra, necessitating the use of 1H-1H COSY NMR for detailed analysis The briarane variant shows numerous correlations between acetoxy or olefinic protons and the aliphatic region In contrast, the eunicellin chemotype is characterized by correlations among oxymethine protons and their connections to the aliphatic region.
44 Figure 27: Typical 1 H NMR spectrum of the 75% acetone/water fraction of the briarane-rich B asbestinum chemotype (400 MHz, CD3OD)
Figure 28: Typical 1 H- 1 H COSY NMR spectrum of the 75% acetone/water fraction of the briarane-rich B asbestinum chemotype (400 MHz, CD3OD)
Figure 29: 1 H NMR spectrum of 11-hydroxybrianthein U (35) (400 MHz, CDCl3)
47 Figure 30: 1 H- 1 H COSY NMR spectrum of 11-hydroxybrianthein U (35) (400
The overlay of the 1 H-1 H COSY NMR spectra reveals key diagnostic regions for the briarane carbon skeleton, comparing the 75% acetone/water fraction of the briarane-rich B asbestinum chemotype (black) with 11-hydroxybrianthein U (red) This analysis was conducted at 400 MHz in CD3OD, highlighting significant spectral features that aid in the identification of briarane compounds.
Application to Location
Briareolate esters are rare compounds with unique chemistry and notable biological activity, with only fourteen known instances reported, primarily sourced from B asbestinum in Tobago Recently, our laboratory discovered briareolate esters from collections along the southeast coast of Florida and the Florida Keys, marking the first report of such findings in this region To determine the influence of geographical location on the chemistry of B asbestinum and to explore the isolation of these uncommon esters, we employed analytical methods on multiple collections from this area.
An analysis of fourteen samples from five collection sites in southeast Florida and the Florida Keys revealed that location significantly influences the chemistry of soft coral Specifically, eunicellins and briaranes were isolated from Key West, the southernmost site, while only briarane/eunicellins and briareolate esters were found further north at Big Pine Key and Hillsboro Ledge In contrast, only briarane/eunicellins were identified off Boca Raton, indicating a trend of increasing eunicellins as one moves northward.
Out of fourteen samples collected, four were identified as briareolate esters, highlighting an unusual occurrence of these compounds The briarane/eunicellin skeleton emerged as the most prevalent, with seven samples categorized under this chemotype Overall, briaranes represented the majority within the briarane/eunicellin category.
51 reported class of secondary metabolites of B asbestinum globally, in southeast
Florida the lesser found eunicellin and the rare briareloate ester are abundant (Table 1.)
Big Pine Key briareolate ester
Big Pine Key briarane/eunicellin
Hillsboro Ledge (Deerfield Beach) briareolate ester
Hillsboro Ledge (Deerfield Beach) briarane/eunicellin
Hillsboro Ledge (Deerfield Beach) briarane/eunicellin
During the development of an analytical method, an unknown compound was discovered in the non-polar 100% acetone fraction of B asbestinum Structure elucidation using NMR data revealed that this compound, named (7E-11E)-3,4-epoxy-7,11,15-cembratriene, had previously been isolated from an unidentified soft coral collected at Canton Island in the South Pacific.
The methanolic extract of B asbestinum underwent solid phase extraction using HP20 resin, followed by column chromatography on HP20ss resin, resulting in the collection of 88 fractions These fractions were subsequently combined according to their similarities, as determined by thin-layer chromatography (TLC).
The compound (7E-11E)-3,4-epoxy-7,11,15-cembratriene, identified as a white powder with a molecular formula of C20H32O, was characterized using HRESIMS, revealing the [M + Na]+ ion at m/z 311.2, which indicates five degrees of unsaturation Analysis of the 13C NMR data confirmed the presence of three C-C double bonds, accounting for three of the unsaturation degrees, while the remaining two were attributed to two rings The structure includes a trisubstituted epoxide with a methyl substituent, indicated by signals from an oxymethine, a quaternary carbon, and a methyl singlet With 20 carbons present, this compound is classified as a cyclic diterpene, a typical structure produced by B asbestinum, featuring three double bonds and an epoxide.
Table 2: 1 H and 13 C NMR Data for 36
The structure of compound 36 was elucidated through a comprehensive analysis of NMR data, revealing key correlations between protons and their corresponding carbon atoms via the HSQC experiment The 1H-1H COSY experiment identified significant correlations, including between H-1 and H-2, as well as H-1 and H-14 These additional correlations, such as H-5 to H-4, contributed to the overall understanding of the molecular framework.
