Induction of New Aromatic Polyketides from the Marine Actinobacterium Streptomyces griseorubiginosus through an OSMAC Approach.

Using the OSMAC (One Strain Many Compounds) approach, the actinobacterium Streptomyces griseorubiginosus, derived from an unidentified cnidarian collected from a reef near Pointe de Bellevue in Réunion Island (France), was subjected to cultivation under diverse conditions. This endeavour yielded the isolation of a repertoire of 23 secondary metabolites (1-23), wherein five compounds were unprecedented as natural products (19-23). Specifically, compounds 19 and 20 showcased novel anthrone backbones, while compound 23 displayed a distinctive tetralone structure. Additionally, compounds 21 and 22 presented an unusual naphtho [2,3-c]furan-4(9H)-one chromophore. Interestingly, the detection of all these novel compounds (19-23) was exclusively achieved when the bacterium was cultured in FA-1 liquid medium supplemented with the epigenetic modifier γ-butyrolactone. The elucidation of the structural features of the newfound compounds was accomplished through a combination of HRESIMS, 1D and 2D NMR spectroscopy, as well as QM-NMR (Quantum Mechanical-Nuclear Magnetic Resonance) methods and by comparison with existing literature. Moreover, the determination of the relative configuration of compound 23 was facilitated by employing the mix-J-DP4 computational approach.

Chemical Stability of Petrichorins.

The structure of petrichorin C1 (4) converted from petrichorin C (3) was determined using NMR spectroscopy and X-ray crystallography. The chemical stability of petrichorins A and C (1 and 3) was investigated by NMR spectroscopy, X-ray crystallography, and calculations.

May the Force (Field) Be with You: On the Importance of Conformational Searches in the Prediction of NMR Chemical Shifts.

NMR data prediction is increasingly important in structure elucidation. The impact of force field selection was assessed, along with geometry and energy cutoffs. Based on the conclusions, we propose a new approach named mix-J-DP4, which provides a remarkable increase in the confidence level of complex stereochemical assignments-100% in our molecular test set-with a very modest increment in computational cost.

Structure and Computational Basis for Backbone Rearrangement in Marine Oxasqualenoids.

Six novel oxasqualenoids (polyether triterpenes) were isolated from the red alga Laurencia viridis. Laurokanols A-E (1-5) comprise an unreported tricyclic core with a [6,6]-spiroketal system. Yucatecone (6) shows a biogenetically intriguing epimerization at C14. Quantum mechanical calculations were used to corroborate their structures and to explain key steps involved in the biogenetic mechanisms proposed for the formation of oxasqualenoids.

Dynamic Nucleophilic Aromatic Substitution of Tetrazines.

A dynamic nucleophilic aromatic substitution of tetrazines (SN Tz) is presented herein. It combines all the advantages of dynamic covalent chemistry with the versatility of the tetrazine moiety. Indeed, libraries of compounds or sophisticated molecular structures can be easily obtained, which are susceptible to post-functionalization by inverse electron demand Diels-Alder (IEDDA) reaction, which also locks the exchange. Additionally, the structures obtained can be disassembled upon the application of the right stimulus, either UV irradiation or a suitable chemical reagent. Moreover, SN Tz is compatible with the imine chemistry of anilines. The high potential of this methodology has been proved by building two responsive supramolecular systems: A macrocycle that displays a light-induced release of acetylcholine; and a truncated [4+6] tetrahedral shape-persistent fluorescent cage, which is disassembled by thiols unless it is post-stabilized by IEDDA.

Synthesis and biological evaluation of crown ether acyl derivatives.

A set of crown ethyl acyl derivatives based on 18-crown-6 moiety was synthesized and evaluated for biological activity. In vitro antiproliferative profiling demonstrated significant activities against HBL-100, HeLa, SW1573 and WiDr human cell lines. The most active compound exhibited GI50 values in the range of 3.7-5.6µM. Antimicrobial evaluation showed that three polyaromatic compounds were active against Staphylococcus aureus (MIC90 values from 8.3µM to 50µM), whereas a (decyloxy)benzene substitution exhibited moderate activity against Candida albicans (MIC90 values 36µM). According to SAR evaluation, the size of the crown ether and the acyl side chain had a significant effect on the bioactivity. Aromatic moieties close to the acyl group led to improved bioactivity as exemplified by some of the tested compounds. These results provide further evidence on the potential of crown ethyl structure as a scaffold for developing new biological probes and lead candidates for drug development.

Oxasqualenoids from Laurencia viridis: Combined Spectroscopic-Computational Analysis and Antifouling Potential.

The chemical study of the red alga Laurencia viridis has led to the isolation of four new polyether triterpenoids: 28-hydroxysaiyacenol B (2), saiyacenol C (3), 15,16-epoxythyrsiferol A (4), and 15,16-epoxythyrsiferol B (5). The structures of 2 and 3 were established mainly by NMR data analysis and comparison with the well-known metabolite dehydrothyrsiferol (1). However, due to the existence of a nonprotonated carbon within the epoxide functionality, stereochemical assignments in 4 and 5 required an in-depth structural study that included NOESY data, J-based configuration analysis, comparison with synthetic models, and DFT calculations. The biological activities of the new metabolites and other related oxasqualenoids were evaluated for the first time against a panel of relevant biofouling marine organisms, and structure-activity conclusions were obtained.

