1. The Role of Intra-structural Hydrogen Bond Interactions in Pre-organization of Enterobactin Reina Chu and Eric Marinez Department of Chemistry and Biochemistry California State University Long Beach

2. Iron is essential for life for many microbes However, the aerobic atmosphere of the earth causes iron to convert to oxyhydroxide polymers that possess very low solubility. – The insolubility of ferric hydroxide limits the concentration of [Fe 3+ ] to about 10 -18 M under normal physiological conditions. Bacteria require 10 5 to 10 6 ferric ions per cell per generation. – This continuous demand for ferric ions led bacteria to develop mechanisms to acquire iron.

3. What is Enterobactin? One mechanism developed by many bacteria is the synthesis and secretion of powerful low molecular weight chelating agents called siderophores that are capable of high affinity binding and transport of ferric irons. Enterobactin is a distinctive siderophore that is secreted by E. coli and many other gram negative bacteria for acquisition of iron. – Formation constant for iron is 10 49. – It has the highest affinity for iron than any other natural substance!

4. Structure of Enterobactin Consists of three L-serine residues that are linked head to tail by ester linkages to form a twelve membered trilactone platform ring. Attached to the trilactone backbone through amide linkages are three catecholate groups (2,3- dihydroxybenzoyl)

5. Enterobactin is Predisposed for Metal Binding The dynamic conformation of free enterobactin enhances its ability to hunt and capture iron atoms by two ways: 1)The ligand free enterobactin conformation assists rapid initial binding of Fe(III) 2)Conformation change caused by deprotonation facilitates the fully encapsulation of iron Hydrogen bonding locks the catechol group into one of two rigid conformations, the interconversion of which is triggered by deprotonation/metal complexation.

6. Ferric Enterobactin Complex The binding of Fe(III) at neutral pH occurs through hexadentate coordination of Fe(III) with six catecholate oxygens. The tri-L-serine lactone backbone induces chirality at the metal center in ferric enterobactin complex. The major chirality of the ferric enterobactin complex is a ∆ conformation (right-handed propeller) in aqueous solution.

7. Ferric Enterobactin Complex is Very Thermodynamically Stable One unique characteristic of enterobactin is that it forms a remarkable thermodynamically stable complex with iron. It prominent stability led to the development of many different synthetic analogues of enterobactin. – However, none of these synthetic siderophores Fe(III) complexes came near the thermodynamic stabilities of Fe(III) enterobactin complex. – Many of these synthetic analogs form Fe(III) complexes that were 10 6 less stable than enterobactin.

8. Pre-organization of Enterobactin Structure Allows Remarkable Stability Much of the thermodynamic properties is due to the rigidity of the trilactone backbone, allowing considerable pre-organization in the ligand-free enterobactin This pre-organization is a major contributing factor of creating the unusual high binding affinity characteristics. The pre-organization allows for strain free binding of the iron molecule. – Therefore, binding occurs due to entropy as well as enthalpy factors.

9. Our Purpose/Hypothesis My research is interested in how the trilactone backbone contributes in maintaining the rigid pre-organized structure. We suspect that the hydrogen bond interactions between the three ester linked oxygens of the triserine lactone backbone and the three amide protons in the catecholate side groups significantly influences the pre- organization of enterobactin.

10. Hypothesis In order to investigate the influence of these hydrogen bond interactions, we eliminate the hydrogen bonds by inserting N-methyls to the three catechol side groups, therefore inhibiting hydrogen bond interactions from occurring between the trilactone backbone and the catecholate groups. If these hydrogen bonds were indeed important in maintaining the pre-organization of enterobactin, then the insertion of methyl groups would alter the pre- organization of the free ligand structure, triggering the catecholate legs to move equatorially.

11. Hypothesis Our proposed conformation for N-methyl enterobactin is pseudoequatorial (Upper figure a and b) rather than pseudoaxial conformation that is seen in the pre-organized enterbactin (Below figure c and d).

12. Specific Aim 1: Reduction Animation Reduction animation of L-serine methyl ester to yield N-methyl-L-serine methyl ester

13. Specific Aim 2: Trimerization of N- methyl-L-serine methyl ester to make enterbactin analog N-methyl-L-serine methyl ester is reacted with triphenylmethyl chloride to make N-methyl-N-trityl-L-serine methyl ester The N-methyl-N-trityl-L-serine methyl ester is cyclooligomerized to N- methyl-L-serine trilactone in refluxing dry xylene and 2,2-dibutyl-1,3,2- dioxastannolane

14. Specific Aim 2: Trimerization of N- methyl-L-serine methyl ester to make enterbactin analog The trityl protecting groups are removed with anhydrous HCl to make the trimer salt The trimer salt is reacted with 2,3-dibenzyloxybenzoyl chloride to make N-methyl-hexabenzylenterobactin

15. Specific Aim 2: Trimerization of N- methyl-L-serine methyl ester to make enterbactin analog Hydrogenolysis on Pd-C produces N-methyl enterobactin

16. Specific Aim 3: NMR Analysis Reveal Conformation of N-methyl Enterobactin Analog The two possible conformations of enterobactin are the pseudoaxial and pseudoequatarial forms and they have different splitting patterns for the seryl protons Since enterobactin exhibits C 3 symmetry, the conformation of the siderophore can be elucidated by analysis of the spin- spin coupling of alpha-methine (H α ) and beta-methylene protons (H β1 and H β2 ) on the seryl units by 1 H NMR.

18. Specific Aim 3: NMR Analysis Reveal Conformation of N-methyl Enterobactin Analog 1 H NMR spectrum of Enterobactin backbone

19. Conclusions Any conformational changes in enterobactin can be speculated by the spin-spin coupling constants of alpha- methine(H α ) and beta-methylene protons (H β1 and H β2 ). Also, any conformational changes would suggest that the pre-organization of the structure has been interfered. Thus, if the insertion of the methyl groups between the trilactone backbone and the N-methyl groups interfered with the pre-organization of enterobactin, then our new N-methyl enterobactin analog would exhibit the H β2 as a pseudo triplet and the H β1 would display doublets of doublets, the proton NMR pattern for pseudoequatorial conformation.

20. Thank You! Any questions?