Synthesis and physiochemical characterization of new siderophore- inspired peptide-chelators with 1- hydroxypridine-2-one (1,2-HOPO) Thesis Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy by Danah Mahdi AlShaer 2020 Supervisor: Prof. Beatriz Garcia de la Torre Co-supervisor: Prof. Fernando Albericio Synthesis and physiochemical characterization of new siderophore-inspired peptide• chelators with 1-hydrnxypridine-2-one (1,2-JB[OPO) 217078895 Danah Mahdi AllSlh.aer A thesis submitted to the School of Health Sciences, College of Health Sciences, University of KwaZulu-Natal, Westville, for the degree of Doctor of Philosophy by research in Pharmaceutical Chemistry. This is the thesis in which the chapters are written as a set of discrete research publications that have followed each journal's format with an overall introduction and final summary. These chapters have been published in internationally recognized, peer-reviewed journals. This is to certify that the contents of this thesis are the original research work of Mrs Danah Mahdi AllSllnaer, carried out under our supervision at the Peptide Sciences Laboratory, Westville campus, University of KwaZulu-Natal, Durban, South Africa. Supervisor: --"-:I-----::... Date: gth December 2020 Date: 8th December 2020 As the candidate's supervisors we agree to the submission of this thesis Table of Contents Abstract……………..…………………………………………………………………..……1 Declaration 1: Plagiarism.……………………………………………………….…………..2 Declaration 2: Publications ….………………………………………………….…...….…..3 Acknowledgment………………………………………..………………...…………….…..5 Aim and objectives……….….……………………………………………………………...6 Chapter 1: (Introduction) Hydroxamate Siderophores: Natural Occurrence, Chemical Synthesis, Iron Binding Affinity and Use as Trojan Horses Against ………..……….. 7 Reprint………………………………………………………………………………………8 Chapter 2: Solid-phase synthesis of peptides containing 1-Hydroxypyridine-2-one (1,2-HOPO) …………………………………………………….………………………. 39 Reprint………………………………………………………………………………………40 Chapter 3: Protocol for efficient solid-phase synthesis of peptides containing 1- hydroxypyridine-2-one (1,2-HOPO) ………………………….………………………. 44 Reprint………………………………………………………………………………………45 Chapter 4: Chapter 4. Synthesis of new peptide-based Ligands with 1,2-HOPO pendant chelators and the thermodynamic evaluation of their iron (III) complexes ………………………………………………………………………………………..……. 53 1. Introduction ….……...………….…….….……………………………………………. 53 2. Results and discussion….……………………………………….….…………………. 55 2.1 Synthesis of Ligands……..………………………………….….…………………. 55 2.2 Physiochemical characterization of ligands and their Fe(III) complexes………… 57 2.2.1 Ligand-Fe (III) complex stoichiometry determination ….…………..……… 57 2.2.2 Determination of pKa values………………...……… ….…………..……… 59 2.2.3 Iron (III) complexation…..………………………..………………………… 61 3. Conclusion….……...………….….…….….……………………………………………. 63 4. Experimental.……...………….….…….….……………………………………………. 63 5. References …………………………………………………….….………..……………. 66 Chapter 5: 2019 FDA TIDES (Peptides and Oligonucleotides) Harvest ……………. 68 Reprint………………………………………………………………………………………69 Chapter 6: 2018 FDA TIDES Harvest …………………………………..……………. 85 Reprint………………………………………………………………………………………86 Chapter 7: 2017 FDA peptide Harvest …………………………………..……………. 92 Reprint………………………………………………………………………………………93 Chapter 8: Conclusion and future perspectives………………………..……………. 103 Supplementary information Abstract Compounds containing hydroxamate moieties (N-hydroxyl amides) in their structure have found a vast range of therapeutic applications such as antibacterial, anti-tumour, anti-immune suppressor, and for iron overloading treatment. Hydroxamate chelators binds to Fe (III) tightly through its electron donating oxygens. The binding strength is maximized in compounds containing three hydroxamic moieties due to the so called “chelate effect”. As all microorganisms require iron for surviving, they develop endogenous siderophores to acquire iron from the surroundings. Siderophores contain hydroxamate, catecholates, α-hydroxy carboxylates groups, among others, in their structures. The acquisition of iron by siderophores in microorganisms goes through specific cycles that includes sequestration of Fe(III), recognition and uptake of the ferri- siderophore through the cell membrane, and then release of the iron in the cytoplasm. Many natural hydroxamate siderophores contain a peptidyl backbone. In this work, peptides containing one or more units of 1,2-hydroxypyridine-N-oxide (1,2-HOPO) have been synthesized. The introduction of these units on the peptides has been done by means of 4- carboxy-1-hydroxypyridil-2-one (1,2-HOPO-4-COOH) using solid-phase peptides synthesis (SPPS) protocols. The obtention of the new “siderophores” containing three 1,2-HOPO units have been done by two different approaches, sequential and convergent. The compounds have been evaluated as potential iron chelators. Thus, the pKa values and the thermodynamic constants of all ligands have been spectrophotometrically determined. The Fe(III) affinities of the two hexadentate ligands (ligand B and ligand C) have been determined by competition experiments against EDTA. The results showed that the iron complex ligand B is stronger that the iron complex EDTA since the last is not be able to replace it. Hence, this new siderophore could be a promising candidate to be used in further therapeutic applications. 