Caratterizzazione del primo biarilitide ulteriormente modificata YVH+OH

Natural products are a large and relevant research topic, which impress not only for their activity, but also for their chemical diversity. In fact novel compounds can have different mechanism of action and interact with novel targets, which makes their study important especially for the medical field. Biarylitides are a newly discovered class of RiPPs (2021). They feature a precursor peptide made of 5 amino acids, where two amino acids are the leader peptide and three are the core peptide. This pentapeptide is coded by 18-bp byTA, to our knowledge the smallest coding gene ever reported, consisting of only 18 base pairs. Closely linked to bytA is bytO, encoding a cytochrome P450 monooxygenase likely responsible for the formation of a cycle consisting of three amino acids with a biaryl bridge. The biaryl bridge is closed between the C5 of histidine and the C6 of tyrosine. Also possible is a biaryl bridge between a nitrogen of the histidine ring and C6 of tyrosine .Amino acids with aromatic side chains at the first and third positions are necessary for the biaryl installment. The middle amino acid can be variable, since it is not involved in the biaryl bridging. Due to the aromatic rings with delocalized electrons, biarylitides have a chromophore with typical absorption maxium at 270 nm and 315 nm. Global bioinformatic analyses showed that gene clusters for biarylitides and their modification are very widespread, at least in actinobacteria , although the role in the metabolism of biarylitides is still unknown. In this study, our main focus is on a specific actinomycete called Saccarothrix variisporea, which have in his gene cluster, the genes for the precursor peptide(MRYVH), for the BytO enzyme and the Beta- hydroxylase, which has shown to be the possible cause of the presence of a precent of YVH peptide hydroxylated. The YVH biarylitide produced by Saccharothrix variisporea 43911 was successfully isolated and detected by LC/MS at 458.2 m/z and HPLC. Its structure is made tyrosine, valine and histidine, where the biaryl bridge is closed between the C5 of histidine and the C6 of tyrosine. A further modification of the structure of the YVH biarylitide by a hydroxy group (-OH) was detected by LC/MS at 474.2 m/z. The plasmid pSET_43911_BH_AT conjugated in Streptomyces coelicolor M1152 synthesized the unmodified biarylitide in large quantities than the hydroxylated one. An optimization of the production is needed to increase the amount of YVH+OH produced and reach the required quantity for the NMR to elucidate its structure and detect the position of the hydroxy group. The wild type Saccharothrix variisporea 43911 was cultivated using different media. Crude samples were taken every two days from the same flask. After two weeks, the cultivation was extracted using methanol and resin HP-20. When the extracts were analyzed by LC/MS to detect the presence of biarylitides at 458,2 m/z and 474,2 m/z, and flash chromatography and HPLC was performed to further detect our compounds and isolate the purified form.