1H NMR Mixture Design-Fingerprints and ASCA Analysis in Ilex paraguariensis: Model Stability in Search of a Global Metabolome.

The effects of experimental repetitions and solvent extractors on the 1H NMR fingerprinting of yerba mate extracts, obtained from two genders and two light environments, were analyzed in-depth by ANOVA-simultaneous component analysis (ASCA). Different solvents were used according to a mixture design based on ethanol, dichloromethane, and hexane and their combinations. The number of experimental repetitions significantly affected the ASCA results. Increasing repetitions led to decreases in the percentage effect variance values and an increase in the percentage residual variance. However, secondary sexual dimorphism, light availability, and their interaction effects became more significant with decreasing p-values at or above the 95% confidence level. The choice of a solvent extractor significantly affects the chemical profile and can lead to distinct conclusions regarding the significance of effect values. Pure solvents yielded different conclusions about the significance of factorial design effects, with each solvent extracting unique metabolites and maximizing information for specific effects. However, the use of binary solvent mixtures, such as ethanol-dichloromethane, proved more efficient in extracting sets of compounds that simultaneously differentiate between different experimental conditions. The mixture design-fingerprint strategy provided satisfactory results expanding the range of extracted metabolites with high percentage of residual variances and low explained percentage effect variances in the ASCA models. Ternary and even higher-ordered mixtures could be good alternative extracting media for work-intensive procedures. Our study underscores the significance of experimental design and solvent selection in metabolomic analysis, improving the accuracy, robustness, and interpretability of metabolomic models, leading to a better understanding of the chemical composition and biological implications of plant extracts.

Green Synthesis of Spiro Compounds with Potential Anticancer Activity through Knoevenagel/Michael/Cyclization Multicomponent Domino Reactions Organocatalyzed by Ionic Liquid and Microwave-Assisted.

In this work a microwave-assisted Knoevenagel/Michael/cyclization multicomponent domino methodology, using ethanol as solvent and the ionic liquid 1-methylimidazolium chloride as catalyst was developed for the synthesis of spiro compounds. The reaction conditions considered ideal were determined from a methodological study varying solvent, catalyst, amount of catalyst, temperature, and heating mode. Finally, the generality of the methodology was evaluated by exploring the scope of the reaction, varying the starting materials (isatin, malononitrile, and barbituric acid). Overall, the twelve spiro compounds were synthesized in good yields (43-98%) and the X-ray structure of compound 1b was obtained. In addition, the in vitro antiproliferative activities of the spirocycles against four types of human cancer cell lines including HCT116 (human colon carcinoma), PC3 (prostate carcinoma), HL60 (promyelocytic leukemia), and SNB19 (astrocytoma) were screened by MTT-based assay. It is noteworthy that spiro compound 1c inhibited the four cell lines tested with the lowest IC50 values: 52.81 µM for HCT116, 74.40 µM for PC3, 101 µM for SNB19, and 49.72 µM for HL60.

Mechanistic investigation of enolate/stabilized vinylogous carbanion-mediated organocatalytic azide (3 + 2) cycloaddition reactions for the synthesis of 1,2,3-triazoles.

Herein, we report a fully detailed mechanistic study involving an organocatalyzed 1,3-dipolar cycloaddition via enolate or stabilized vinylogous carbanion intermediates and azide for the synthesis of 1,2,3-triazoles. A detailed investigation of the elementary steps, intermediates, and transition states of the two organocatalyzed metal-free click reactions is supported by DFT calculations and 1H NMR monitoring experiments, providing detailed profiles for both reaction mechanisms. Distortion-interaction activation-strain (DIAS) analysis was also employed to further elucidate the regioselectivity in both reactions.

Proteomic characterization of the fibroin-based silk fibers produced by weaver ant Camponotus textor.

The fibroin-based silk fibers of weaver ants are an alternative biomaterial to be investigated and explored for potential biomedical applications. In this context, the silk fibers from the nest of the weaver ant Camponotus textor was solubilized and fractionated by gel permeation. The different fractions were collected, pooled and submitted to analysis with a series of biochemical methods, nuclear magnetic resonance (NMR) spectroscopy, analytical proteomic strategies, and data treatment with bioinformatic tools to perform the structural characterization of the fibroin-based silk fibers produced by the ant. Our data demonstrated the identification of one fibroin proteoform in the ant silk fibers. The protein chracterized as a glycoprotein with MW around 40 kDa and presenting 66% (w/w) of total sugars attached to it through O-linked carbohydrates. The 3D of protein was modeled, revealing a structure predominantly constituted of coiled-coil secondary units in the whole model, featuring at least four superhelices (arrangement with multiple α-helices). The scientific outcomes reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based polymers for biomedical applications. BIOLOGICAL SIGNIFICANCE: The present investigation significantly expanded knowledge regarding to the fibroin-based silk fibers from weaver ants, contributing to improvements in our understanding of the properties and characteristics of these silk fibers. For example, as reported here, carbohydrates were detected in the ants' silk for the first time presenting the fibroin as a glycoprotein. Moreover, the 3D structure provided new insights into the secondary structures considering the whole model of the protein.

Enantiodiscrimination by matrix-assisted DOSY NMR.

