| Summary: | Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2014. === Cataloged from PDF version of thesis. === Includes bibliographical references. === Chapter 1. Introduction to Iron Homeostasis and Siderophores Iron is an essential nutrient for almost all living organisms. This Chapter presents an overview of iron homeostasis in human and bacteria, as well as the biology and chemistry of siderophores and siderophore conjugates. Siderophores are small-molecule iron chelators synthesized by bacteria for scavenging iron from the environment. Their iron-binding properties, structures, transport machineries and biosynthesis are discussed with a focus on enterobactin and salmochelins, which are the two siderophores studied in this thesis. The notion of conjugating functional molecules to siderophores has been actively studied for decades. The syntheses and applications of siderophore conjugates are summarized, with an emphasis on siderophore-antibiotic conjugates. New antimicrobial strategies targeting bacterial iron acquisition are also described. A summary of project goals and thesis organization is presented at the end of this Chapter. Chapter 2. Siderophore-mediated Cargo Delivery to the Cytoplasm of Escherichia coli and Pseudomonas aeruginosa: Syntheses of Monofunctionalized Enterobactin Scaffolds and Evaluation of Enterobactin-cargo Conjugate Uptake This Chapter describes the design and syntheses of monofunctionalized enterobactin (Ent, L- and Disomers) scaffolds. A family of ten Ent-cargo conjugates is synthesized to probe the extent to which the Gram-negative ferric Ent uptake and processing machinery recognizes, transports, and utilizes derivatized Ent scaffolds. The delivery of Ent-cargo conjugates is evaluated by a series of growth recovery assays. The results demonstrate that the Escherichia coli K12 and Pseudomonas aeruginosa PAO 1 Ent transport machinery identifies and delivers select Ent-cargo conjugates to the cytoplasm, and P. aeruginosa PAO exhibits greater promiscuity than E. coli K12 for the uptake and utilization of the conjugates. This work affords a new native siderophore platform amendable for synthetic modification and cargo attachment. It also indicates that cargo size affects the delivery efficiency, which influences the conjugate design for Ent-mediated delivery strategies described in the following Chapters. Chapter 3. Enterobactin-Mediated P-Lactam Delivery into Gram-negative Bacteria The design, synthesis, and characterization of two enterobactin-antibiotic conjugates, where the [beta]-lactam antibiotics ampicillin (Amp) and amoxicillin (Amx) are linked to a monofunctionalized enterobactin scaffold via a stable polyethyleneglycol linker are reported. Under conditions of iron limitation, these siderophore-modified antibiotics provide greatly enhanced antibacterial activity compared to the parent [beta]-lactams against Escherichia coli strains that include several pathogens. The mechanism of the antimicrobial activity is probed and the results reveal that the improvement is due to Ent-mediated delivery. Strain-specific killing and faster time-kill kinetics are also observed for the conjugates. These studies demonstrate that the native enterobactin platform provides a means to effectively deliver antibacterial cargo across the outer membrane permeability barrier of Gram-negative pathogens that utilize enterobactin for iron acquisition. Chapter 4. Stability Evaluation of Acyloxymethyl/Acyloxyethyl Ester Linker for the Design of Enterobactin-fluoroquinolone Conjugates Fluoroquinolones are widely used antibiotics that target DNA gyrase. Prior studies with siderophore-fluoroquinolone conjugates suggest that a release step is required following cytosolic entry for the conjugates to exhibit antimicrobial activity. We design and synthesize enterobactin-fluoroquinolone conjugates harboring several acyloxymethyl/acyloxyethyl ester-based labile linkers and the stability of these likers and the conjugates are studied. These linkers exhibit relatively short half-lives and afford premature release of the antibiotics under our experimental conditions, which renders them not suitable for siderophore-based antibiotic delivery. This conclusion is also supported by the Ent-independent antimicrobial activity observed for the enterobactin-fluoroquinolone conjugates harboring the labile linkers. Appendix 1. Chemoenzymatic Syntheses of Enterobactin-antibiotic Conjugates and Studies of Antimicrobial Activity In this Appendix, cargo attachment to enterobactin by using chemoenzymatic reactions is described. This approach affords conjugates containing a 10-mer peptide as the linker between enterobactin and the cargo. A series of enterobactin conjugates harboring antimicrobial peptides and fluoroquinolone antibiotics are reported, and their antimicrobial activity against K coli is evaluated. Unfortunately, none of these conjugates afford enhanced activity compared to the unmodified antimicrobial agents, which may result from impropriate cargo selection or linker design. Appendix 2. NMR, HPLC and UV-Vis Characterizations of the Reported Compounds. === by Tengfei Zheng. === Ph. D.
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