The connectivity of the 14-member ring was established through HMBC correlations, specifically between H-6, H-8 to H-9, H-9 to H-10, H-10 to H-11, and H-13 to H-14 Additionally, correlations were observed from H-4 to C-3, H-9 to C-7 and C-8, and H-13 to C-14 The position of the methyl group at position 20 was confirmed by HMBC correlations from H-20 to C-13.
11 and a COSY correlation from H-20 to H-11 The methyl substituent at position
Correlations observed in the COSY and HMBC experiments confirmed the placement of various substituents in the compound Specifically, H-19 showed connections to C-6, C-7, C-8, and C-9, while H-7 correlated with H-19 The positioning of the final methyl group at C-12 was substantiated through correlations between H-20 and H-11, as well as H-20's connections to C-11 and C-13 Additionally, the isopropene substituent was assigned to C-1 based on COSY correlations from H-14 and H-16 to H-17.
The connectivity of the compound was confirmed through various NMR experiments, specifically noting the relationships between C-15, C-16, and C-17, as well as H-16 and H-17 The presence of an epoxide at C-3 and C-4 was established through COSY correlations from H-4 to C-3, alongside HMBC correlations from H-18 to both C-3 and C-4 Ultimately, these NMR analyses enabled a clear and unambiguous determination of the structure of compound 36.
Table 3: Selected 1 H- 1 H COSY and 1 H- 13 C HMBC Correlations for 36
57 Figure 33: 13 C NMR Spectrum of 36 (400 MHz, CD3OD)
58 Figure 34: 1 H- 1 H COSY NMR Spectrum of 36 (400 MHz, CD3OD)
59 Figure 35: HSQC NMR Spectrum of 36 (400 MHz, CD3OD)
60 Figure 36: HMBC NMR Spectrum of 36 (400 MHz, CD3OD)
General Experimental Procedures
All 1D and 2D NMR spectra were obtained on a Varian MercuryPlus 400 MHz NMR spectrometer, and all chemical shifts are expressed in parts per million (δ) relative to tetramethylsilane
Solid phase extraction (SPE) was performed using HP20 resin (3.0 g) in a
The semi-preparative HPLC separations were performed using a 25 mL syringe-barrel column and a 10-port manifold on a Shimadzu HPLC system This system included a Shimadzu D6U-20AF online degasser, a Shimadzu LC-20AT quaternary solvent delivery system, an ELSD-LTII detector, a SPDA-M20A photodiode array detector, and a FRC-10A fraction collector The flow was split in a ratio of 1:20 to both the ELSD and the fraction collector, with system control managed by EZStart chromatography software.
Collection of Animal Material
Between August 2009 and June 2011, samples of Briareum asbestinum were collected from depths of 45 to 75 feet off the southeast coast of Florida and the Florida Keys, approximately one mile from shore The samples were promptly frozen after collection to preserve their integrity.
Sclerite Analysis
Sclerite analysis commenced with 0.5 g of dry animal material, which was combined with 20.0 mL of 0.1M nitric acid in a test tube The mixture was gently heated in a warm water bath until complete dissolution of the solid material occurred, yielding a yellow solution After settling, the bottom layer was extracted for microscopic examination.
Solid Phase Extraction
Organic extracts were fractionated through a solid phase extraction 10-port vacuum manifold and concentrated on polymeric HP20 using a Savant vacuum centrifuge system The HP20 was then moved to a 25 mL syringe-barrel SPE column, which was washed with 20 mL of water before being eluted drop-wise with 15 mL fractions of a 40% acetone/water solution.
2) 75% acetone/water, and 3) 100% acetone The eluent was collected in 20 mL scintillation vials and dried in a vacuum centrifuge.
Thin Layer Chromatography
Thin layer chromatography began with extracts of the raw animal material from four different specimens of Briareum asbestinum with known chemotype
The 75% and 100% fractions were dissolved in MeOH to a concentration of 20 mg/mL
The solution was then spotted onto alumina backed silica gel plates and allowed to dry The first system used for separation was 20% EtOAc/hexane The
63 next system employed was 5% MeOH/CH2Cl2 Finally, a 10% MeOH/CH2Cl2 system gave satisfactory separation Each time, the TLC was recovered using a 10% H2SO4 spray with applied heat.
Development of High Performance Liquid Chromatography Method
Semi-preparative HPLC separations were conducted using a Shimadzu HPLC system, featuring a quaternary solvent delivery system, evaporative light scattering detector, and photodiode array detector A 5 mg sample of the 75% acetone/water fraction underwent separation on reversed phase columns, starting with a Hamilton PRP reverse phase column (10 x 250 mm, part number 79496) This was followed by a Phenomenex C18 column (10 x 250 mm, part number 00G-4041-N0), and the final selection was a Phenomenex Curo-Sil-PFP column.
