Simplified and enhanced VCD analysis of cyclic peptides guided by artificial intelligence.

Cyclic peptides are privileged structures in medicinal chemistry; however, their solution-state structure characterization is difficult. Vibrational circular dichroism (VCD) spectroscopy is a powerful alternative to NMR, but requires challenging calculations. We present a VCD approach guided by a genetic algorithm, which is simple, more effective, and has a higher conformer resolution.

Pushing the boundaries of VCD spectroscopy in natural product chemistry.

Vibrational circular dichroism (VCD) is one of the most powerful techniques to assess the stereochemistry of chiral molecules in solution state. The need for quantum chemical calculations to interpret experimental data, however, has precluded its widespread use by non-experts. Herein, we propose the search and validation of IR and VCD spectral markers to circumvent the requirement of DFT calculations allowing for absolute configuration assignments even in complex mixtures. To that end, a combination of visual inspection and machine learning based methods is used. Monoterpene mixtures are selected for this proof-of-concept study.

Absolute Configuration and Antileishmanial Activity of (-)-Cyclocolorenone Isolated from Duguetia lanceolata (Annonaceae).

BACKGROUND: The fractionation of the n-hexane phase of the EtOH extract from the leaves of Duguetia lanceolata (Annonaceae) led to the identification of the sesquiterpene (-)-cyclocolorenone. OBJECTIVES: Chemical characterization, including determination of the absolute stereochemistry, and in vitro evaluation of antileishmanial activity of the sesquiterpene (-)-cyclocolorenone, isolated from D. lanceolata, were carried out. METHODS: (-)-Cyclocolorenone was isolated from D. lanceolata leaves using different chromatographic steps and its structure was defined by analysis of NMR and ESI-HRMS data. Additionally, the absolute configuration of (-)-cyclocolorenone was ambiguously assigned by means of vibrational circular dichroism (VCD). Antileishmanial activity of (-)-cyclocolorenone was evaluated on promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. The integrity of the cell membrane of L. (L.) amazonensis was analyzed using the SYTOX green probe. RESULTS: (-)-(1R,6S,7R,10R)-Cyclocolorenone displayed activity against promastigotes and amastigotes forms of L. (L.) amazonensis with IC50 of 4.54 and 28.44 µM, respectively. Furthermore, this compound was non-toxic in J774 macrophage cells (CC50 > 458.71 µM) with a selectivity index > 100 (promastigotes) and > 32.2 (amastigotes). Additionally, (-)-cyclocolorenone was observed to target the parasite cell membrane. CONCLUSION: Obtained data suggested that (-)-cyclocolorenone, in which absolute configuration was determined, can be considered as a scaffold for the development of new drugs for the treatment of leishmaniasis.

Absolute configuration of cytotoxic anthraquinones from a Brazilian cave soil-derived fungus, Aspergillus sp. SDC28.

Microbial strains isolated from extreme and understudied environments, such as caves, are still poorly investigated for the production of bioactive secondary metabolites. Investigation of the ethyl acetate extract from the growth medium produced by the soil-derived fungus Aspergillus sp. SDC28, isolated from a Brazilian cave, yielded two anthraquinones: versicolorin C (1) and versiconol (2). The complete assignment of nuclear magnetic resonance and mass spectroscopic data of 1 and 2 was performed for the first time. Moreover, the yet unreported absolute configuration of both compounds was unambiguously established by analysis of experimental and theoretical electronic circular dichroism data. Vibrational circular dichroism was also applied to confirm the absolute stereochemistry of 2. Compounds 1 and 2 showed cytotoxic activity against human ovarian cancer cells (OVCAR3).

Conformational preferences induced by cyclization in orbitides: a vibrational CD study.

