C-Type lectin receptors are calcium dependent proteins able to selectively recognize and bind to carbohydrates exposed by invading pathogens and damaged cells, thus acting as important first-line host defence. However, C-type lectins activity can be also responsible for the pathogenesis of infections and inflammatory diseases, thus representing a class of promising therapeutic targets. Protein-carbohydrate interactions are generally weak interactions, with dissociation constants lying in the millimolar range. This problem has been solved by Nature exploiting multivalency: C-type lectins are usually oligomeric receptors interacting with polyglycosylated surfaces, thus concomitant multiple interactions lead to high affinity binding. This thesis work has been devoted to the synthesis and identification of potent C-type lectin antagonists. In particular, our strategy was based on the combination of glycomimetic compounds as active and enzymatically stable ligands and on their multivalent presentation. The first part of the thesis work was focused on the synthesis of stabilised versions of previously developed antagonists for the Mannose Binding Lectin (MBL), a C-type lectin involved in the reperfusion damage after acute ischemic stroke. Tetravalent glycodendrons were synthesized following an efficient modular approach, which allowed functionalization at the dendron focal point and ready access to higher valency compounds, as demonstrated with the synthesis of a 16-valent glycodendrimer. All the compounds were fully characterized and showed complete water solubility and remarkable stability in water solution and under chromatographic purification (both direct and reverse phase). The binding affinity of the glycodendons and glycodendrimer was assessed towards the murine isoform MBL-C by an SPR competition assay, confirming high binding affinity and valency-dependent avidity. A second part of the work was addressed to the synthesis of inhibitors for the Dendritic Cell-Specific ICAM-Grabbing Non-Integrin (DC-SIGN), a C-type lectin exploited by several pathogens to initiate infections. In the development of new DC-SIGN antagonists, a rational design approach was adopted. Driven by the high affinity reached by divalent and hexavalent DC-SIGN chelating agents, a cross-shaped 12-valent glycodendrimer able to perform as a tetradentate ligand and simultaneously bind to the four DC-SIGN binding sites was designed and successfully synthesized and characterized. The affinity towards DC-SIGN is still under study. Finally, the development of a methodology aimed to identify potent C-type lectin glycomimetic ligands was undertaken in collaboration with the group of Dr. Niels C. Reichardt at CIC biomaGUNE (San Sebastian, Spain). A small library of glycomimetics was synthesized exploiting glycan microarray technology. In particular, the use of recently described MALDI-TOF MS detectable microarrays allowed us the on-chip synthesis of unnatural glycan through enzymatic glycosylation with clickable modified sugar donors followed by Cu(I) catalyzed Azido Alkyne Cycloaddition (CuAAC). In the attempt of applying this strategy to the generation of LewisX trisaccharide mimics, the synthesis of a clickable Fucose-GDP sugar donor was accomplished. This derivative proved to be readily accepted by α-1,3-fucosyltransferase from H. pylori and the synthesis of LewisX mimics is currently on going at CIC biomaGUNE.
Le lectine di tipo C sono recettori calcio dipendenti in grado di riconoscere in modo selettivo e di legarsi a carboidrati esposti da patogeni e cellule danneggiate, agendo quindi come un’importante meccanismo di prima difesa. Tuttavia, lectine di tipo C possono essere responsabili della patogenesi di infezioni e malattie infiammatorie, costituendo così un promettente bersaglio terapeutico. Le interazioni proteina-carboidrato sono generalmente deboli, con costanti di dissociazione nell’ordine del millimolare. Tale problema di bassa ffinità è stato risolto in Natura ricorrendo alla multivalenza: lectine di tipo C sono solitamente recettori oligomerici che interagiscono con superfici poliglicosilate, di conseguenza concomitanti interazioni multiple determinano un legame ad alta affinità. Questo lavoro di tesi è stato dedicato alla sintesi e identificazione di forti antagonisti di lectine di tipo C. In particolare, la strategia adottata si è basta sulla combinazione di composti glicomimetici come ligandi attivi e resistenti all’azione di enzimi e sulla loro presentazione multivalente. Una prima parte del seguente lavoro è stato incentrato sulla stabilizzazione di antagonisti precedentemente sviluppati contro la Mannose Binding Lectin (MBL) , una lectina di tipo C implicata nel danno da riperfusione in seguito ad ictus emorragico. Glicodendroni tetravalenti sono stati sintetizzati tramite un approccio efficiente e modulare. La strategia adottata ha permesso la funzionalizzazione a livello del punto focale del dendrone consentendo un rapido accesso a composti a valenza superiore, come dimostrato dalla sintesi di un glicodendrimero 16-valente. È stato possibile effettuare una caratterizzazione di tutti i dendroni e dendrimeri, che si sono rivelati solubili in acqua ed estremamente stabili sia in soluzione acquosa, sia durante purificazione tramite cromatografia (sia fase diretta che fase inversa). L’affinità dei composti è stata verificata nei confronti dell’isoforma murina MBL-C tramite un saggio SPR competitivo, che ha confermato una alta capacità di legare la proteina fortemente dipendente dalla valenza dei ligandi. Un’ulteriore parte del lavoro è stato volto alla sintesi di inibitori della Dendritic Cell-Specific ICAM-Grabbing Non-Integrin (DC-SIGN), una lectina di tipo C sfruttata da vari agenti patogeni per dare inizio ad infezione. Nuovi antagonisti di DC-SIGN sono stati sviluppati tramite una progettazione razionale. Vista l’alta affinità raggiunta da agenti chelanti divalenti ed esavalenti, è stato progettato e sintetizzato un glicodendrimero dodecavalente a forma di croce capace di legare simultaneamente i quattro siti di riconoscimento di DC-SIGN. Studi per valutarne l’affinità nei confronti di DC-SIGN sono attualmente in corso. Infine, in collaborazione col gruppo del Dr. Niels C. Reichardt presso CIC biomaGUNE (San Sebastian, Spain) è stato intrapreso lo sviluppo di una nuova metodologia volta all’identificazione di nuovi ligandi glicomimetici per lectine di tipo C. Una piccola libreria di composti glicomimetici è stata sintetizzata sfruttando la tecnologia dei microarray di glicani. In particolare, l’utilizzo di microarray analizzabili via MALDI-TOF MS ha permesso la sintesi on-chip di glicani non naturali tramite glicosilazione enzimatica con donatori funzionalizzabili tramite Cu(I) catalyzed Azido Alkyne Cycloaddition (CuAAC). Al fine di generare una libreria di mimetici del trisaccaride LewisX tramite tale metodologia, è stata eseguita la sintesi di un Fucosio-GDP derivato contenente un gruppo azidico. Come da noi dimostrato, questo composto viene accettato come donatore di legame glicosidico in modo eccellente dall’enzima α-1,3-fucosiltransferasi da H. pylori e la sintesi di mimetici di LewisX è tuttora in corso presso il centro di ricerca CIC biomaGUNE.