10 x 250 mm (part number 00G-4012-N0) A post column fixed flow splitter was used to split the flow in a ratio of 1:20 to the ELSD and fraction collector, respectively.
Protocol for NMR Analysis
NMR spectra were obtained on a Varian Inova 400 NMR spectrometer at
400 MHz in CD3OD Proton chemical shifts were referenced to the residual
CD3OD signals ( δH 4.98 and 3.31) and carbon chemical shifts were referenced to the center peak of CD3OD at δC 49.15
After extraction and fractionation as described previously, the resulting fractions were diluted in 600 àL CD3OD and placed in a 5 mm NMR tube for analysis.
Isolation of 3,4-epoxy-7,11,15-cembratriene
Using a specific extraction technique, 0.5657 g of crude extract was obtained and processed through a #11 MPLC column packed with HP20, which was washed with acetone The crude extract was dissolved in methanol and loaded onto the column, employing a MeCN/H2O gradient at a flow rate of 8.0 mL/min The gradient started at 60% for the first 10 minutes, increasing to 100% from 10 to 70 minutes, followed by an isocratic phase for an additional 10 minutes A total of 88 tubes were collected, and thin-layer chromatography (TLC) was performed on each The tubes were pooled into fractions A through S, with specific ranges assigned to each fraction Each fraction was then dissolved in 600 µL of CD3OD for 1H NMR analysis, revealing that Fraction R contained 3,4-epoxy-7,11,15-cembratriene.
OD)
The identification of the briareolate ester chemotype can be enhanced by utilizing 1 H NMR and 1 H-1 H COSY techniques Notably, a significant correlation between H-6 and H-7 is observed in both briareolate esters, providing valuable insights for structural analysis.
30 with the long range correlations observed from both H-6 and H-7 to the olefinic methyl signal H3-16 at δH 1.73 in briareolate ester L
Figure 18: Typical 1 H- 1 H COSY NMR spectrum of the 75% acetone/water fraction of the briareolate ester containing B asbestinum chemotype indicating cross peaks characteristic for the briareolate ester chemotype (400 MHz, CD3OD)
2.7 Analysis of the Briarane/Eunicellin Containing Chemotype
Diterpenoids from the briarane chemotype exhibit complex 1H NMR spectra due to their high oxygenation levels The 1H NMR spectrum of the 75% acetone/water fraction from the briarane and eunicellin-containing B asbestinum chemotype indicates that 1H-1H COSY may be more effective for distinguishing this chemotype Notable compounds isolated from this group include the briarane diterpenoid briarein G (33) and the eunicellin 11-acetoxy-4-deoxyasbestinin B (34) To identify diagnostic signals and correlations for these compounds, 1H and 1H-1H COSY analyses of briarein G (33) and 11-acetoxy-4-deoxyasbestinin B (34) were conducted for comparative purposes.
The 1 H NMR spectrum of briarein G (33) reveals a notable presence of deshielded proton signals within the range of 5.0 to 6.0 ppm Many of these signals show correlations with one another as well as with the aliphatic region of the spectrum.
1H- 1 H COSY spectrum (Figure 22) The proton signals are from the large number
Briarane diterpenoids are characterized by 32 acetylated oxygenated methines or olefinic bonds, with a significant diagnostic feature being the signal from H-7, which is a stable component of the briarane structure Additionally, C-6 is either attached to a heteroatom or forms a double bond with C-5, leading to the presence of the methine proton H-6 H-7 resonates at δH, highlighting its importance in the analysis of these compounds.
5.00 and occurs as a doublet, being split by one proton at position 6
An overlay of the 1 H- 1 H COSY spectrum of the 75% acetone/water fraction of the briarane and eunicellin containing B asbestinum chemotype and briarein
Key correlations diagnostic for the briarane carbon skeleton were identified, particularly notable between 4.5 and 6.0 ppm, a range not observed in other chemotypes A significant correlation at δH 5.30 and 3.75 indicates a coupling between H-2 and H-3 Additionally, numerous correlations between the aliphatic region and the acetylated or olefinic region further enhance the diagnostic importance of this spectrum.
33 Figure 19: Typical 1 H NMR spectrum of the 75% acetone/water fraction of the briarane and asbestinin containing B asbestinum chemotype (400 MHz, CD3OD)
Figure 20: Typical 1 H- 1 H COSY NMR spectrum of the 75% acetone/water fraction of the briarane and eunicellin containing B asbestinum chemotype (400
35 Figure 21: 1 H NMR spectrum of briarein G (33) (400 MHz, CDCl3)
36 Figure 22: 1 H- 1 H COSY NMR spectrum of briarein G (33) (400 MHz, CD3OD)
The overlay of the 1 H-1 H COSY NMR spectra reveals significant diagnostic regions for the briarane carbon skeleton in the 75% acetone/water fraction of the B asbestinum chemotype (black) compared to briarein G (red), analyzed at 400 MHz in CD3OD.
