Nucleotides are the building blocks of DNA and RNA and consist of a nitrogenous base, a sugar moiety, and from one to three phosphate groups. They are involved in several cellular processes such as DNA and RNA synthesis, cellular signaling, enzyme regulation, and metabolism. Given their involvement in many biological processes, over the years the interest in modified nucleoside analogues has grown considerably. Modified nucleosides and nucleotides are able to mimic their endogenous counterparts in order to exploit cellular metabolism, and thus serve as substrates for a variety of nucleotide-dependent biological processes, for instance undergoing incorporation into DNA and RNA leading to termination of chain elongation, accumulation of mutations, or induction of apoptosis. Modified nucleosides are prodrugs that enter the cell through nucleosides transporters (NTs) and need to be converted into their 5’-triphosphate forms to be biologically active. Since the first phosphorylation step is often inefficient for non-natural analogues, different strategies have been applied to bypass this rate-limiting step. A common approach consists in the synthesis of nucleoside phosphonates, which are mimics of nucleoside monophosphates characterized by a chemically and enzymatically stable P-C-O bond that can be activated by kinases to their mono- and diphosphate forms. In this Thesis project, the attention has been focused on a class of nucleoside analogues: namely 4’-thionucleosides, in which a sulfur atom replaces the furanose endocyclic oxygen of native nucleosides, resulting in a thiohemiaminal bond (S-C-N) that connects the sugar moiety and the base, instead of the canonical hemiaminal bond (O-C-N). This bond has a greater stability towards chemical and enzymatic hydrolysis. Thionucleosides are interesting molecules owing to their antiviral, antitumor, and antibiotic activity. Furthermore, they are of particular interest to generate 4’-thionucleic acids, which are biologically equivalent to natural biopolymers and were proven to undergo a sequence of replication, transcription, and translation both in vitro and in artificial cells. So far, the phosphonate concept has not been applied to 4’-thionucleosides, therefore in this Thesis a synthetic route has been developed to obtain 4’-thionucleoside phosphonates. The target molecule selected for the synthesis is β-L-4’-thionucleoside phosphonate with uracil as nucleobase. First, a thio glycosyl donor was prepared in six steps from commercially available D-mannose. Then, the thiosugar underwent a Lewis-acid catalysed glycosylation reaction with diisopropyl hydroxymethylphosphonate to afford the phosphonate sugar intermediate that afterwards was oxidized to its sulfoxide derivative. Subsequentially, the sulfoxide underwent a reaction of deprotection followed by a selective acetylation. Lastly, in the near future we will attempt the installation of the uracil on the sulfoxide by a Pummerer reaction, that due to time constraints falls beyond the scope of the Thesis. All intermediates of the synthesis were characterized by using analytical techniques such as NMR spectroscopy and mass spectrometry (MS). To conclude, although the yield of some reaction was low, the development of this pathway can provide information for the preparation of 4’-thionucleoside phosphonates, which to date, has not yet been described in the literature.
I nucleotidi sono subunità del DNA e dell’RNA e sono costituiti da una base azotata, uno zucchero e da uno a tre gruppi fosfato. I nucleotidi sono coinvolti in molti processi cellulari come la sintesi del DNA e RNA, la segnalazione cellulare, la regolazione enzimatica e il metabolismo. Dato il loro coinvolgimento in molti processi biologici, negli anni l’interesse verso analoghi nucleosidici modificati è cresciuto notevolmente. I nucleosidi e nucleotidi modificati sono in grado di mimare quelli endogeni, sfruttando il metabolismo cellulare e fungendo da substrati per molti processi nucleotide-dipendenti. Possono, per esempio, essere incorporati nel DNA e RNA portando alla terminazione dell’allungamento della catena, all’accumulo di mutazioni o all’induzione dell’apoptosi. I nucleosidi modificati sono profarmaci che entrano nella cellula attraverso specifici trasportatori chiamati NTs e che devono essere convertiti nella loro forma 5’-trifosfato per essere biologicamente attivi. Poiché spesso i nucleosidi non naturali non subiscono la prima fosforilazione, diverse strategie sono state applicate per superare questo step limitante. Un approccio comune consiste nella sintesi di nucleosidi fosfonati, che mimano i nucleosidi monofosfato e che sono caratterizzati da un legame chimicamente e enzimaticamente stabile P-O-C, che può essere attivato dalle chinasi nella forma mono- e difosfato. In questo progetto di Tesi, l’attenzione si è focalizzata su una classe di analoghi nucleosidici: i 4’-tionucleosidi. Questi sono nucleosidi modificati nei quali un atomo di zolfo sostituisce l’ossigeno endociclico, dando origine a un legame tioemiaminale (S-C-N) che connette lo zucchero alla base, a differenza del canonico legame emiaminale (O-C-N). Il legame tioemiaminale tra la base e lo zucchero è più stabile e