Università degli Studi di Milano, 2017-03-28

A purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) was successfully exploited to catalyze the “one-pot, one-enzyme” regio- and stereoselective transfer of ?-D-ribose from a proper sugar donor (7-methylguanosine iodide) to a library of 6-substituted purine acceptors, resulting in the “in batch” synthesis of 24 ribonucleosides. Transglycosylation conversions confirmed the broad tolerance and the potential of AhPNP as biocatalyst, providing the necessary information to undertake the preparative synthesis of 6-modified purine nucleosides. [1] AhPNP was then immobilized in a stainless steel column resulting in a stable and active bioreactor (AhPNP-IMER, Immobilized Enzyme Reactor) that, upon on-line connection to a semi-preparative HPLC system, was used to run transglycosylations in a flow mode. In such a set-up, biotransformation, on-line monitoring and product purification occurred in a single integrated platform, thus allowing the preparation of five nucleoside analogues at a mg scale (52-89% yield). [2] As a step forward, a “one-pot, two-enzyme” strategy was applied by coupling AhPNP-IMER with an analogous bioreactor based on a uridine phosphorylase from Clostridium perfringens (CpUP), immobilized in a monolith column. The on-line apparatus obtained by connecting CpUP-IMER and AhPNP-IMER in series was tested in the synthesis of adenosine, 2’-deoxyadenosine and arabinosyladenine from uridine, 2’-deoxyuridine and arabinosyluracyl as sugar donors, respectively. The corresponding nucleobases were transformed into the products in 90-95% conversion over 1 h for the ribosyl and 2’-deoxyribosyl derivatives, and 20% conversion after 5 h for arabinosyladenine. [3] Furthermore, a new LC-ESI-MS/MS method was set up to evaluate the inhibition activity of 8-substituted purine ribonucleosides toward the PNP from Mycobacterium tuberculosis (MtPNP), as well as the selectivity against the microbial enzyme with respect to the corresponding human one (HsPNP). The corresponding enzymatic assay, based on the phosphorolysis of inosine, proved to be very convenient in terms of time as well as of target amount. A small library of seven 8-substituted purine ribonucleosides were screened, not exerting any significant effect up to 1 mM, with 8-bromoguanosine and 8-methylaminoguanosine being the only exceptions at 500 mM as weak inhibitors. [4] Finally, the chemical synthesis of a series of 8- and N2-substituted inosinic and guanylic acids as potential ligands of the human GPR17 receptor was carried out, starting from studies aided by molecular modeling on a homology model of the target. The molecules were prepared by 5’-phosphorylation of properly 8- and N2-modified/protected inosine or guanosine. Owing to the scarce nucleophilicity of the exocyclic NH2 group of guanosine, the 2-position of the purine ring was activated as a bromo derivative, whose displacement with the proper amine afforded the desired N2-alkylated products. On the contrary, N2-acylations were carried out through nitrogen functionalization with a proper acyl chloride or anhydride. An additional 2’,3’-O-isopropylidene group was inserted in all the N2-functionalized nucleotides. Binding assays on GPR17 will be carried out. [1] D. Ubiali, C. F. Morelli, M. Rabuffetti, G. Cattaneo, I. Serra, T. Bavaro, A. M. Albertini, G. Speranza Curr. Org. Chem. 2015, 19, 2220-2225; [2] E. Calleri, G. Cattaneo, M. Rabuffetti, I. Serra, T. Bavaro, G. Massolini, G. Speranza, D. Ubiali Adv. Synth. Catal. 2015, 357, 2520-2528; [3] G. Cattaneo, M. Rabuffetti, G. Speranza, T. Kupfer, B. Peters, G. Massolini, D. Ubiali, E. Calleri Submitted 2017; [4] G. Cattaneo, D. Ubiali, E. Calleri, M. Rabuffetti, G. C. Hofner, K. T. Wanner, M. C. De Moraes, L. K. B Martinelli, D. S. Santos, G. Speranza Anal. Chim. Acta 2016, 943, 89-97.

diritti: info:eu-repo/semantics/openAccess
In relazione con info:eu-repo/semantics/altIdentifier/hdl/2434/481437
tutor: G. Speranza; co-tutor: G. Massolini; coordinatore: M. Pizzotti
Settore CHIM/06 - - Chimica Organica

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD17002720