Molecular recognition is an essential component of all chemical and biological processes and defined as the capacity to recognize and bind complementary specific molecules in complex mixtures. It plays a primary role in many processes, some of which are linked to the onset of many processes, as well as immune and hormonal response, enzymatic catalysis, protein synthesis, duplication and translation of nucleic acids. In the past few decades, a significant goal for science and technologies has been to discover materials that can mimic the molecular recognition of natural receptors, while overcoming their limitations. An example of technology that addresses this challenge is the Molecular Imprinting Technology (MIT) which provides polymeric materials, the so-called MIPs (Molecularly Imprinted Polymers), with antibody-like recognition characteristics. This is the reason why they are also termed as “Plastic Antibodies” or “Antibody Mimics”. The first section of my work offered a general overview on MIPs and imprinting strategies. In particular, I examined the advantages and disadvantages of each imprinting method and the recent progress concerning the use of MIPs as selective receptors. Second part of the thesis describes design, development and characterization of a highly cooperative system for imprinting of Sialic Acid, a key biomarker for a diversity of cancer types, and detailed studies of the key factors involved in the recognition mechanism. The glycospecific MIPs were produced using a ter-molecular imprinting approach with cooperative covalent/non-covalent binding. Specifically, “bulk” polymers were prepared using Sialic Acid as Tetrabutylammonium salt (SA-TBA) as template (T), N-3,5-bis(trifluoromethyl)-phenyl-N’-4-vinylphenylurea (FM1), 4-vinylphenylboronic acid (FM2), 2-aminoethyl methacrylate hydrochloride (FM3) as functional monomers, ethylene glycol dimethacrylate (EGDMA) as crosslinker, methanol as porogen and N,N’-azo-bis(2,4-dimethyl)valeronitrile (ABDV) as thermal initiator. The aim is to demonstrate that potential binding sites can be produced, by the imprinting of higher-order complexes of sialic-acid which involves three cooperatively orthogonally binding functional monomers, FM1, FM2 and FM3. This outcome was supported by the design of a small MIP combinatorial library. The relative contribution and synergic interaction of these monomers was examined in more detail by HPLC and NMR experiments. This approach yielded powerful hosts for Sialic Acid recognition, resulting in increased imprinting efficiency and showed the relevance of a correct match of counterions with respect to the monomer-template interactions. Therefore, for increasing binding affinity and specificity in homogeneous solution but also in heterogeneous polymer solution systems, it has been observed that is advantageous to investigate what type of counterion encourages the interaction between the urea-based monomer and the Sialic Acid. After obtaining MIPs with high selectivity and imprinting factor, the cross-reactivity of the imprinting polymers was examined and analyzed with different saccharides targets to prove their specificity, in order to demonstrate MIPs' strong affinity for sialylated targets, highlighting the overall low binding of other non-target saccharides. The overall idea is to underline the importance of a multifactor optimization approach to synthesize moderately performing MIPs endowed with high imprinting efficiency. The latter reflect the degree cooperative interactions between the template and orthogonally interacting functional monomers and depends on the preparation conditions.
Il riconoscimento molecolare è una componente fondamentale in numerosi processi chimici e biologici, viene definito come la capacità di riconoscere e legare molecole specifiche all’interno di miscele complesse. Negli ultimi decenni, un obiettivo significativo per la scienza e le tecnologie è stato quello di progettare materiali in grado di imitare il riconoscimento molecolare dei recettori naturali, superandone i limiti. Un esempio di tecnologia che affronta questa sfida è la Molecular Imprinting Technology (MIT) che permette di produrre recettori artificiali di tipo polimerico, chiamati MIP (Molecularly Imprinted Polymers), con caratteristiche di riconoscimento simili a anticorpi, i quali sono capaci di mimare il naturale processo di riconoscimento molecolare. La sezione iniziale della mia tesi offre una panoramica generale dei MIPs e dei metodi principali di sintesi e di imprinting. Il mio progetto di tesi è focalizzato sulla progettazione, la sintesi e la caratterizzazione di un sistema altamente cooperativo per l’imprinting dell’Acido Sialico, un noto biomarker per numerosi tipi di cancro. Nello specifico, mi sono concentrata su uno studio dettagliato dei fattori chiave che sono coinvolti nel meccanismo di riconoscimento molecolare dei MIPs. I MIPs glicospecifici sono stati prodotti seguendo un approccio di imprinting cooperativo mediante la produzione di legami covalenti e non-covalenti. Il tipo di sintesi utilizzata è la “sintesi in bulk” in cui l’Acido Sialico come sale di Tetrabutilammonio è il templato e i tre monomeri utilizzati sono N-3,5-bis(trifluorometil)-fenil-N'-4-vinifenilurea (FM1), l’Acido 4-vinilifenilboronico (FM2) e 2-aminoetile metacrilato cloridrato (FM3). Inoltre, è stato usato l’etilenglicole dimetacrilato (EGDMA) come cross-linker e il 2’2-azo-bis-(2,4-dimetilvaleronitrile) (ABDV) come iniziatore termico. Un’analisi dettagliata mediante HPLC e H1-NMR ha permesso la caratterizzazione dell’interazione sinergica dei tre monomeri, con lo scopo