Orbitides are bioactive head-to-tail natural cyclic peptides from plant species. Their bioactivity is intrinsically related to the main conformations adopted in solution, whose correct characterization represents an important bottleneck for medicinal chemistry applications. To date, NMR spectrosocopy has been the most frequently used technique to assess the secondary structure of orbitides. Despite the amount of structural information commonly available from NMR, its time scale frequently results in a limited conformational ensemble with a single mean structure, which may not represent the bioactive conformation. Additionally, problems with inter-residue NOE/ROE signals can reduce the accuracy and confidence of the 3D assignments. Vibrational circular dichroism (VCD), on the other hand, has been demostrated as a powerful tool to probe the stereostructure of chiral molecules, including peptides and proteins, with enhanced sensitivity to individual conformations in the condensed phase. Herein, we present the first VCD stereochemical investigation of orbitides. By combining IR/VCD experiments in ACN-d3 and ACN-d3/D2O mixtures with DFT calculations in different levels of theory we were able to determine the solution-state conformational behavior, as well as the main structural restraints induced by cyclization, of the seven-residue orbitide pohlianin A and its linear precursor. VCD results indicated inverse γ-turns as the most prevalent structural motif for the linear precursor in partially aqueous solution, while type I and type VI ß-turns were induced by the cyclization process, along with some classic γ-turns. In addition to the conformation of the cyclic peptide already described from NMR data, two previously unidentified conformations with distinct secondary structures were found to significantly populate the sample. This conformational discriminatory power of VCD for both linear and cyclic peptides in different solvent systems may lead to more accurate structural characterization of turn-rich peptidic natural products and help the design of conformationally-tailored peptides for more precise structure-activity relationship results.

Tackling Stereochemistry in Drug Molecules with Vibrational Optical Activity.

Chirality plays a crucial role in drug discovery and development. As a result, a significant number of commercially available drugs are structurally dissymmetric and enantiomerically pure. The determination of the exact 3D structure of drug candidates is, consequently, of paramount importance for the pharmaceutical industry in different stages of the discovery pipeline. Traditionally the assignment of the absolute configuration of druggable molecules has been carried out by means of X-ray crystallography. Nevertheless, not all molecules are suitable for single-crystal growing. Additionally, valuable information about the conformational dynamics of drug candidates is lost in the solid state. As an alternative, vibrational optical activity (VOA) methods have emerged as powerful tools to assess the stereochemistry of drug molecules directly in solution. These methods include vibrational circular dichroism (VCD) and Raman optical activity (ROA). Despite their potential, VCD and ROA are still unheard of to many organic and medicinal chemists. Therefore, the present review aims at highlighting the recent use of VOA methods for the assignment of the absolute configuration of chiral small-molecule drugs, as well as for the structural analysis of biologics of pharmaceutical interest. A brief introduction on VCD and ROA theory and the best experimental practices for using these methods will be provided along with selected representative examples over the last five years. As VCD and ROA are commonly used in combination with quantum calculations, some guidelines will also be presented for the reliable simulation of chiroptical spectra. Special attention will be paid to the complementarity of VCD and ROA to unambiguously assess the stereochemical properties of pharmaceuticals.

Antitrypanosomal Lactones from Nectandra barbellata.

Twigs of Nectandra barbellata were extracted using a solution of the ionic liquid 1-butyl-3-methylimidazolium bromide (BMImBr) in H2O, assisted by microwave (MAE). After successive chromatographic steps, one sesquiterpene, costic acid, and three new related lactones, (R)-3(7)-Z-3-hexadec-21-enylidene-5-(hydroxymethyl)tetrahydrofuran-2-one (1), (R)-3(7)-Z-3-hexadecylidene-5-(hydroxymethyl)tetrahydrofuran-2-one (2), and (R)-3(7)-Z-3-docosylidene-5-(hydroxymethyl)tetrahydrofuran-2-one (3), were isolated. After structural elucidation using IR, UV, HRESIMS, NMR, ECD, and VCD, compounds 1-3 were tested against trypomastigote forms of Trypanosoma cruzi. The mechanism of action of bioactive isolated compounds was studied using different fluorescent-based approaches to investigate alterations of the plasma membrane, permeability/electric potential (ΔΨp), reactive oxygen species levels, mitochondria (electric membrane potential, ΔΨm/ATP levels), Ca2+ levels, and pH of the acidocalcisomes. In addition, in silico studies predicted no resemblance to pan assay interference compounds (PAINS).

Phytotoxic Neocassane Diterpenes from Eragrostis plana.

