Assessment of the Long-Range NMR C,H Coupling of a Series of Carbazolequinone Derivatives.

Synthesis, the complete 1H- and 13C-NMR assignments, and the long-range C,H coupling constants (nJC,H) of some hydrogen-deficient carbazolequinones, assessed by a J-HMBC experiment, are reported. In these molecules, the protons, used as entry points for assignments, are separated by several bonds with non-protonated atom carbons. Therefore, the use of long-range NMR experiments for the assignment of the spectra is mandatory; we used HSQC and HMBC. On the other hand, the measured heteronuclear (C,H) coupling constants 2J to 5J) allow us to choose the value of the long-range delay used in the HMBC experiment less arbitrarily in order to visualize a desired correlation in the spectrum. The chemical shifts and the coupling constant values can be used as input for assignments in related chemical structures.

Combining the Power of J Coupling and DP4 Analysis on Stereochemical Assignments: The J-DP4 Methods.

A systematic study to include 3 JHH couplings into DP4 formalism ( J-DP4) led to the development of three alternative strategies. The d J-DP4 (direct) approach involves a new DP4-like equation including an additional probability term given by 3 JHH. The i J-DP4 (indirect) approach explores the original DP4 method with a restricted conformational search. Despite both strategies performing better than DP4, their combined use (i J/d J-DP4) provided the best results, with a 2.5-fold performance improvement at similar or lower computational cost.

Discovery of New E-Selectin Inhibitors by Virtual Screening, Fluorescence Binding Assays, and STD NMR Experiments.

E-selectin is an endothelial protein that participates in the adhesion of metastatic cancer cells, and is therefore a relevant target for antitumor therapeutic intervention. In this work, virtual screening was used to identify new E-selectin inhibitors from a subset of drug-like molecules retrieved from the ZINC database, including the physiological ligand sLe(x) as reference structure (PDB ID: 1G1T). Four hits were chosen and subjected to molecular dynamics simulations and fluorescence binding assays, which led to the determination of experimental dissociation constants between 333 and 1012 µm. The candidate with the highest affinity was studied by saturation transfer difference (STD) NMR experiments and complete relaxation and conformational exchange matrix analysis of saturation transfer (CORCEMA-ST), aimed at identifying the preferable binding mode with E-selectin. Our results revealed that this new inhibitor binds more strongly than sLe(x) in the E-selectin binding site, in good agreement with simulation predictions. These properties will prove valuable for the future design of drugs that target E-selectin.

Macrocyclization of Peptide Side Chains by the Ugi Reaction: Achieving Peptide Folding and Exocyclic N-Functionalization in One Shot.

The cyclization of peptide side chains has been traditionally used to either induce or stabilize secondary structures (ß-strands, helices, reverse turns) in short peptide sequences. So far, classic peptide coupling, nucleophilic substitution, olefin metathesis, and click reactions have been the methods of choice to fold synthetic peptides by means of macrocyclization. This article describes the utilization of the Ugi reaction for the side chain-to-side chain and side chain-to-termini macrocyclization of peptides, thus enabling not only access to stable folded structures but also the incorporation of exocyclic functionalities as N-substituents. Analysis of the NMR-derived structures revealed the formation of helical turns, ß-bulges, and α-turns in cyclic peptides cross-linked at i, i + 3 and i, i + 4 positions, proving the folding effect of the multicomponent Ugi macrocyclization. Molecular dynamics simulation provided further insights on the stability and molecular motion of the side chain cross-linked peptides.

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.

A multicomponent conjugation strategy to unique N-steroidal peptides: first evidence of the steroidal nucleus as a ß-turn inducer in acyclic peptides.

Constraining small peptides into specific secondary structures has been a major challenge in peptide ligand design. So far, the major solution for decreasing the conformational flexibility in small peptides has been cyclization. An alternative is the use of topological templates, which are able to induce and/or stabilize peptide secondary structures by means of covalent attachment to the peptide. Herein a multicomponent strategy and structural analysis of a new type of peptidosteroid architecture having the steroid as N-substituent of an internal amide bond is reported. The approach comprises the one-pot conjugation of two peptide chains (or amino acid derivatives) to aminosteroids by means of the Ugi reaction to give a unique family of N-steroidal peptides. The conjugation efficiency of a variety of peptide sequences and steroidal amines, as well as their consecutive head-to-tail cyclization to produce chimeric cyclopeptide-steroid conjugates, that is, macrocyclic lipopeptides, was assessed. Determination of the three-dimensional structure of an acyclic N-steroidal peptide in solution proved that the bulky, rigid steroidal template is capable of both increasing significantly the conformational rigidity, even in a peptide sequence as short as five amino acid residues, and inducing a ß-turn secondary structure even in the all-s-trans isomer. This report provides the first evidence of the steroid skeleton as ß-turn inducer in linear peptide sequences.

