Aminoglycosides have been widely recognized as critically important antimicrobials for treatment of multidrug resistant bacteria. As presented in Chapter 1, though effective against a wide range of Gram-positive and Gram-negative bacteria, aminoglycosides can induce reversible nephrotoxicity and irreversible ototoxicity and can be deactivated by aminoglycoside modifying enzymes (AMEs). Chapter 2 details the synthesis of 5-O-furanosylated apramycin derivatives, prepared with the goal of increasing antibacterial activity while circumventing susceptibility to the aminoglycoside phosphoryl transferases (APH(3’,5’’)), all while maintaining the parent’s low toxicity. Ribosyl derivatives performed well compared to apramycin but suffered from APH(3’,5’’) susceptibility. Erythrosyl and 5-amino-5-deoxy-β-D-ribosyl derivatives overcame this hurdle and displayed greater activity and comparable toxicity to the parent. The most promising derivative, 5-O-[5-Amino-3-O-(2-aminoethyl)-5-deoxy-β-D-ribofuranosyl]apramycin, exhibited twofold decreased toxicity in cochlear explant studies and 2.5-fold increased efficacy in vivo compared to apramycin, and can be synthesized from the parent in six linear steps. Chapter 3 discusses systematic 5’’-modifications of multiple 4,5-aminoglycosides in an attempt to generalize the results observed among the apralogs. Though most modifications were well-tolerated in neomycin, 5’’-deoxy, 5’’-amino, 5’’-acetamido, and erythrosyl derivatives of paromomycin, ribostamycin, and propylamycin all exhibited strongly reduced antibacterial activity. Installation of a 5’’-formamide, however, resulted in increased activity and reduced toxicity. These results will aid in the design of next-generation aminoglycoside antibiotics. Chapter 4 presents the glycosylation reaction, a nucleophilic substitution between a glycosyl donor and acceptor and examines how glycosyl donor reactivity is significantly influenced by side chain conformation, where the most reactive gauche,gauche (gg) conformation strongly stabilizes the transition state. Chapter 5 illustrates through crystallographic analysis that glycosidases and glycosyltransferases have evolved to maximize reactivity of their substrates through side chain restriction. Glucoside, mannoside, and ulosonide processing enzymes strongly favor the most reactive gg conformation, while α-galactoside processing enzymes enforce the second-most reactive gauche,trans (gt) conformation, and β-galactoside processing enzymes favor the least reactive trans,gauche (tg) conformation. Some glycosidases maximize reactivity by binding their substrates in a superarmed conformation that strongly stabilizes the transition state. The results of this study will pave the way for the design of improved conformationally-locked inhibitors of carbohydrate-processing enzymes.