GLYCOMIMETIC ANTAGONISTS OF C-TYPE LECTINS: IMPROVING LIGAND POTENCY AND MULTIVALENT PRESENTATION / G. Goti ; tutor: A. Bernardi; coordinatore: E. Licandro. DIPARTIMENTO DI CHIMICA, 2017 Mar 17. 29. ciclo, Anno Accademico 2016. [10.13130/g-goti_phd2017-03-17].
GLYCOMIMETIC ANTAGONISTS OF C-TYPE LECTINS: IMPROVING LIGAND POTENCY AND MULTIVALENT PRESENTATION
G. Goti
2017
Abstract
C-Type lectin receptors are calcium dependent proteins able to selectively recognize and bind to carbohydrates exposed by invading pathogens and damaged cells, thus acting as important first-line host defence. However, C-type lectins activity can be also responsible for the pathogenesis of infections and inflammatory diseases, thus representing a class of promising therapeutic targets. Protein-carbohydrate interactions are generally weak interactions, with dissociation constants lying in the millimolar range. This problem has been solved by Nature exploiting multivalency: C-type lectins are usually oligomeric receptors interacting with polyglycosylated surfaces, thus concomitant multiple interactions lead to high affinity binding. This thesis work has been devoted to the synthesis and identification of potent C-type lectin antagonists. In particular, our strategy was based on the combination of glycomimetic compounds as active and enzymatically stable ligands and on their multivalent presentation. The first part of the thesis work was focused on the synthesis of stabilised versions of previously developed antagonists for the Mannose Binding Lectin (MBL), a C-type lectin involved in the reperfusion damage after acute ischemic stroke. Tetravalent glycodendrons were synthesized following an efficient modular approach, which allowed functionalization at the dendron focal point and ready access to higher valency compounds, as demonstrated with the synthesis of a 16-valent glycodendrimer. All the compounds were fully characterized and showed complete water solubility and remarkable stability in water solution and under chromatographic purification (both direct and reverse phase). The binding affinity of the glycodendons and glycodendrimer was assessed towards the murine isoform MBL-C by an SPR competition assay, confirming high binding affinity and valency-dependent avidity. A second part of the work was addressed to the synthesis of inhibitors for the Dendritic Cell-Specific ICAM-Grabbing Non-Integrin (DC-SIGN), a C-type lectin exploited by several pathogens to initiate infections. In the development of new DC-SIGN antagonists, a rational design approach was adopted. Driven by the high affinity reached by divalent and hexavalent DC-SIGN chelating agents, a cross-shaped 12-valent glycodendrimer able to perform as a tetradentate ligand and simultaneously bind to the four DC-SIGN binding sites was designed and successfully synthesized and characterized. The affinity towards DC-SIGN is still under study. Finally, the development of a methodology aimed to identify potent C-type lectin glycomimetic ligands was undertaken in collaboration with the group of Dr. Niels C. Reichardt at CIC biomaGUNE (San Sebastian, Spain). A small library of glycomimetics was synthesized exploiting glycan microarray technology. In particular, the use of recently described MALDI-TOF MS detectable microarrays allowed us the on-chip synthesis of unnatural glycan through enzymatic glycosylation with clickable modified sugar donors followed by Cu(I) catalyzed Azido Alkyne Cycloaddition (CuAAC). In the attempt of applying this strategy to the generation of LewisX trisaccharide mimics, the synthesis of a clickable Fucose-GDP sugar donor was accomplished. This derivative proved to be readily accepted by α-1,3-fucosyltransferase from H. pylori and the synthesis of LewisX mimics is currently on going at CIC biomaGUNE.File | Dimensione | Formato | |
---|---|---|---|
phd_unimi_R10403.pdf
Open Access dal 09/09/2018
Descrizione: Giulio Goti PhD Thesis
Tipologia:
Tesi di dottorato completa
Dimensione
14.03 MB
Formato
Adobe PDF
|
14.03 MB | Adobe PDF | Visualizza/Apri |
Pubblicazioni consigliate
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.