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.

Combined nuclear magnetic resonance spectroscopy and molecular dynamics study of growth hormone releasing hexapeptide GHRP-6 and a cyclic analogue.

The Growth Hormone Releasing Hexapeptide, GHRP-6 was the first of a family of synthetic peptides that enhance the release of the Growth Hormone by the pituitary gland in a dose-dependent manner. Since its discovery, it has been used as a benchmark and starting point in numerous researches aiming to obtain new drugs. Complete resonance assignment of GHRP-6 NMR spectra in both open and cyclic forms are reported, showing some differences to random coil chemical shifts. Connectivities observed in the ROESY spectra indicate spatial proximity between the aromatic residues side-chains in both molecules, as well as between residues DPhe5 and Lys6 sidechains. An ensemble of 10 structures was generated for each one of the molecules, showing RMSD values indicative of nonrandom structures. Molecular Dynamics simulations, both with and without explicit solvent, were carried out for GHRP-6 and its cyclic analogue. Conformational analysis performed on the trajectories showed a nonrandom structure with a well preserved backbone. The presence of geometrical patterns resembling those typical of π-π interactions in both peptides, suggest that this kind of interactions may be relevant for the biological activity of GHRP-6. Same conclusion can be drawn from the spatial proximity of residues DPhe5 and Lys6 sidechains.

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.

On the unusual 2J(C2-H(f)) coupling dependence on syn/anti CHO conformation in 5-X-furan-2-carboxaldehydes.

A remarkable difference for (2)J(C(2)-H(f)) coupling constant in syn and anti conformers of 5-X-furan-2-carboxaldehydes (X = CH(3), Ph, NO(2), Br) and a rationalization of this difference are reported. On the basis of the current knowledge of the Fermi-contact term transmission, a rather unusual dual-coupling pathway in the syn conformer is presented. The additional coupling pathway resembles somewhat that of the J(H-H) in homoallylic couplings, which are transmitted by hyperconjugative interactions involving the pi(C=C) electronic system. The homoallylic coupling pathway can be labeled as sigma*(C-H) <-- pi(C=C) --> sigma*(C-H). In the present case, this additional coupling pathway, using an analogous notation, can be labeled as sigma*(C(2)-C(C)) <-- LP(1)(O(1))...LP(2)(O(C)) --> sigma*(C(C)-H(f)) (sigma*(C(2)-C(C))) where O(1) and O(C) stand for the ring and carbonyl O atoms, respectively. This additional coupling pathway is not activated in the anti conformers since both oxygen lone pairs do not overlap.

In vitro cytotoxicity of norditerpenoid alkaloids.

Forty-three norditerpenoid alkaloids isolated from Aconitum, Delphinium and Consolida species have been evaluated for their cytotoxic effects on the tumor cell lines CT26 (murine colon adenocarcinoma), SW480 (human colon adenocarcinoma), HeLa (human cervical adenocarcinoma), SkMel25 (human melanoma) and SkMel28 (human malignant melanoma) with several multidrug resistance mechanisms and the non-tumor cell line CHO (Chinese hamster ovary cells). Neoline (5), 8-O-methylcolumbianine (6), 1,14-diacetylcardiopetaline (9), 18-O-demethylpubescenine (13), 14-deacetylpubescenine (14), pubescenine (15), 14-deacetylajadine (25), lycoctonine (26), browniine (28), delphatine (29), dehydrotakaosamine (34), and ajadelphinine (37) exhibited selective cytotoxicity to cancerous versus non-cancerous cells. Some of these compounds had an irreversible effect on SW480 (5, 15, 25, 26, and 34), HeLa (15, 34, and 37) and SkMel25 (15 and 34) cell lines. In order to gain insights into the mechanism of irreversible cytotoxic action of these compounds we compared the cell viability by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and the acid phosphatase (AP) methods. Our results suggest that the effects of these compounds could be related to the inhibition of ATP production.

Structural diversity and defensive properties of norditerpenoid alkaloids.