Five new neocassane-like diterpenes (1-5) were isolated from the roots of Eragrostis plana. Their structures were elucidated by spectroscopic data, and the absolute configurations of the representative compounds neocassa-12(17),15-dien-3-one (2) and neocassa-12(13),15-diene-3,14-dione (5) were assigned by vibrational circular dichroism. The phytotoxic activity of compounds 3, 4, and 5 was evaluated against the germination and growth of Ipomoea grandifolia and Euphorbia heterophylla. Compound 4 exhibited the most potent activity for growth variables, resulting in the smallest radicle length of I. grandifolia and in the smallest aerial part length of E. heterophylla. Compound 5 reduced the germination speed index more effectively than the herbicide glyphosate (Roundup).

Cytotoxic sesquiterpene lactones from Campuloclinium macrocephalum (=Eupatorium macrocephalum).

Three undescribed germacranolide sesquiterpene lactones, named macrocephalides A-C, along with known steroids, triterpenes and flavonoids were isolated from the aerial parts of Campuloclinium macrocephalum. The structures of the undescribed compounds were elucidated with basis on their 1D and 2D-NMR, and HR-ESI-MS data. Their absolute configurations were assigned by comparison of experimental and calculated electronic circular dichroism (ECD) spectra. Additionally, macrocephalides A-C were evaluated for their in vitro cytotoxic activities against nine human cancer cell lines. Macrocephalides A and B exhibited moderate to potent cytotoxic activity, inhibiting 50% of cell growth (GI50) at concentrations ranging from 0.576 to 6.37 µM.

Sesquiterpene Lactones from Calea pinnatifida: Absolute Configuration and Structural Requirements for Antitumor Activity.

This work describes the chromatographic fractionation of the aerial parts of Calea pinnatifida and the structural characterization and determination of the absolute configuration of the isolated compounds as well as their antitumor potential. The HPLC fractionation of the CH2Cl2 phase of the MeOH extract from the leaves of C. pinnatifida led to the isolation of two related sesquiterpene lactones (STLs): calein C (1) and calealactone B (2). Additionally, during the purification process, a derivative of calein C (3) was formed as a product of the Michael addition of MeOH. The structures of Compounds 1-3 were established based on spectroscopic and spectrometric data, while the absolute stereochemistry was established by vibrational circular dichroism. In order to evaluate the effect of the conjugated double bonds on the cytotoxic activity of STLs, Compounds 1-3 were tested against anaplastic (KTC-2) and papillary (TPC-1) thyroid carcinoma cells. Calein C was the most active of the STLs, and displayed activity against both KTC-2 and TPC-1. On the other hand, the calein C derivative (3) was the least cytotoxic of all the compounds tested. These results are promising and suggest the importance of studying sesquiterpene lactones isolated from C. pinnatifida in terms of antitumor activity, especially considering the effects of α,ß-unsaturated carbonyl systems.

Structure Elucidation and Absolute Configuration Determination of Nortriterpenoids from Picramnia glazioviana.

In this study, HPLC-PDA-HRMS-SPE-NMR data were used for initial analysis of the CH2Cl2 fraction of an EtOH extract of the leaves of Picramnia glazioviana. The HRMS, UV, and NMR data obtained from the HPLC-PDA-HRMS-SPE-NMR analysis were used to direct semipreparative HPLC isolation toward nortriterpenoids, which resulted in the isolation of 18 new and highly oxygenated nortriterpenoids (1-3, 5-10, 12-19, and 21), named picravianes C-T. Their structures were determined on the basis of analysis of UV, HRMS, and 2D NMR spectroscopic data, including determination of the relative configuration on the basis of coupling pattern analysis and nuclear Overhauser effect correlations. The absolute configurations of compounds 7, 9, 10, 14, 15, 17, 18, 19, and 21 were assigned using electronic circular dichroism data, and the cytotoxicity of compounds 6, 10, 14, 16, 17, 18, 19, and 21 was evaluated against MDA-MB-231 triple-negative breast cancer, SKBR-3 Her2-overexpressing breast cancer, and A549 lung cancer cells lines. The isolated compounds contain a hitherto undescribed modification of the terminal backbone and/or E-ring, and a possible biosynthetic pathway for their formation is proposed.

Water-Soluble Glutamic Acid Derivatives Produced in Culture by Penicillium solitum IS1-A from King George Island, Maritime Antarctica.