High-resolution NMR is an essential technique for structure determination; however, stereochemistry assignment is still an obstacle. Several methods are known to overcome this limitation but usually at high costs or using derivatizations. Here we describe the use of different solvating agents to virtually discriminate the enantiomers of 15 analytes using 1H and 19F-{1H} DOSY NMR.

Unexpected behavior of the 3 JCH coupling constant in unsaturated compounds.

The experimental and theoretical behavior of the (OC)CCH 3 JCH coupling constant is investigated for a series of α,ß-unsaturated compounds (1 to 8), and for some of them, the well-known relationship (3 JCHcis  < 3 JCHtrans ) was observed. However, for some compounds, close values for 3 JCHcis and 3 JCHtrans couplings were observed, and for aldehydes group containing compounds (7 and 8E), an inversion order is observed (3 JCHcis  > 3 JCHtrans ). In all cases where the 3 JCHcis  < 3 JCHtrans relationship it is not followed, a polar group or electronegative atom oriented in opposite direction (s-trans) to the HCC hydrogen is present, suggesting that conformational preference of such polar group or atoms are important factor on the behavior of 3 JCH couplings. Taking all of these in consideration, a new Karplus-type equation was proposed for 3 JCH couplings in α,ß-unsaturated compounds, which can be used for configurational and conformational assignment on trisubstituted double bond derivatives.

Cobalt-Catalyzed Stereoselective Synthesis of 2,5- trans-THF Nitrile Derivatives as a Platform for Diversification: Development and Mechanistic Studies.

A straightforward protocol integrating a sustainable approach for the synthesis of new 2,5- trans-THF nitrile derivatives enabling an easy diversification of its side chain scaffolds is described. The reaction tolerated different aromatic and alkyl substituents, affording the corresponding 2,5- trans-THFs in high diastereoselectivity. A detailed mechanistic study using DFT calculation reveals details of the ligand-exchange step, suggesting an inner-sphere syn attack to form the 2,5- trans stereochemistry as the most likely pathway, excluding the previous cation radical intermediate. The formation of a Co-C intermediate is suggested on the basis of the homolytic cleavage to give the previously proposed free carbon radical intermediate.

Revisiting NMR through-space J(FF) spin-spin coupling constants for getting insight into proximate F---F interactions.

At present times it is usual practice to mark biological compounds replacing an H for an F atom to study, by means of (19)F NMR spectroscopy, aspects such as binding sites and molecular folding features. This interesting methodology could nicely be improved if it is known how proximity interactions on the F atom affect its electronic structure as gauged through high-resolution (19)F NMR spectroscopy. This is the main aim of the present work and, to this end, differently substituted peri-difluoronaphthalenes are chosen as model systems. In such compounds are rationalized some interesting aspects of the diamagnetic and paramagnetic parts of the (19)F nuclear magnetic shielding tensor as well as the transmission mechanisms for the PSO and FC contributions to (4)JF1F8 indirect nuclear spin-spin coupling constants.

Structure determination of a tetrahydro-beta-carboline of arthropod origin: a novel alkaloid-toxin subclass from the web of spider Nephila clavipes.

The orb-web spiders are polyphagous animals in which the web plays a very important role in the capture of preys; oily droplets usually cover the capture-web of the spider Nephila clavipes and seem to be of great importance for prey capture. The knowledge of the chemical composition of these droplets is necessary to understand the function of this adhesive material in web mechanics and prey capture. A novel subclass of spider toxins, tetrahydro-beta-carboline, was identified among the weaponry of compounds present inside of oily droplets. This type of alkaloid is not common among the natural compounds of spider toxins. Apparently, when the prey arthropods get caught by the spider web, their bodies are covered with many adhesive oily droplets, which disrupt delivering the tetrahydro-beta-carboline to the direct contact with the prey integument. Toxicity assays demonstrated a potent lethal effect of the alkaloid toxin to the spider preys; topical applications of the tetrahydro-beta-carboline at first caused clear signs of neurotoxicity, followed by the death of preys. The structure of the major component, a tetrahydro-beta-carboline, among the alkaloid toxins was elucidated by means of UV spectrophotometry, ESI mass spectrometry, 1H-NMR spectroscopy, and high-resolution mass spectrometry. The structure of the natural toxin was determined as 1-(2-guanidinoethyl)-1,2,3,4-tetrahydro-6-hydroxymethyl)-beta-carboline; the investigation of the pharmacological properties and neurotoxic actions of this compound may be used in the future as reference for the development of new drugs to be applied at level of pest control in agriculture.

Structure determination of an organometallic 1-(diazenylaryl)ethanol: a novel toxin subclass from the web of the spider Nephila clavipes.

A novel chemical subclass of toxin, [1-(3-diazenylphenyl)ethanol]iron, was identified among the compounds present in the web of the spider Nephila clavipes. This type of compound is not common among natural products, mainly in spider-venom toxins; it was shown to be a potent paralytic and/or lethal toxin applied by the spider over its web to ensure prey capture only by topical application. The structure was elucidated by means of ESI mass spectrometry, 1H-NMR spectroscopy, high-resolution (HR) mass spectrometry, and ICP spectrometry. The structure of [1-(3-diazenylphenyl)ethanol]iron and the study of its insecticidal action may be used as a starting point for the development of new drugs for pest control in agriculture.