Advancement and investigation of cellular delivery vehicles using guanidinoglycosides for the transport of biologically relevant cargos are reported. Insertion of the PEG-modified phospholipid DSPE-PEG-OMe 2000 and a lipophilic guanidinoneomycin derivative on the surface of liposomes diminishes uptake of the nanoassemblies and is dependent on the amount of DSPE-PEG-OMe inserted on the liposome surface. To illustrate this, liposomes with guanidinoneomycin on the bilayer surface (i.e., GNeosomes) were treated with varying amounts of the PEG-modified phospholipid and subsequently analyzed to study the effects on liposomal size, zeta potential, and cellular uptake. By systematically protecting GNeosomes through inserting varying amounts of PEG chains into liposomal bilayers, methodical control over cellular uptake is obtained.

Additionally, an analysis of modified amino- and guanidino-glycosides derived from kanamycin, tobramycin, and neomycin in native and mutant CHO cells is examined using confocal microscopy and flow cytometry, illustrating the significance of multivalency for mammalian cell internalization of carriers that specifically interact with cell surface heparan sulfate proteoglycans.

ABSTRACT OF THE DISSERTATION

Synthesis and Analysis of Fluorescent Antibiotics Containing Emissive Adenosine Surrogates

by

Kaivin Hadidi

Doctor of Philosophy

University of California, San Diego, 2022

Professor Yitzhak Tor, Chair

The synthesis, photophysical analysis, and biological investigation of puromycin analogues containing fluorescent adenosine derivatives are discussed. We analyzed the photophysical properties of two novel fluorophores, N,N-dimethylthieno[3,4-d]pyrimidin-4-amine and isothiazolo[4,3-d]pyrimidine-7(6H)-thione. While the photophysical properties of these two compounds were found to be lackluster, substitution of the dimethylamine substituents for modified and unmodified azetidines were found to significantly augment the brightness of the fluorophores by nearly three orders of magnitude in the thienopurines and two orders of magnitude in the isothiazolo derivatives. We then synthesized C-nucleoside analogues of puromycin employing a thieno[3,4-d]pyrimidine heterocycle in place of the native purine, along with other derivatives employing azetidine and 2,2-difluoroazetidine at the 6 position (following native purine numbering). A convergent synthesis utilizing lithium halogen exchange between a halogenated thiophene-based purine analogue and a benzyl protected 3’-deoxy-3’-azido-ribolactone produced a desired C-glycoside with reactive handles at the 3’position of the sugar (for peptide coupling) and at the 6-position of the modified purine (for SnAr reactions) providing synthetic accessibility to numerous fluorescent antibiotic scaffolds. Biological evaluation of the synthesized antibiotics indicates similar bioactivity of the novel fluorophores compared to native puromycin, both in in vitro protein expression systems and bactericidal assays. Treatment of the modified derivatives in HEK293T cells indicated localization of the compounds with ribosomes following fixation immunofluorescence treatments. One derivative, the 2,2-difluoroazetidine modified analogue, exhibited fluorescence in live cells and neurons at sub-cytotoxic concentrations, one of the first and few times a fluorescent nucleoside derivative has been used in this context given the effect of size restriction on the chromophores in question.