A new method of screening was developed to generate 770 organic and water-soluble fractions from extracts of nine species of marine sponges, from the growth media of 18 species of marine-derived fungi, and from the growth media of 13 species of endophytic fungi. The screening results indicated that water-soluble fractions displayed significant bioactivity in cytotoxic, antibiotic, anti-Leishmania, anti-Trypanosoma cruzi, and inhibition of proteasome assays. Purification of water-soluble fractions from the growth medium of Penicillium solitum IS1-A provided the new glutamic acid derivatives solitumine A (1), solitumine B (2), and solitumidines A-D (3-6). The structures of compounds 1-6 have been established by analysis of spectroscopic data, chemical derivatizations, and vibrational circular dichroism calculations. Although no biological activity could be observed for compounds 1-6, the new structures reported for 1-6 indicate that the investigation of water-soluble natural products represents a relevant strategy in finding new secondary metabolites.

An epigenetic modifier induces production of 3-(4-oxopyrano)-chromen-2-ones in Aspergillus sp. AST0006, an endophytic fungus of Astragalus lentiginosus.

Incorporation of the epigenetic modifier suberoylanilide hydroxamic acid (SAHA) into a potato dextrose broth culture of the endophytic fungus Aspergillus sp. AST0006 affected its polyketide biosynthetic pathway providing two new 3-(4-oxopyrano)-chromen-2-ones, aspyranochromenones A (1) and B (2), and the isocoumarin, (-)-6,7-dihydroxymellein (3). Eight additional metabolites (4-11) and two biotransformation products of SAHA (12-13) were also encountered. The planar structures and relative configurations of the new metabolites 1-2 were elucidated with the help of high-resolution mass, 1D and 2D NMR spectroscopic data and the absolute configurations of 1-3 were determined by comparison of experimental and calculated ECD data. Possible biosynthetic pathways to 1 and 2 are presented.

Diterpenoids with inhibitory activity of nitrite production from Croton floribundus.

ETHNOPHARMACOLOGICAL RELEVANCE: Croton floribundus Spreng. (Euphorbiaceae), popularly known as Capixinguí, stands out due to its widespread use in traditional medicine to treat wounds, syphilis, hemorrhoids, eye diseases and as a purgative. AIM OF THE STUDY: To characterize clerodanes diterpenes from C. floribundus and to evaluate the effects of the fraction and diterpenes (1-5) on inhibition of nitrite production. MATERIALS AND METHODS: The hydroethanolic root extract of C. floribundus was fractionated on a solid phase extraction column to obtain the fraction named Fr80%. From this, five compounds were obtained and characterized. The absolute configuration of compound 1 was determined by a combination of electronic and vibrational circular dichroism spectroscopies. Additionally, compounds 1-5 were evaluated for their inhibitory effects on nitrite production induced by lipopolysaccharide (LPS) in RAW 264 macrophage cell. RESULTS: Five clerodane diterpenoids were characterized, and the absolute stereochemistry of 1 was established as 3R,4R,5R,8R,9R,10S,12S. The IC50 values obtained through inhibition of nitrite production were 28.52 ±â€¯2.21 µM (1), 40.26 ±â€¯2.79 µM (2), 25.47 ±â€¯2.16 µM (3), 35.78 ±â€¯2.93 µM (4) and 40.58 ±â€¯4.78 µM (5). In the tested concentrations, the samples presented low toxicity in macrophages. CONCLUSIONS: Four new diterpenes were characterized from C. floribundus, these being croflorins A-D (1-4) and a known halimane (5). These compounds exhibited inhibitory effect on nitrite production.

Stereochemistry of spongosoritins: beyond optical rotation.

Stereochemical determinations based solely on the comparison of optical rotation (OR) measured at a single wavelength may commonly result in misassignments. Herein, we use vibrational and electronic CD, NMR, OR, and DFT calculations, to confirm the absolute configuration (AC) of the cytotoxic marine polyketides spongosoritin A (1) and 9,10-dihydrospongosoritin A (2) as (-)-(6R,8R) and (-)-(6R,8S), respectively. The AC of natural 1 and 2 has so far relied upon comparisons of OR values only. Besides showing the dependence of OR on achiral structural features, such as E/Z double bond geometries, an IR spectral marker is provided to help distinguish the geometric isomers of related molecules.