resistente agli enzimi degradativi, rispetto al legame emiaminale. I tionucleosidi sono interessanti molecole con attività antivirale, antitumorale e antibiotica. In aggiunta a questo, sono di particolare interesse per lo sviluppo di oligonucleotidi (ONs) che portano alla generazione di acidi 4’-tionucleici che sono biologicamente equivalenti ai biopolimeri naturali e possono essere impiegati nei processi di replicazione, trascrizione e trasduzione. Fino ad oggi il concetto di fosfonato non è ancora stato applicato ai 4’-tionucleosidi. In questo progetto di Tesi è stata sviluppata una via sintetica per l’ottenimento di un 4’-tionucleoside fosfonato. La molecola target selezionata per la sintesi è il β-L-4’-tionucleoside fosfonato con l’uracile come base azotata. Innanzitutto, è stato preparato il donatore tioglicosidico in sei step a partire dal D-mannosio disponibile in commercio. Successivamente, lo zucchero contenente lo zolfo è stato sottoposto a una reazione di glicosilazione catalizzata da diisopropil idrossimetilfosfonato per ottenere l’intermedio che presenta lo zucchero fosfonato. Questo è stato successivamente sottoposto ad una reazione di ossidazione che ha consentito l’ottenimento del corrispondente sulfossido. Infine, il sulfossido è stato selettivamente deprotetto e acetilato. Per mancanza di tempo non è stata effettuata la coniugazione dell’uracile con il sulfossido mediante la reazione di Pummerer. Per concludere, anche se la resa di alcune reazioni risulta bassa, lo sviluppo di questo pathway sintetico può fornire informazioni per la preparazione di 4’-thionucleosidi fosfonati, che ad oggi non è ancora stata descritta in letteratura.
Verso la sintesi di 4’-tio nucleosidi fosfonati
LO PRESTI, MARIANNA
2021/2022
Abstract
Nucleotides are the building blocks of DNA and RNA and consist of a nitrogenous base, a sugar moiety, and from one to three phosphate groups. They are involved in several cellular processes such as DNA and RNA synthesis, cellular signaling, enzyme regulation, and metabolism. Given their involvement in many biological processes, over the years the interest in modified nucleoside analogues has grown considerably. Modified nucleosides and nucleotides are able to mimic their endogenous counterparts in order to exploit cellular metabolism, and thus serve as substrates for a variety of nucleotide-dependent biological processes, for instance undergoing incorporation into DNA and RNA leading to termination of chain elongation, accumulation of mutations, or induction of apoptosis. Modified nucleosides are prodrugs that enter the cell through nucleosides transporters (NTs) and need to be converted into their 5’-triphosphate forms to be biologically active. Since the first phosphorylation step is often inefficient for non-natural analogues, different strategies have been applied to bypass this rate-limiting step. A common approach consists in the synthesis of nucleoside phosphonates, which are mimics of nucleoside monophosphates characterized by a chemically and enzymatically stable P-C-O bond that can be activated by kinases to their mono- and diphosphate forms. In this Thesis project, the attention has been focused on a class of nucleoside analogues: namely 4’-thionucleosides, in which a sulfur atom replaces the furanose endocyclic oxygen of native nucleosides, resulting in a thiohemiaminal bond (S-C-N) that connects the sugar moiety and the base, instead of the canonical hemiaminal bond (O-C-N). This bond has a greater stability towards chemical and enzymatic hydrolysis. Thionucleosides are interesting molecules owing to their antiviral, antitumor, and antibiotic activity. Furthermore, they are of particular interest to generate 4’-thionucleic acids, which are biologically equivalent to natural biopolymers and were proven to undergo a sequence of replication, transcription, and translation both in vitro and in artificial cells. So far, the phosphonate concept has not been applied to 4’-thionucleosides, therefore in this Thesis a synthetic route has been developed to obtain 4’-thionucleoside phosphonates. The target molecule selected for the synthesis is β-L-4’-thionucleoside phosphonate with uracil as nucleobase. First, a thio glycosyl donor was prepared in six steps from commercially available D-mannose. Then, the thiosugar underwent a Lewis-acid catalysed glycosylation reaction with diisopropyl hydroxymethylphosphonate to afford the phosphonate sugar intermediate that afterwards was oxidized to its sulfoxide derivative. Subsequentially, the sulfoxide underwent a reaction of deprotection followed by a selective acetylation. Lastly, in the near future we will attempt the installation of the uracil on the sulfoxide by a Pummerer reaction, that due to time constraints falls beyond the scope of the Thesis. All intermediates of the synthesis were characterized by using analytical techniques such as NMR spectroscopy and mass spectrometry (MS). To conclude, although the yield of some reaction was low, the development of this pathway can provide information for the preparation of 4’-thionucleoside phosphonates, which to date, has not yet been described in the literature.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15679