The intracellular delivery of molecules to modulate signaling pathways and gene expression is a powerful approach to control stem cell fate decision. For applications in gene therapy and regenerative medicine, the use of genetic material and viral vectors raise concerns because stem cells persist throughout life, and long-term effects of uncontrolled genetic modifications could affect the cellular progeny. An alternative is to deliver directly peptides or proteins using cell-permeable peptides (CPPs) which have the ability of crossing the plasma membrane and carrying cargos into cells. CPPs can therefore be used to deliver factors to direct stem cells proliferation, survival and differentiation. This thesis describes an approach to control stem cell fate based on the delivery of a CPP-conjugated bioactive peptide. A first significant contribution from this work is the development of a flow cytometric assay to accurately quantify the uptake of a panel of CPPs. This study revealed that HIV-transactivator of transcription (TAT) and Antennapedia (Antp) offered the highest level of translocation in different cell types. The uptake was improved by treating the cells with a single, low-voltage electrical pulse that selectively enhances the amount of TAT-conjugated peptides and proteins delivered by at least an order of magnitude, without causing cellular toxicity or apoptosis. Subsequently, flow cytometry, confocal microscopy, capillary electrophoresis and mass spectrometry were used to examine the intracellular fate of TAT-conjugated peptides in order to define the parameters that limit their bioactivity and point to specific sequence modifications that can improve their efficacy. The advances described in this thesis were applied to the development of TAT-eIF2B, a peptide-inhibitor of glycogen synthase kinase-3 (GSK-3). TAT-eIF2B was found to be specific for GSK-3 and had a significant positive effect on the formation of neurospheres in embryonic stem cell cultures and on the survival of myeloid progenitors in cytokine-starved fetal liver cell cultures. On the other hand, GSK-3 inhibition reduced the number of neurospheres generated by human olfactory neuroepithelium cells due to lower proliferation and increased neuronal differentiation. In summary, this work describes the development of a peptide-based technology to deliver bioactive cargoes in cells, and it demonstrates its utility for modulating the activity of a master regulator of stem cell fate decision.

The intracellular delivery of molecules to modulate signaling pathways and gene expression is a powerful approach to control stem cell fate decision. For applications in gene therapy and regenerative medicine, the use of genetic material and viral vectors raise concerns because stem cells persist throughout life, and long-term effects of uncontrolled genetic modifications could affect the cellular progeny. An alternative is to deliver directly peptides or proteins using cell-permeable peptides (CPPs) which have the ability of crossing the plasma membrane and carrying cargos into cells. CPPs can therefore be used to deliver factors to direct stem cells proliferation, survival and differentiation. This thesis describes an approach to control stem cell fate based on the delivery of a CPP-conjugated bioactive peptide. A first significant contribution from this work is the development of a flow cytometric assay to accurately quantify the uptake of a panel of CPPs. This study revealed that HIV-transactivator of transcription (TAT) and Antennapedia (Antp) offered the highest level of translocation in different cell types. The uptake was improved by treating the cells with a single, low-voltage electrical pulse that selectively enhances the amount of TAT-conjugated peptides and proteins delivered by at least an order of magnitude, without causing cellular toxicity or apoptosis. Subsequently, flow cytometry, confocal microscopy, capillary electrophoresis and mass spectrometry were used to examine the intracellular fate of TAT-conjugated peptides in order to define the parameters that limit their bioactivity and point to specific sequence modifications that can improve their efficacy. The advances described in this thesis were applied to the development of TAT-eIF2B, a peptide-inhibitor of glycogen synthase kinase-3 (GSK-3). TAT-eIF2B was found to be specific for GSK-3 and had a significant positive effect on the formation of neurospheres in embryonic stem cell cultures and on the survival of myeloid progenitors in cytokine-starved fetal liver cell cultures. On the other hand, GSK-3 inhibition reduced the number of neurospheres generated by human olfactory neuroepithelium cells due to lower proliferation and increased neuronal differentiation. In summary, this work describes the development of a peptide-based technology to deliver bioactive cargoes in cells, and it demonstrates its utility for modulating the activity of a master regulator of stem cell fate decision.

Cyclization reactions play an important role in synthesizing a significant number of small molecule scaffolds for various purposes, including drug discovery. However, the application of cyclization reactions in the modifications of biomolecules in a single step is still limited. This dissertation reports a stereoselective thiomorpholine ring formation reaction to site-selectively modify N-terminal cysteines of unprotected peptides or proteins in a single step. We showed that α-fluoroketone molecules afford the cyclization reaction with the beta amino thiol group of N-terminal cysteine. Both chemo and stereo-selectivity of this reaction have been studied using 2D NMR analyses. Cysteine located at the Nterminal of a short protein (VHP protein) has been modified site-selectively with a fluorescein isothiocyanate (FITC) containing an α-fluoroketone linker to demonstrate the applicability of this reaction in modifying biomolecules. This chemistry has the potential to generate homogeneous and stable antibody-drug conjugates (ADCs) for the treatment of cancer. This dissertation also demonstrates a fluorine-thiol displacement reaction (FTDR) to synthesize various macrocyclic and stapled peptides, which renders medicinally privileged peptides with improved biological properties such as binding affinities and cell membrane permeability. The cyclization of fluoroacetamide containing peptides with benzenedimethane thiol linkers enhances peptides' alpha helicity. These FTDR stapled peptides exhibit better cellular uptake compared to the classic ring-closing metathesis (RCM) stapled peptides. Compared to the proteoglycan-aided cell penetration by peptides stapled with RCM, the preliminary mechanistic studies of our FTDR-stapled peptides revealed that our thiolated linkers allowed peptides to enter cells in multiple pathways. Taken together, our FTDR-based stapling approach may provide a novel class of cell-permeable peptides that might open a new window to probe intracellular targets. This dissertation reports another cyclization reaction between hydrazine and carbamate, synthesized from 7-hdyroxy coumarin derivatives. We demonstrate that the secondary nitrogen of hydrazine is much more reactive than the primary nitrogen for intramolecular cyclization reactions. Hydrazine affords urea bonds upon the substitution of a coumarin moiety of carbamates to generate 6 or 7 membered cyclic scaffolds. The exocyclic free amine might allow facile generation of a library of compounds. In addition, further optimization of this reaction might allow using these hydrazine containing molecules in monitoring the real-time activity of histone deacetylases (HDACs). / Chemistry