Botryane terpenoids produced by Nemania bipapillata, an endophytic fungus isolated from red alga Asparagopsis taxiformis - Falkenbergia stage.

A chemical study of the EtOAc extract of Nemania bipapillata (AT-05), an endophytic fungus isolated from the marine red alga Asparagopsis taxiformis - Falkenbergia stage, led to the isolation of five new botryane sesquiterpenes, including the diastereomeric pair (+)-(2R,4S,5R,8S)-(1) and (+)-(2R,4R,5R,8S)-4-deacetyl-5-hydroxy-botryenalol (2), (+)-(2R,4S,5R,8R)-4-deacetyl-botryenalol (3), one pair of diastereomeric botryane norsesquiterpenes bearing an unprecedented degraded carbon skeleton, (+)-(2R,4R,8R)-(4) and (+)-(2R,4S,8S)-(5), which were named nemenonediol A and nemenonediol B, respectively, in addition to the known 4ß-acetoxy-9ß,10ß,15α-trihydroxyprobotrydial (6). Their structures were elucidated using 1D and 2D NMR, HRESIMS and comparison with literature data of similar known compounds. The absolute configurations of 2, 3 and 4 were deduced by comparison of experimental and calculated electronic circular dichroism (ECD) spectra, while those of 1 and 5 were assigned from vibrational circular dichroism (VCD) data. Compound 4 weakly inhibited acetylcholinesterase, whereas compound 1 inhibited both acetylcholinesterase and butyrylcholinesterase. Compounds 1, 3, 5 and 6 were tested against two carcinoma cell lines (MCF-7 and HCT-116), but showed no significant citotoxicity at tested concentrations (IC50 > 50 µM).

Enantioselective ene-reduction of E-2-cyano-3-(furan-2-yl) acrylamide by marine and terrestrial fungi and absolute configuration of (R)-2-cyano-3-(furan-2-yl) propanamide determined by calculations of electronic circular dichroism (ECD) spectra.

This work reports the green organic chemistry synthesis of E-2-cyano-3(furan-2-yl) acrylamide under microwave radiation (55 W), as well as the use of filamentous marine and terrestrial-derived fungi, in the first ene-reduction of 2-cyano-3-(furan-2-yl) acrylamide to (R)-2-cyano-3-(furan-2-yl)propanamide. The fungal strains screened included Penicillium citrinum CBMAI 1186, Trichoderma sp. CBMAI 932 and Aspergillus sydowii CBMAI 935, and the filamentous terrestrial fungi Aspergillus sp. FPZSP 146 and Aspergillus sp. FPZSP 152. A compound with an uncommon CN-bearing stereogenic center at the α-C position was obtained by enantioselective reactions mediated in the presence of the microorganisms yielding the (R)-2-cyano-3-(furan-2-yl) propanamide 3a. Its isolated yield and e.e. ranged from 86% to 98% and 39% to 99%, respectively. The absolute configuration of the biotransformation products was determined by time-dependent density functional theory (TD-DFT) calculations of electronic circular dichroism (ECD) spectra. Finally, the tautomerization of 2-cyano-3-(furan-2-yl) propanamide 3a to form an achiral ketenimine was observed and investigated in presence of protic solvents.

New Benzaldehyde and Benzopyran Compounds from the Endophytic Fungus Paraphaeosphaeria sp. F03 and Their Antimicrobial and Cytotoxic Activities.

Three new benzaldehyde derivatives, sporulosaldeins A - C (1: -3: ), and 3 new benzopyran derivatives, sporulosaldeins D - F (4: -6: ), were discovered from an endophytic fungus, Paraphaeosphaeria sp. F03, which was isolated from Paepalanthus planifolius leaves. Compounds 1: -6: were elucidated by 1- and 2-dimensional nuclear magnetic resonance experiments and high-resolution mass spectrometry analysis. The absolute configuration of compound 5: was determined through the comparison of experimental and calculated electronic circular dichroism data. Compounds 1: -6: were found to exhibit antifungal activity with minimum inhibitory concentration (MIC) values of 7.8 - 250 µg/mL and racemic mixture of compound 6: exhibited weak cytotoxicity against MCF-7 and LM3 with IC50 values of 34.4 and 39.2 µM, respectively.