The main objective of this review is to provide a comprehensive survey of methods used for stereoselective construction of carbon-nitrogen bonds during the total synthesis of nitrogen-containing natural products that have appeared in the literature since 2000. The material is organized by specific reaction in order of decreasing number of applications in natural product synthesis. About 800 total syntheses of natural products with stereogenic carbon-nitrogen bonds described since 2000 have been reviewed.

Targeted metabolomics aims to provide a new approach to investigate metabolites and gather both qualitative and quantitative information. We describe a protocol for extraction and analysis of plant metabolites, specifically 13 secondary metabolites (antioxidants) using liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS), with high linearity (R2 > 0.99) and reproducibility (0.23-6.23 R%) with low limits of detection (>0.001 ng/mL) and quantification (>0.2 ng/mL). The protocol was applied to study the antioxidant response of cucumber plants exposed to nanocopper pesticide. Dose-dependent changes in antioxidant concentrations were found, and 10 antioxidants were significantly consumed to scavenge reactive oxygen species, protecting plants from damage. Levels of three antioxidants were up-regulated, as a response to the depletion of the other antioxidants, signaling activation of the defense system. We demonstrated that the reported LC-MS/MS method provides a quantitative analysis of antioxidants in plant tissues, for example to investigate interactions between plants and nanomaterials.

Mechanistic understanding of the interaction of copper-based nanomaterials with crops is crucial for exploring their application in precision agriculture and their implications on plant health. We investigated the biological response of soybean (Glycine max) plants to the foliar application of copper hydroxide nanowires (CNWs) at realistic exposure concentrations. A commercial copper based-fungicide (Kocide), dissolved copper ions, and untreated controls were used for comparison to identify unique features at physiological, cellular, and molecular levels. After 32 d of exposure to CNW (0.36, 1.8, and 9 mg CNW/plant), the newly developed tissues accumulated significantly high levels of Cu (18−60 μg/g) compared to Kocide (10 μg/g); however, the rate of Cu translocation from the site of CNW treatment to other tissues was slower compared to other Cu treatments. Like Kocide, CNW exposure at medium and high doses altered Co, Mn, Zn, and Fe accumulation in the tissues and enhanced photosynthetic activities. The proteomic and metabolomic analyses of leaves from CNW-treated soybean plants suggest a dose-dependent response, resulting in the activation of major biological processes, including photosynthesis, energy production, fatty acid metabolism, lignin biosynthesis, and carbohydrate metabolism. In contrast to CNW treatments, Kocide exposure resulted in increased oxidative stress response and amino acid metabolism activation.

The increasing usage of nanopesticides in agriculture poses a concern to plant crops due to unknown implications of engineered nanomaterials (ENMs). Targeted metabolomics can provide both qualitative and quantitative information at a molecular level to investigate the response of plants to emerging environmental stressors, such as nanopesticides. Here we describe a detailed protocol for the extraction and analysis of plant metabolites, specifically 23 amino acids in plants, using hydrophilic interaction liquid chromatography coupled to triple quadrupole mass spectrometry (HILIC-LC-MS/MS). Sufficient separation of 23 amino acids was achieved, without the need for derivatization, on an HILIC column with an MS/MS detector in a single run of 12 min with high sensitivity, selectivity and robustness and low LOD (0.005-15 ng/mL) and LOQ (0.02-50 ng/mL). A simple and efficient method to effectively extract amino acids from plant tissues was developed with a high recovery rate (80-120%). The protocol was then applied to determine the levels of amino acids in cucumber plants exposed to various environmentally-relevant levels of nano copper (nCu at 0, 200, 400, and 800 mg/kg soil; harvested in 60 days). Dose-dependent changes in amino acid levels were found; 13 amino acids were upregulated due to nCu stress, particularly, tyrosine increased 6.1, 8.2, and 11.0 fold after exposure to 200, 400 and 800 mg/kg nCu, respectively. The change in amino acid levels suggests an active defense response of the cucumber plant to nCu stress. We demonstrate that the HILIC-LC-MS/MS method is an effective and efficient technique to analyze underivatized amino acids in plant samples.

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