The 1 H NMR spectrum of the asbestinin compound 11-acetoxy-4-deoxyasbestinin B (34) reveals a reduced number of deshielded protons, indicating that eunicellins possess lower levels of oxygenation and olefinic characteristics Notably, several signals observed between 3.5 and 4.0 ppm are linked to the presence of two ether functionalities, specifically attributed to the methine hydrogens H-2 and H-9, as well as the methylene protons H-16α and H-16β.
The 1 H-1 H COSY spectrum overlay of the 75% acetone/water fraction from the eunicellin and briarane-containing B asbestinum chemotype, including 11-acetoxy-4-deoxyasbestinin B (34), revealed crucial correlations for identifying the eunicellin carbon skeleton Notably, a significant correlation exists at δH 2.25 and 5.25 between H-6 and H-7, along with another correlation between H-16α and H-16β at δH 3.60 and 4.25 These correlations in the oxymethine region further characterize this specific chemotype.
39 Figure 24: 1 H NMR spectrum of 11-acetoxy-4-deoxyasbestinin B (34) (400 MHz,
40 Figure 25: 1 H- 1 H COSY NMR spectrum of 11-acetoxy-4-deoxyasbestinin B (34)
The overlay of the 1 H-1 H COSY NMR spectra reveals significant insights into the 75% acetone/water fraction of the briarane and eunicellin-rich B asbestinum chemotype (black) compared to 11-acetoxy-4-deoxyasbestinin B (red) This analysis highlights key regions that are diagnostic for the eunicellin carbon skeleton, utilizing a 400 MHz frequency in CD3OD solvent.
2.8 Analysis of the Briarane-rich Chemotype
The final chemotype extracted from B asbestinum exclusively features the briarane skeleton Figure 27 illustrates the 1H NMR spectrum of the 75% acetone/water fraction from this chemotype, revealing a complex spectrum characterized by numerous downfield signals attributed to oxygenated compounds and double bonds.
11-hydroxybrianthein U (35) is another notable briarane compound, analyzed using 1H NMR and 1H-1H COSY spectra for comparison with the 75% acetone/water fraction of the briarane-rich chemotype Similar to briarein G (33), this molecule exhibits multiple olefinic and acetylated bonds in the downfield region of the 1H NMR spectrum, along with numerous correlations between 5.50 and 6.50 ppm in the 1H-1H COSY Additionally, H-7 resonates as a doublet at approximately δH 5.00.
An overlay of the 1 H-1 H COSY spectrum for the 75% acetone/water fraction of the briarane-rich B asbestinum chemotype and 11-hydroxybrianthein U (35) revealed critical correlations essential for identifying the briarane carbon skeleton Similar to previous findings, numerous correlations were observed.
The diagnostic analysis of this chemotype reveals significant couplings in the downfield region, particularly between the aliphatic and downfield areas Notably, a correlation is observed between δH 2.75 (H-10) and δH 5.80 (H-9), as well as between δH 3.60 (H-13) and δH 4.60 (H-12), both of which are indicative of the chemotype's characteristics.
In summary, the briareolate ester chemotype can be readily identified using 1H NMR, which reveals four distinct resonances linked to the conjugated diene in either (E,Z) or (Z,Z) configurations Additionally, the presence of a briareolate ester is further validated by the correlations observed between these protons and the methyl substituent at C-16.
The briarane/eunicellin chemotype exhibits intricate 1H NMR spectra, which are analyzed using 1H-1H COSY NMR techniques In the briarane chemotype, numerous correlations are observed among acetoxy or olefinic protons, extending to the aliphatic region Conversely, the eunicellin chemotype is characterized by correlations among oxymethine protons, also linking to the aliphatic region.
44 Figure 27: Typical 1 H NMR spectrum of the 75% acetone/water fraction of the briarane-rich B asbestinum chemotype (400 MHz, CD3OD)
Figure 28: Typical 1 H- 1 H COSY NMR spectrum of the 75% acetone/water fraction of the briarane-rich B asbestinum chemotype (400 MHz, CD3OD)
Figure 29: 1 H NMR spectrum of 11-hydroxybrianthein U (35) (400 MHz, CDCl3)
47 Figure 30: 1 H- 1 H COSY NMR spectrum of 11-hydroxybrianthein U (35) (400