1 Declaration 1- Plagiarism I, Danah Mahdi AlShaer, declare that, 1. The research report in this thesis, except where othe1wise indicated, is my original work. 2. This thesis has not been submitted forany degree or examination at any other university. 3. This thesis does not contain other person's data, pictures, graphs, or other information, unless specificallyacknowledged as being sourced from other persons. 4. This thesis does not contain other person's writing, unless specificallyacknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written, but the general informationattributed to them has been referenced. b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks and referenced. 5. This thesis does not contain text, graphics or tables copied and pasted from the intern,t, unless specificallyacknowledged, and the source being detailed in the thesis and in the references sections. Signed -----------�----------------- 2 Declaration 2- Publication List of publications originated from this Thesis 1. Al Shaer, D.; Al Musaimi, O.; de la Torre, B, G.; Albericio, F. Hydroxamate siderophores: Natural occurrence, chemical synthesis, iron binding affinity and use as Trojan horses against pathogens. Eur. J.Med.Chem. 2020, 208. 10.1016/j.ejmech.2020.112791. (Al Shaer D., and Al Musaimi, O. contributed to searching for information and in writing the manuscript, the remaining authors are supervisors). 2. Al Shaer, D. ; Albericio, F.; de la Torre, B, G. Solid-phase synthesis of peptides containing 1-Hydroxypyridine-2-one (1,2-HOPO). Tetrahedron Lett. 2020, 61. 10.1016/j.tetlet.2020.152299. (Al Shaer, D. contributed in the synthesis, analysis, characterization of all compounds in this work, the remaining authors are supervisors). 3. Al Shaer, D.; de la Torre, B, G.; Albericio, F. Protocol for efficient solid-phase synthesis of peptides containing 1-hydroxypyridine-2-one (1,2-HOPO). MethodsX 2020, 7. 10.1016/j.mex.2020.101082. (Al Shaer, D. contributed in the synthesis, analysis, characterization of all compounds in this work, also in writing the manuscript, the remaining authors are supervisors). 4. Al Shaer, D. ; Albericio, F.; de la Torre, B, G. Synthesis of new peptide-based Ligands with 1,2-HOPO pendant chelators and the thermodynamic evaluation of their iron (III) complexes (submitted manuscript). (Al Shaer, D. contributed to the synthesis, analysis, characterization of all compounds in this work, the remaining authors are supervisors). 5. Al Shaer, D.; Al Musaimi, O.; Albericio, F.; de la Torre, B, G. 2019 FDA TIDES (Peptides and Oligonucleotides) Harvest. Pharmaceuticals (Basel). 2020, 13, doi:10.3390/ph13030040. (Al Shaer D., and Al Musaimi, O. contributed equally to searching for information and in writing the manuscript, the remaining authors are supervisors. 6. Al Shaer, D.; Al Musaimi, O.; Albericio, F.; de la Torre, B, G. 2018 FDA Harvest. Pharmaceuticals (Basel). 2019, 12, doi: 10.3390/ph12020052. 3 (Al Shaer D., and Al Musaimi, O. contributed equally to searching for information and in writing the manuscript, the remaining authors are supervisors. 7. Al Musaimi, O.; Al Shaer, D.; de la Torre, B.G.; Albericio, F. 2017 FDA peptide harvest. Pharmaceuticals (Basel) 2018, 11. 10.3390/ph11020042. (Al Shaer D., and Al Musaimi, O. contributed equally to searching for information and in writing the manuscript, the remaining authors are supervisors). 4 Acknowledgements My heartful thanks and praises to almighty God “Allah” for giving me the health, patience and strength to move on in life, and for all his blessings. Then, special thanks to my supervisors Prof. Beatriz de la Torre and Prof. Fernando Albericio for their continuous support, guidance, inspiration, and motivation all throughout my PhD study, my gratitude cannot be expressed by words, THANK YOU BOTH. Next, many thanks and appreciation go to my colleague and husband Dr. Othman Al Musaimi, and all my group mates Dr. Anamika, Dr. Ashish, Dr. Edikarlos, Dr. Al Hassan, Dr. Srinivas, Nandhini, Amit, Amanda,