RESUMO
Many peptidic natural products, such as lasso peptides, cyclic peptides, and cyclotides, are conformationally constrained and show biological stability, making them attractive scaffolds for drug development. Although many peptides can be synthesized and modified through chemical methods, knot-like lasso peptides such as microcin J25 (MccJ25) and their analogues remain elusive. As the chemical space of MccJ25 analogues accessible through purely biological methods is also limited, we proposed a hybrid approach: flow-based chemical synthesis of non-natural precursor peptides, followed by in vitro transformation with recombinant maturation enzymes, to yield a more diverse array of lasso peptides. Herein, we established the rapid, flow-based synthesis of chemically modified MccJ25 precursor peptides (57 amino acids). Heterologous expression of enzymes McjB and McjC was extensively optimized to improve yields and facilitate the synthesis of multiple analogues of MccJ25, including the incorporation of non-canonical tyrosine and histidine derivatives into the lasso scaffold. Finally, using our chemoenzymatic strategy, we produced a biologically active analogue containing three d-amino acids in the loop region and incorporated backbone N-methylations. Our method provides rapid access to chemically modified lasso peptides that could be used to investigate structure-activity relationships, epitope grafting, and the improvement of therapeutic properties.
Assuntos
Peptídeos , Peptídeos/química , Peptídeos/síntese química , BacteriocinasRESUMO
Push-pull technology (PPT) employs mixed cropping for sustainable intensification: an intercrop repels or suppresses pests of the focal crop (push), while a trap crop attracts pests out of the field (pull), where they may be targeted for control. Underlying chemical-ecological mechanisms have been demonstrated in controlled settings, primarily for some of the best-established cereal PPT systems developed in east Africa. Yet, many questions remain regarding mechanisms, and strategies to adapt PPT for different crops and locations. We conducted a systematic review of scientific literature on PPT and related practices for biological control of pests of food and fodder. Of 3335 results, we identified 45 reporting on chemistry of trap- or intercropping systems for pest control, of which 30 focused on cereals or African pests. Seven of these reported primary chemical data: measurements from glasshouse and laboratory studies (5), or of field-collected samples (2). From these 30, we provide a database of compounds, discussing degrees of evidence for their mediation of push-pull. We depict hypothesized spatial distributions of selected compounds in PPT fields from physical properties and emission/exudation rates, and design of the east African cereal PPT system, and discuss influences on activity in field settings likely to affect success.
RESUMO
Mass spectrometry-based plant metabolomics is frequently used to identify novel natural products or study the effect of specific treatments on a plant's metabolism. Reliable sample handling is required to avoid artifacts, which is why most protocols mandate shock freezing of plant tissue in liquid nitrogen and an uninterrupted cooling chain. However, the logistical challenges of this approach make it infeasible for many ecological studies. Especially for research in the tropics, permanent cooling poses a challenge, which is why many of those studies use dried leaf tissue instead. We screened a total of 10 extraction and storage approaches for plant metabolites extracted from maize leaf tissue across two cropping seasons to develop a methodology for agroecological studies in logistically challenging tropical locations. All methods were evaluated based on changes in the metabolite profile across a 2-month storage period at different temperatures with the goal of reproducing the metabolite profile of the living plant as closely as possible. We show that our newly developed on-site liquid-liquid extraction protocol provides a good compromise between sample replicability, extraction efficiency, material logistics, and metabolite profile stability. We further discuss alternative methods which showed promising results and feasibility of on-site sample handling for field studies.