Stereochemical determination of five-membered cyclic ether acetogenins using a spin-spin coupling constant approach and DFT calculations.

Five-membered rings are of particular interest, due to their presence in some of the most common molecules in chemistry and biology. Despite their apparent simplicity, the structural resolution of these rings is complex, due to their inherent conformational flexibility. Here, we describe an application of a recently reported simple and efficient NMR protocol based on the measurement of spin-spin coupling constants to achieve the challenging relative configurations of five new halogenated C15 tetrahydrofuranyl-acetogenins, marilzafurollenes A-D (1-4) and 12-acetoxy-marilzafurenyne (5), isolated from the red alga, Laurencia marilzae. Although DFT chemical shift calculations were used to connect remote stereocenters, the NMR-based approach seems advantageous over computational techniques in this context, as the presence of halogens may interfere with reliable calculations.

Stereochemistry of complex marine natural products by quantum mechanical calculations of NMR chemical shifts: solvent and conformational effects on okadaic acid.

Marine organisms are an increasingly important source of novel metabolites, some of which have already inspired or become new drugs. In addition, many of these molecules show a high degree of novelty from a structural and/or pharmacological point of view. Structure determination is generally achieved by the use of a variety of spectroscopic methods, among which NMR (nuclear magnetic resonance) plays a major role and determination of the stereochemical relationships within every new molecule is generally the most challenging part in structural determination. In this communication, we have chosen okadaic acid as a model compound to perform a computational chemistry study to predict 1H and 13C NMR chemical shifts. The effect of two different solvents and conformation on the ability of DFT (density functional theory) calculations to predict the correct stereoisomer has been studied.

Computationally Assisted Analysis of NMR Chemical Shifts as a Tool in Conformational Analysis.

A key to understanding the properties of functional molecules is to determine their conformation in solution. A conformational analysis procedure that relies on quantum mechanical calculations and the widely used DP4+ probability was evaluated to decipher the structural information encoded in NMR chemical shifts. The results underscore the potential utility of using NMR chemical shifts in advancing conformational analysis studies of complex molecules in solution.

Self-association of okadaic acid: structural and pharmacological significance.

Okadaic acid (OA) has been an invaluable pharmacological tool in the study of cellular signaling. The great affinity of this polyether for its targets together with its high specificity to inhibit certain protein phosphatases enables the differential study of these proteins. Crystallographic structures of protein phosphatases in complex with OA show a 1:1 protein to toxin ratio. Nevertheless, it has been found that OA is able to self-associate under certain conditions although very little is known about the importance of this phenomenon. Here we review the available knowledge on the latter topic and we report on the existence of an unusual self-associated tetrameric form. The structure of these oligomers is proposed based on spectroscopic data and molecular modeling calculations.

OSMAC Approach and Cocultivation for the Induction of Secondary Metabolism of the Fungus Pleotrichocladium opacum.

The application of an OSMAC (One Strain-Many Compounds) approach on the fungus Pleotrichocladium opacum, isolated from a soil sample collected on the coast of Asturias (Spain), using different culture media, chemical elicitors, and cocultivation techniques resulted in the isolation and identification of nine new compounds (8, 9, 12, 15-18, 20, 21), along with 15 known ones (1-7, 10, 11, 14, 19, 22-25). Compounds 1-9 were detected in fungal extracts from JSA liquid fermentation, compounds 10-12 were isolated from a solid rice medium, whereas compounds 14 and 15 were isolated from a solid wheat medium. Addition of 5-azacytidine to the solid rice medium caused the accumulation of compounds 16-18, whereas adding N-acetyl-d-glucosamine triggered the production of two additional metabolites, 19 and 20. Finally, cocultivation of the fungus Pleotrichocladium opacum with Echinocatena sp. in a solid PDA medium led to the production of five additional natural products, 21-25. The structures of the new compounds were elucidated by HRESIMS and 1D and 2D NMR as well as by comparison with literature data. DP4+ and mix-J-DP4 computational methods were applied to determine the relative configurations of the novel compounds, and in some cases, the absolute configurations were assigned by a comparison of the optical rotations with those of related natural products.

Biosynthetic studies on water-soluble derivative 5c (DTX5c).

The dinoflagellate Prorocentrum belizeanum is responsible for the production of several toxins involved in the red tide phenomenon known as Diarrhetic Shellfish Poisoning (DSP). In this paper we report on the biosynthetic origin of an okadaic acid water-soluble ester derivative, DTX5c, on the basis of the spectroscopical analysis of ¹³C enriched samples obtained by addition of labelled sodium [l-¹³C], [2-¹³C] acetate to artificial cultures of this dinoflagellate.

