Monash University

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Aza-macrocyclic neoemycin B conjugates and their metal complexes as novel RNA targeting agents

posted on 2017-01-24, 00:10 authored by Kong, Bopha
This thesis describes the synthesis of novel neomycin B-tacn (1,4,7-triazacyclonone) and -cyclen (1,4,7,10-tetraazacyclododecane) conjugates, the assessment of the ability of these derivatives and their Cu(II)/ Zn(II) complexes to bind and/or cleave the bacterial ribosomal A-site and the HIV-1 transactivation response (HIV-1 TAR) element, and the evaluation of their antibiotic activities against various Gram-positive and Gram-negative bacterial strains. Six novel neomycin B-tacn/cyclen conjugates, varying in the length of the alkyl spacer between the macrocyclic unit and neomycin B, were prepared by multi-steps syntheses. Tacn and cyclen were initially protected with Boc following literature procedures, leaving one secondary amine free to undergo further reaction. Alkyl esters of varying chain lengths were then introduced onto the free secondary amine, and were subsequently hydrolysed under basic conditions to their corresponding carboxylic acids. In addition, neomycin B was modified at the 5″ position by first converting the primary hydroxyl group into an azide, followed by Pd/C mediated hydrogenation to an amine group. The modified neomycin B bearing the primary amine functionality at the 5″ position was subsequently coupled to the macrocycles bearing the carboxylic group using a standard peptide coupling procedure. Global trifluoroacetic acid (TFA)-mediated Boc-deprotection yielded the final products as their TFA salts, which were purified by high performance liquid chromatography (HPLC) and characterised using nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy and mass spectrometry techniques. Neomycin B-macrocyclic conjugates and their Zn(II) and Cu(II) complexes were tested for their ability to bind to the bacterial ribosomal A-site and the HIV-1 TAR ribonucleic acid (RNA). The conjugates showed subtle enhancement in their binding ability to the A-site over the parent neomycin B. The Zn(II)-cyclen complexes further enhanced the A-site binding, whereas the Cu(II)-tacn complexes were less effective than neomycin B. From the pH dependence studies, it was found that the binding affinity of the conjugates was greater at lower pH. This was attributed to an increase in the charge of conjugate relative to neomycin B, which arises from the partial protonation of the amine groups on the macrocyclic ring. The neomycin B-macrocyclic conjugates and their metal complexes displayed greater binding affinity for the HIV-1 TAR RNA compared to the A-site. This may be due to the fact that the A-site is highly discriminatory of the structure and conformation of the aminoglycoside ligand, while TAR is an inherently less selective RNA target than the A-site and can readily adjust its structure to interact with different aminoglycosides. Both the macrocycles and their metal complexes appear to enhance the binding of neomycin B to TAR RNA, with the Zn(II) neomycin B-cyclen complexes exhibiting the highest affinity. This may be the result of the strong interactions between the Zn(II) ions and the uracil bases within the RNA. This explanation is supported by the fact the Zn(II) neomycin B-cyclen complexes were found to bind more strongly to TARU3 than TARC3, which have three uracil or three cytosine bases in the bulge binding site, respectively. TAR cleavage studies showed that all the neomycin B-macrocyclic conjugates and their metal complexes induce site-specific hydrolysis in the same manner as neomycin B. However, the addition of metal ions did not enhance the hydrolysis of these conjugates. The greatest extent of cleavage under physiological conditions was observed for the neomycin B-cyclen conjugate 38, followed by Zn(II)-38, however, their rate constants for TAR cleavage (2.6±0.9 × 10-⁶ s-¹ and 2.5±0.6 × 10-⁶ s-¹, respectively) were found to be similar to that observed for neomycin B (2.3±1.2 × 10-⁶ s-¹). The preliminary assessments of the antibacterial activities showed the conjugates 37, 39, and 40 to be slightly more effective than neomycin B in inhibiting the growth of the resistant strain of the Gram-negative, P. aeruginosa (ATCC27853). However, their antibiotic activities against the other tested bacterial strains were lower than that of neomycin B.


Campus location


Principal supervisor

Leone Spiccia

Year of Award


Department, School or Centre



Doctor of Philosophy

Degree Type



Faculty of Science

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