Precursor-Directed Biosynthesis and Fluorescence Labeling of Clickable Microcystins

Abstract

Microcystins, cyclic nonribosomal heptapeptides, are the most well-known cyanobacterial toxins. They are exceptionally well studied, but open questions remain concerning their physiological role for the producing microorganism or their suitability as lead compounds for anticancer drug development. One means to study specialized metabolites in more detail is the introduction of functional groups that make a compound amenable for bioorthogonal, so-called click reactions. Although it was reported that microcystins cannot be derivatized by precursordirected biosynthesis, we successfully used this approach to prepare clickable microcystins. Supplementing different azide-or terminal alkyne containing amino acid analogues into the cultivation medium of microcystin-producing cyanobacteria strains, we found that these strains differ strongly in their substrate acceptance. Exploiting this flexibility, we generated more than 40 different clickable microcystins. We conjugated one of these derivatives with a fluorogenic dye and showed that neither incorporation of the unnatural amino acid analogue nor attachment of the fluorescent label significantly affects the cytotoxicity against cell lines expressing the human organic anion transporting polypeptides 1B1 or 1B3. Using time-lapse microscopy, we observed that the fluorescent microcystin is rapidly taken up into eukaryotic cells expressing these transporters.

High Plasticity of the Amicetin Biosynthetic Pathway in Streptomyces sp. SHP 22-7 Led to the Discovery of Streptcytosine P and Cytosaminomycins F and G and Facilitated the Production of 12F-Plicacetin

“…We hypothesized that we could exploit the extensive substrate flexibility of AmiF and AmiR to generate optimized or clickable new-to-nature analogues by feeding either fatty acids or benzoic acid analogues within the framework of a precursor-directed feeding approach . In order to determine an optimal feeding time point, a growth curve was established and the production rate was monitored.…”

Recent Advances in Bioorthogonal Click Chemistry for Enhanced PET and SPECT Radiochemistry

“…Since Sharpless and co-workers first proposed the concept of the click reaction in 2003, bioorthogonal click chemistry has been extensively developed in the communities of chemical biology and nuclear medicine. , Different from traditional genetic-based approaches, the bioorthogonal chemistry-triggered strategy not only benefits from fast reaction kinetics, high selectivity, and favorable yields but also is compatible with biological systems and complex physiological environments. Therefore, bioorthogonal chemistry can tackle the challenges of post-translational events and underlying mechanisms by the manipulation and modification of biomolecules in biological processes. , In light of these facts, numerous clickable handle-modified biomolecules have been leveraged for in vivo biological imaging and radiolabeling techniques of diverse targets (e.g., receptors, − enzymes, − transporters, , glycoproteins, , and nucleic acids − ), biomaterial science, , targeting therapy, , drug development, − and nanotechnology − over the past two decades.…”

“…Therefore, bioorthogonal chemistry can tackle the challenges of post-translational events and underlying mechanisms by the manipulation and modification of biomolecules in biological processes. 3,4 In light of these facts, numerous clickable handlemodified biomolecules have been leveraged for in vivo biological imaging and radiolabeling techniques of diverse targets 5 (e.g., receptors, 6−8 enzymes, 9−12 transporters, 13,14 glycoproteins, 15,16 and nucleic acids 17−19 ), biomaterial science, 20,21 targeting therapy, 22,23 drug development, 24−26 and nanotechnology 27−35 over the past two decades.…”

Biosynthetic diversification of non-ribosomal peptides through activity-based protein profiling of adenylation domains

“…PDB and mutasynthesis have frequently been applied to the diversification of natural products, including NRPs. [17][18][19][20] The A-domains of NRPSs frequently display the ability to catalyze adenylation toward structurally related amino acids. 21 A better understanding of the substrate tolerance of the A-domains of NRPSs in advance using the ABPP-NRPS strategy can facilitate efficient modification of NRPs by the PDB and mutasynthesis strategies.…”