ETD

• Glycoconjugated Europium complexes

In the last decades, metal complexes based on lanthanide trivalent ions demonstrated to be a new valuable approach as diagnostic and therapeutic agents. Their unique photophysical properties, especially in emission, are connected to their electronic configuration, in which intraconfigurational f-f electronic transitions are characterized by large Stokes shift, long lived photoluminescence, narrow emission bands and long decay times. In addition, for bio-imaging application they possess high photochemical stability in biological medium with a low susceptibility to photobleaching processes and high brightness. Because of this last property, lanthanide complexes constitute suitable and versatile agents in bioassays and luminescence microscopy. Nevertheless, the f-f transitions are prohibited according to the Laporte rule and require the presence of a chromophoric ligand to be populated. The ligand acts as an antenna capable of absorbing energy from the UV excitation wavelength and transfers it to the lanthanide ion enabling its photoluminescence. Due to its role, the antenna moiety represents a crucial part of the molecule as it must guarantee excellent stability, strong luminescence, high biocompatibility, and interaction ability towards biological targets.
However, these antennas often present highly conjugated structures containing aryl-alkynyl or aryl-pyridyl groups, which are distinguished by a strong apolar character, therefore altering the ability of these agents to fulfill a series of requirements that they must present as imaging agents first water solubility and biocompatibility. Beyond solubility, cellular delivery represents a further challenge: to overcome this limitation, in literature different strategies have been investigated, including conjugation with biomolecular vectors such as antibodies, peptides, nucleic acids or carbohydrates. Surprisingly, there are just few examples of glycoconjugated lanthanide complexes with respect to other bioconjugates, often limited to MRI contrast agents or luminescent complexes applied to in vitro essays rather than in vivo bioimaging experiments (our goal). In particular, the glycoconjugation approach is often applied to metal complexes to improve critical properties including water solubility and biocompatibility, but most of all their selectivity of action. The presence of sugar moieties represents a possible interaction site between the probe and biological structures such as GLUTs: these D-Glucose (and other D-monosaccharides) transporters are overexpressed in cancerous cells as a consequence to their dysregulated glucose metabolism compared to healthy cells (Warburg effect), then glycoconjugated molecular entities exploit this condition to be selectively internalized. Beyond cancer research, glycoconjugates luminescent probes are also attractive to target bacteria-related biofilms, which are characterized by a biological environment rich of carbohydrate-targeting Lectins, and their recognition process can impart selectivity of action to the glycoconjugated probe.
During the last years, in our laboratory a series of glycoconjugated luminescent lanthanide complexes has been developed as selective probes for in vivo two-photon (2P) microscopy bioimaging of tumors. These complexes present a well-defined design, consisting of an azamacrocycle such as triazacyclononane (TACN) or cyclen (tetraazacyclododecane) based complex with 2P excitable chromophoric antennas, decorated with a flexible PEG spacer (to distance the sugar moiety from the antenna for a more efficient biological recognition) bearing the monosaccharide unit.
The glycosidic portion has been introduced via direct glycosylation reaction or more recently via click chemistry, by means of Copper-(I)-catalyzed azide-alkyne cycloaddition (CuAAC), between propargyl glycosides and a Europium complex bearing the azido functionalities, making this latter approach extremely versatile. A series of differently glycoconjugated complexes has been prepared transferring their remarkable optical properties in water medium, in addition to their stability and negligible cytotoxicity. Their selective uptake in cancer cells or staining capability of xenografted tumor in zebrafish embryos has been pointed out.
The aim of my thesis is the synthesis of new glycoconjugated Europium complexes bearing α—D-Glucopyranosidic and β—D-Galactopyranosidic pendants, through a CuAAC reaction directly on azido functionalized Europium complex. This allowed the expansion of a library of glycoconjugated Europium complexes to study their sugar-dependent uptake mechanisms across different tumor cell lines, both in vitro and in vivo, and potentially contributing to the development of more selective diagnostic and therapeutic agents. The sugar portion of α D-Glucose was obtained through glycosylation reaction with propargyl alcohol, followed by acetylation, anomers separation and deacetylation. The sugar portion of β D-Galactose was obtained through the stereoselective glycosylation of its trichloroacetimidate derivative with propargyl alcohol. Finally, I also synthesized a more complex TACN-based Europium bioconjugate, in which the different antennas carry distinct bioactive molecules: in particular I realized a complex with two antennas bearing an α D-Mannopyranosidic portion, and the third antenna functionalized with a bioactive molecule, namely a compound that stimulates GLP 1 release through TRPA1 channels based on a diaza-oxa-[3.3.1]-bicyclic compound. This luminescent complex will be used to trace the intracellular localization of the bioactive molecule to gain insights on the mechanism of action.