Isolamento e purificazione di prodotti marini naturali e in vitro. Patellamidi: metaboliti secondari di Lissoclinum patella e del suo simbionte.

Lissoclinum patella is a tunicate endemic into the tropical Indo-Pacific ocean, which arbors in the cloacal cavities the cyanobacterial symbiont Prochloron sp.. This bacterium produces, among the others, interesting cyclic peptides as secondary-metabolites. Patellamides, a specific group of azole-based cyclic peptides found in Lissoclinum patella extract, is the main focus of our research. Naturally, these molecules are biosynthesized in a ribosome-dependent manner and are subsequently modified via post-translational mechanisms. Our group try to produce these azole-based octapeptides using engineered enzymes from patellamide pathway, reacting with a pre-propeptide and leading to the production of the final cyclic peptide. The main advantage of this method is that, mutating the encoding sequence for the pre-propeptide, it's possible to obtain a large variety of different cyclic peptides. The experimental results obtained through purification, isolation and structure elucidation of several in vitro products confirm the possibility to produce cyclic octapeptides using this technique. However, all the cyclic peptides produced contain thiazoline rings, while in natural patellamides it's observed the oxidation of thiazoline to thiazole rings. Furthermore, in order to obtain consistent results from the biological assays, it's fundamental to have a valid method to estimate the quantity of each in vitro product. During my internship, we considered and investigated several approaches to quantitation, including HPLC-UV, HPLC-UV-MS, HPLC-MS. All these methods have their own strengths and weaknesses, but none of them appear to be precise and accurate enough for our purpose. The best option that we currently find is based on HPLC-ICP-MS method, which will be developed in the next future.