We have tested the insect antifeedant and toxic activity of 43 norditerpenoid alkaloids on Spodoptera littoralis and Leptinotarsa decemlineata including eserine (physostigmine), anabasine, and atropine. Antifeedant effects of the test compounds were structure- and species-dependent. The most active antifeedants to L. decemlineata were 1,14-diacetylcardiopetaline (9) and 18-hydroxy- 14-O-methylgadesine (33), followed by 8-O-methylconsolarine (12), 14-O-acetyldelectinine (27), karakoline (7), cardiopetaline (8), 18-O-demethylpubescenine (13), 14-O-acetyldeltatsine (18), takaosamine (21), ajadine (24), and 8-O-methylcolumbianine (6) (EC50 < 1 microg/cm2). This insect showed a moderate response to atropine. S. littoralis had the strongest antifeedant response to 24, 18, 14-O-acetyldelcosine (19), and delphatine (29) (EC50 < 3 microg/cm2). None of the model substances affected the feeding behavior of this insect. The most toxic compound to L. decemlineata was aconitine (1), followed by cardiopetalidine (10) (% mortality > 60), 14-deacetylpubescenine (14), 18-O-benzoyl-18-O-demethyl-14-O-deacetylpubescenine (17), 14-O-acetyldelcosine (19), 14-deacetylajadine (25) and methyllycaconitine (30) (% mortality > 45). Orally injected S. littoralis larvae were negatively affected by 1, cardiopetaline (8), 10, 1,14-O-acetylcardiopetalidina (11), 12, 14, 1,18-O-diacetyl-19-oxo-gigactonine (41), olivimine (43), and eserine in varying degrees. Their antifeedant or insecticidal potencies did not parallel their reported nAChR binding activity, but did correlate with the agonist/antagonist insecticidal/antifeedant model proposed for nicotininc insecticides. A few compounds [14, tuguaconitine (38), 14-demethyldelboxine (40), 19, dehydrodelsoline (36), 18-O-demethylpubescenine (13), 41, 9, and delcosine (23)] had selective cytotoxic effects to ward insect-derived Sf9 cells. None were cytotoxic to mammalian CHO cells and none increased Trypanosoma cruzi mortality. The selective cytotoxic effects of some structures indicate that they can act on biological targets other than neuroreceptors.

Biosynthetic studies of the DSP toxin skeleton.

Marine toxins have drawn wide interest because their economical impact and disastrous effect upon the shellfish industry and public health in many parts of the world. One of the most interesting group of substances of marine toxins, from structural and pharmacological points of view are polyether compounds, which generally present a great diversity in size and potent biological activities. The subject of this work was about to biosynthesis of okadaic acid skeleton as leader as DSP toxins. Its biosynthesis attracts considerable attention since the carbon skeleton has been shown to be synthesised via an unusual route. In this paper we report on stable isotope incorporation experiments on DSP toxin in artificial cultures of dinoflagellate. The comparison of the degrees of incorporation in these samples measured by different methods led to contradictory results. This implies that further experimental data is needed in order to propose a logical biogenetic scheme.

Self-association of okadaic acid upon complexation with potassium ion.

Okadaic acid (OA) is a toxin responsible for diarrhetic shellfish poisoning and is an extremely useful tool for studying processes that are regulated by phosphorylation, although the exact mechanism of action is still undetermined. We report on a study that proved the existence of OA in an unusual dimeric form when complexed with potassium ion. The proposed structure of this dimer is based on spectroscopic and conformational studies.

Absolute configuration and complete assignment of 13C NMR data for new sesquiterpenes from Maytenus chiapensis.

Five new dihydro-beta-agarofuran sesquiterpenes (1-5) were isolated from the leaves of Maytenus chiapensis. The structures of 1-5 were determined by means of 1D and 2D NMR techniques. A semiselective HMBC technique was applied in order to obtain complete (13)C NMR assignments. Absolute configurations were determined by CD studies and chemical correlations and on biogenetic grounds. Compound 4 showed weak activity against a multidrug-resistant Leishmania tropica line.

Alkaloids from Consolida oliveriana.

Four new diterpenoid alkaloids, olivimine (1), olividine (2), 8-O-methylcolumbianine (3), and 7alpha-hydroxycossonidine (4), were isolated from the aerial parts of Consolida oliveriana. Compounds 1 and 2, 3, and 4 belong to the lycoctonine-azomethine, aconitine, and hetisine types, respectively, and their structures were elucidated by spectral data interpretation.