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Synthesis, Characterization and Iron-Acquisition of Hydroxamic Acid Derivatives -Functional studies based on the subuni

Synthesis, Characterization and Iron-Acquisition of Hydroxamic Acid Derivatives -Functional studies based on the subunits of amphiphilic siderophores. Linda M. Johnson University of North Carolina at Chapel Hill Class of 2003 Dr. John T. Groves/ Minkui Luo Princeton University May 6, 2004

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Synthesis, Characterization and Iron-Acquisition of Hydroxamic Acid Derivatives -Functional studies based on the subuni

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  1. Synthesis, Characterization and Iron-Acquisition of Hydroxamic Acid Derivatives -Functional studies based on the subunits of amphiphilic siderophores Linda M. Johnson University of North Carolina at Chapel Hill Class of 2003 Dr. John T. Groves/ Minkui Luo Princeton University May 6, 2004 Mercer County Community College

  2. Iron – An Important Element • 4th most abundant element in the world • An important nutrient for the body. • Helps with growth and development in the body, especially in children. • Iron is forever cycled from a liver storehouse in a protein called ferritin.

  3. Lack “iron” in such an iron-abundant planet ? The Paradox of the Limited Abundance of Iron • Extremely limited bioavailability. • Bacteria battles to acquire iron to obtain nutrients that to help contribute to growth

  4. Siderophores • Small weight compounds that have a high affinity for Fe (III). • Help with Iron Transport. • Three subunits of siderophore’s chelating groups.

  5. Pseudobactin Albrecht-Gary A. M.; Crumbliss A. L. Metal Ions in Biological Systems1998, 35, 239 A Common Strategy of Bacteria --- Iron-Delivery Shuttle, Siderophore

  6. Water-phase Iron-acquisition Kinetic of Citrate-based Amphiphilic Siderophore Apo-Acinetoferin Apo-Rhizobactin Apo-SAF

  7. Purpose of Experiment • To examine and compare the structures of siderophores’ sub-units-hydroxamic acids. • To understand how hydroxamic acids facilitate the iron chelating process of siderophores by measuring their iron-acquisition rate.  

  8. Synthesis of N1-BOC propane diamine Compound 1 Compound 2 Compound 3

  9. Synthesis of Compound II & III • Synthesized Boc-protected amine • Use Flash Column Chromatography (FCC) to help separate compound. • Perform Thin Layer Chromatography (TLC) to help locate the final product. • Check purity of compound with 1H-NMR.

  10. Five substrates used to react with the amine: • Acetyl Chloride • Cinnamoyl Chloride • Butyryl Chloride • Crotonyl Chloride • Hydrocinnamoyl Chloride

  11. Procedure of Analog Synthesis • Performed test reactions with each halide. • Check TLC and take 1H-NMR for each reaction. • Once reaction is successful, upscale amount of material for future experiments.

  12. Reaction Between Amine and Acetyl Chloride • Equation: • Results: • 1st attempt: No distillation or either substrate nor solvent. Some reaction occurred, but it was not complete, even after adding 10% base to help push reaction. • 2nd attempt: Distilled Chloride and Solvent (CH2Cl2). Reaction completed. Rxn. time estimate: ~0.5 hr. CH2Cl2/Reflux

  13. Equations for Other Hydroxamic Acid Analogs With Butyryl Chloride o With Crotonyl Chloride o With Hydrocinnamoyl Chloride o

  14. Reaction Between Amine and Cinnamoyl Chloride X • Equation: • Result: Did not completely react even after distilling both chloride and solvent. • 2 ways attempted to push reaction - No heat, extra base added - With heat and extra base added.

  15. Procedure (Cont.) • Once the analogs were synthesized, solvent was removed with rotavapor and oil pump • 1H-NMR, 13C-NMR and Mass Spec. were taken of each derivative to check purity.

  16. 1H-NMR of

  17. Four Final Analogs Used for Iron Acquisition Measurement

  18. Procedure for Measuring the Iron-Acquisition Rate • Iron stock solution was made (.6394g/200ml distilled water) • 10mM Buffer solution -Ammonium Fe(III) Citrate (FAC) w/Hepes buffer • Used SF 61 DX2 Mixing Stopped-Flow Machine • Conditions: - pH of buffer solution: 7.4 - Temp-37oC

  19. Results of iron acquisition of hydroxamate analogs

  20. Results and Conclusions • There was a general curve that formulated for each analog. • The shorter the substrate, the higher the iron acquisition rate. • Having a unsaturated substrate also increased the iron-acquisition rate.

  21. Future Studies Adjust conditions to successfully react the following: To continue to further examine the chelating process of iron acquisition due to hydroxamic acid structure.

  22. Acknowledgements Dr. John T. Groves Princeton University Department of Chemistry Minkui Luo Dr. Groves’ Research Team Mercer County Community College Prof. Helen Tanzini Family and Friends THANK YOU ALL FOR YOUR SUPPORT! 

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