|dc.contributor.author||Telfer, Thomas James||-|
Desferrioxamine B (DFOB) is a linear hydroxamate-type siderophore trimer that forms a high-affinity (Kd 1031 M) hexadentate complex with Fe(III). Streptomyces pilosus produces DFOB as its native siderophore for iron acquisition from the extracellular environment. The DesABCD enzyme cluster is responsible for DFOB biosynthesis, catalysing the N-hydroxylation of 1,5-diaminopentane (DP) (DesB), N-hydroxy-acetylation or N-hydroxy-succinylation to form N-acetyl-N-hydroxy-DP (AHDP) or N-succinyl-N-hydroxy-DP (SHDP) (DesC), and condensation between one AHDP and two SHDP molecules (DesD). DFOB is used clinically for the treatment of chronic iron overload. The hydrophilicity (logP 2.10), short plasma half-life (t1/2 20 min), and poor cell membrane permeability of DFOB necessitate subcutaneous infusion on 60 – 70 h week1 dose regimes, and limit the pool of iron that can be accessed. A high Fe(III) concentration in the substantia nigra catalyses the reactive oxygen species-mediated death of dopaminergic neurons in Parkinson’s disease (PD). DFOB has minimal blood-brain barrier (BBB) permeability but would have applications in PD for Fe(III) removal if it could be improved. This study aimed to generate analogues of DFOB with improved physicochemical properties, such as increased lipophilicity and BBB permeability, as new potential iron overload or PD treatments. Two methods to produce new DFOB analogues were explored: semi-synthetic chemistry and precursor-directed biosynthesis (PDB).
Eighteen DFOB conjugates with adamantyl- or other polycyclic cage-based ancillary fragments were synthesised and evaluated as potential PD treatments. All compounds had significantly increased logP values (0.15-2.82) over DFOB (2.29). The structure-activity relationship revealed compounds with methyl-substituted adamantyl- (1-3) and 1-pentylbicyclo[2.2.2]octane-based (17) ancillary fragments as the most protective in
models of oxidative stress where PD-relevant SK-N-BE2-M17 neuroblastoma cells were exposed to paraquat or H2O2. These compounds performed well in dose-dependent screens (EC50 10 μM). The ancillary fragments of these successful conjugates had close to zero dipole moments (0.01-0.06), small surface area:volume ratios (0.60-0.61) and logP values in the range 2.0-2.6. The promise of these compounds support progressing to in vivo PD models.
S. pilosus cultures in a medium optimised for DFOB production were augmented with 1,4-diamino-2E-butene (E-DBE) in PDB experiments to generate novel unsaturated DFOB analogues. Three classes of species were produced where the number of DP to E-DBE molecules incorporated were 2:1 (uDFOA1), 1:2 (uDFOA2), or 0:3 (uDFOA3) in contrast to the 3:0 of DFOB. Three constitutional isomers of each uDFOA1 and uDFOA2 were produced, as determined by MS/MS fragmentation. The distribution of these species were uDFOA1 >> uDFOA1 > uDFOA1 and uDFOA2 > uDFOA2 >> uDFOA2, where 0 represents DP and 1 represents E-DBE at the N-acetylated terminus, internal region, or amine terminus of the trimer. Dimeric precursors assembled from one AHDP and one SHDP molecule were also detected, with relative concentrations dDFX[00-] >> udDFX[10-] > udDFX[01-], where ‘-’ represents a vacant position. Equivalent SHDP-SHDP dimers were not identified. These results were paralleled with DB augmented cultures, where DB competed against DP in DFOB assembly. The distribution of these trimeric products and dimeric precursors suggested the following preferred DesD-catalysed biosynthetic sequence: (i) activation of SHDP; (ii) condensation with AHDP to form AHDP-SHDP; (iii) SHDP activation; and (iv) condensation with AHDP-SHDP to form DFOB. The production of unsaturated DFOB analogues allowed insight into DFOB biosynthesis and provided reagents for downstream olefin semi-synthesis to expand structural diversity. Unsaturated DFOB
analogues retained Fe(III) affinity, were successfully reduced and deuterated in model experiments, and retained the potential for modification at the free amine group.
Fluorinated DFOB analogues were generated using PDB with 1,4-diamino-2-fluorobutane (DFB) augmentation. This system was more complex than the E-DBE system. Identified alongside trimeric DFOB analogues and dimeric precursors were DFOB analogues with two hydroxamic acid groups and three amide groups, produced when DesC acted directly on DFB. Analogues assembled from a combination of DFB, DB, and DP were also identified. R-DFB (78%) was incorporated into DFOB analogues more readily than S-DFB (33%), demonstrating that the DesBCD enzyme cascade is enantioselective.
Overall this study has detailed the synthesis of a variety of novel analogues of DFOB through conjugation to adamantyl- or other polycyclic cage-based ancillary fragments, generation of unsaturated or fluorinated analogues of DFOB using PDB, and production of deuterated analogues by semi-synthesis on unsaturated DFOB analogues. Many of these novel analogues have potential as therapeutics. The established PDB and downstream semi-synthesis methodology has broad utility for the generation of analogues of natural products other than DFOB.||en_AU|
|dc.publisher||University of Sydney||en_AU|
|dc.publisher||Sydney Medical School||en_AU|
|dc.publisher||Discipline of Pharmacology||en_AU|
|dc.rights||The author retains copyright of this thesis. It may only be used for the purposes of research and study. It must not be used for any other purposes and may not be transmitted or shared with others without prior permission.||en_AU|
|dc.title||Analogues of Desferrioxamine B Produced using Semi-Synthetic Chemistry and Precursor-Directed Biosynthesis||en_AU|
|dc.type.pubtype||Doctor of Philosophy Ph.D.||en_AU|
|dc.description.disclaimer||Access is restricted to staff and students of the University of Sydney . UniKey credentials are required. Non university access may be obtained by visiting the University of Sydney Library.||en_AU|
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