Osteoprotective effect of the marine alkaloid norzoanthamine on an osteoporosis model in ovariectomized rat.

Norzoanthamine (NZ), an alkaloid that has been isolated from the marine cnidiaria Zoanthus sp., has been shown an interesting anti-osteoporotic activity. Although its mechanism of action is not yet clear, it seems that it is different from those of currently used drugs making it particularly interesting. Previous studies have been carried out mostly in vitro. Herein, we present an in vivo study that allows to check the real potential of NZ as a protector substance by direct application into ovariectomized rat bone using a sustained delivery system. Histological and histomorphometric results in ovariectomized rats showed higher bone quality as a result of greater number of trabeculae and osteogenic activity in the group implanted with NZ, compared to controls. In contrast with the untreated controls, NZ-treated groups showed a balanced osteoblast/osteoclast number ratio, similar to that found in the normal bone. These results suggest that NZ could be useful as adjunct to other osteoporosis treatments, but probably its main therapeutic role would be as preventive therapy against bone deterioration.

ML-J-DP4: An Integrated Quantum Mechanics-Machine Learning Approach for Ultrafast NMR Structural Elucidation.

A new tool, ML-J-DP4, provides an efficient and accurate method for determining the most likely structure of complex molecules within minutes using standard computational resources. The workflow involves combining fast Karplus-type J calculations with NMR chemical shifts predictions at the cheapest HF/STO-3G level enhanced using machine learning (ML), all embedded in the J-DP4 formalism. Our ML provides accurate predictions, which compare favorably alongside with other ML methods.

Squamins C-F, four cyclopeptides from the seeds of Annona globiflora.

Four cyclic octapeptides, squamins C-F, were isolated from the seeds of Annona globiflora Schltdl. These compounds share part of their amino acid sequence, -Pro-Met(O)-Tyr-Gly-Thr-, with previously reported squamins A and B. Their structures were determined using NMR spectroscopic techniques together with quantum mechanical calculations (QM-NMR), ESI-HRMS data and a modified version of Marfey's chromatographic method. All compounds showed cytotoxic activity against DU-145 (human prostate cancer) and HeLa (human cervical carcinoma) cell lines. Clearly, A. globiflora is an important source of bioactive molecules, which could promote the sustainable exploitation of this undervalued specie.

On the configuration of five-membered rings: a spin-spin coupling constant approach.

Five-membered rings are clearly among the most common structural motifs found in chemistry and biology. Nevertheless, the configuration of conformationally mobile five-membered rings is often difficult to assign from nuclear magnetic resonance (NMR) data. A simple, reliable, and efficient approach for the stereochemical analysis of five-membered rings based on the measurement of NMR coupling constants is presented. Density functional theory calculations using representative conformations of the full conformational space available to rings with different substitution patterns were used to identify differences between the accessible coupling constant values for cis and trans relative orientations of the substituents. The calculations were assessed experimentally using NMR data obtained from a number of models. This approach can be easily used to analyze different five-membered rings, such as oxolanes, cyclopentanes, furanosides and pyrrolidines, and their relative configuration can be determined without the need for making further conformational considerations.

New polyether triterpenoids from Laurencia viridis and their biological evaluation.

The red seaweed Laurencia viridis is a rich source of secondary metabolites derived from squalene. New polyethers, such as iubol (2), 22-hydroxy-15(28)- dehydrovenustatriol (3), 1,2-dehydropseudodehydrothyrsiferol (4), and secodehydrothyrsiferol (5) have been isolated and characterized from this alga. The structures were determined through the interpretation of NMR spectroscopic data and the relative configuration was proposed on the basis of NOESY spectrum and biogenetic considerations. All new compounds exhibited significant cytotoxic activity against a panel of cancer cell lines.

Are Computational Methods Useful for Structure Elucidation of Large and Flexible Molecules? Belizentrin as a Case Study.

Quantum mechanical NMR methods are progressively becoming decisive in structure elucidation. However, problems arise using low-level calculations for complex molecules, whereas methods using higher levels of theory are not practical for large molecules. This report outlines a synergistic effort employing computationally inexpensive quantum mechanical NMR calculations with conformer selection incorporating 3JHH values as a way to solve the structure of large, complex, and highly flexible molecules using readily available computational resources with belizentrin as a case study.

Prorocentroic Acid, a Neuroactive Super-Carbon-Chain Compound from the Dinoflagellate Prorocentrum hoffmannianum.

Prorocentroic acid (PA) was isolated from the dinoflagellate Prorocentrum hoffmannianum. Relative configurations for its 35 asymmetric centers were determined by analysis of NMR data including heteronuclear couplings and quantum mechanical calculations. PA was tested by using murine cortical neurons grown on microelectrode arrays. Long-term exposure to subtoxic concentrations induced a significant reorganization of neuronal signaling, mainly by changes in the bursting activity. The observed effects could be due to the activation of a plasticity process.