di ottenere MIPs con prestazioni migliori. L’obiettivo è stato quello di dimostrare che l'imprinting dell’Acido Sialico con i tre monomeri funzionali legati in modo ortogonale, nello specifico FM1, FM2 e FM3 i quali sono” hosts” più potenti per il templato in questione, si traduce in una maggiore efficienza di imprinting.Questo risultato è stato dimostrato mediante la sintesi e la caratterizzazione di una piccola libreria combinatoriale di MIPs. Tale approccio ha permesso di ottenere potenti “host” per il riconoscimento dell'Acido Sialico, i quali presentano una forte interazione da parte dei controioni rispetto alle interazioni ioniche monomero-templato. Pertanto, per aumentare l'affinità e la specificità di legame del MIP in soluzione, è stato osservato che è vantaggioso verificare quale tipo di controione favorisca l'interazione tra il monomero ureidico e l'Acido Sialico. Perciò la scelta del monomero che interagirà con la funzione ureidica dell’altro monomero che legherà il templato, è di fondamentale importanza per ottenere un legame forte e stabile. Lo studio sul comportamento dei singoli monomeri funzionali che interagiscono ortogonalmente, dimostra chiaramente che è possibile produrre potenziali siti di legame sfruttando monomeri ad azione cooperativa. Inoltre, dopo aver ottenuto MIPs con elevata selettività, usando un Plate-Reader è stata indagata anche la “cross-reactivity” dei polimeri con diversi derivati saccaridici. Dagli esperimenti effettuati si può dedurre che l’idea generale del mio progetto di tesi è quella di sottolineare l’importanza di un approccio di ottimizzazione multifattoriale e ortogonale, tramite una scelta accurata dei monomeri, per sintetizzare MIPs con elevate capacità di legame verso il templato per cui sono stati sintetizzati
Sintesi e caratterizzazione di Molecularly Imprinted Polymers per il riconoscimento dell'Acido Sialico.
VERDUCI, ELENA
2019/2020
Abstract
Molecular recognition is an essential component of all chemical and biological processes and defined as the capacity to recognize and bind complementary specific molecules in complex mixtures. It plays a primary role in many processes, some of which are linked to the onset of many processes, as well as immune and hormonal response, enzymatic catalysis, protein synthesis, duplication and translation of nucleic acids. In the past few decades, a significant goal for science and technologies has been to discover materials that can mimic the molecular recognition of natural receptors, while overcoming their limitations. An example of technology that addresses this challenge is the Molecular Imprinting Technology (MIT) which provides polymeric materials, the so-called MIPs (Molecularly Imprinted Polymers), with antibody-like recognition characteristics. This is the reason why they are also termed as “Plastic Antibodies” or “Antibody Mimics”. The first section of my work offered a general overview on MIPs and imprinting strategies. In particular, I examined the advantages and disadvantages of each imprinting method and the recent progress concerning the use of MIPs as selective receptors. Second part of the thesis describes design, development and characterization of a highly cooperative system for imprinting of Sialic Acid, a key biomarker for a diversity of cancer types, and detailed studies of the key factors involved in the recognition mechanism. The glycospecific MIPs were produced using a ter-molecular imprinting approach with cooperative covalent/non-covalent binding. Specifically, “bulk” polymers were prepared using Sialic Acid as Tetrabutylammonium salt (SA-TBA) as template (T), N-3,5-bis(trifluoromethyl)-phenyl-N’-4-vinylphenylurea (FM1), 4-vinylphenylboronic acid (FM2), 2-aminoethyl methacrylate hydrochloride (FM3) as functional monomers, ethylene glycol dimethacrylate (EGDMA) as crosslinker, methanol as porogen and N,N’-azo-bis(2,4-dimethyl)valeronitrile (ABDV) as thermal initiator. The aim is to demonstrate that potential binding sites can be produced, by the imprinting of higher-order complexes of sialic-acid which involves three cooperatively orthogonally binding functional monomers, FM1, FM2 and FM3. This outcome was supported by the design of a small MIP combinatorial library. The relative contribution and synergic interaction of these monomers was examined in more detail by HPLC and NMR experiments. This approach yielded powerful hosts for Sialic Acid recognition, resulting in increased imprinting efficiency and showed the relevance of a correct match of counterions with respect to the monomer-template interactions. Therefore, for increasing binding affinity and specificity in homogeneous solution but also in heterogeneous polymer solution systems, it has been observed that is advantageous to investigate what type of counterion encourages the interaction between the urea-based monomer and the Sialic Acid. After obtaining MIPs with high selectivity and imprinting factor, the cross-reactivity of the imprinting polymers was examined and analyzed with different saccharides targets to prove their specificity, in order to demonstrate MIPs' strong affinity for sialylated targets, highlighting the overall low binding of other non-target saccharides. The overall idea is to underline the importance of a multifactor optimization approach to synthesize moderately performing MIPs endowed with high imprinting efficiency. The latter reflect the degree cooperative interactions between the template and orthogonally interacting functional monomers and depends on the preparation conditions.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/20.500.14239/13593