Transcript Reina-Chu - California State University, Long Beach

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
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 [Fe3+] to about 10-18 M under
normal physiological conditions.
• Bacteria require 105 to 106 ferric ions per cell
per generation.
– This continuous demand for ferric ions led bacteria
to develop mechanisms to acquire iron.
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 1049.
– It has the highest affinity for iron than any other
natural substance!
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 (2,3dihydroxybenzoyl)
Enterobactin is Predisposed for Metal
Binding
• Hydrogen bonding
locks the catechol group
into one of two rigid
conformations, the
interconversion of which
is triggered by
deprotonation/metal
complexation.
• 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
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.
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 106 less stable than
enterobactin.
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.
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 preorganization of enterobactin.
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 preorganization of the free ligand structure, triggering the
catecholate legs to move equatorially.
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).
Specific Aim 1: Reduction Animation
Reduction animation of L-serine methyl ester to yield N-methyl-L-serine methyl ester
Specific Aim 2: Trimerization of Nmethyl-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 Nmethyl-L-serine trilactone in refluxing dry xylene and 2,2-dibutyl-1,3,2dioxastannolane
Specific Aim 2: Trimerization of Nmethyl-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
Specific Aim 2: Trimerization of Nmethyl-L-serine methyl ester to make
enterbactin analog
Hydrogenolysis on Pd-C produces N-methyl enterobactin
Specific Aim 3: NMR Analysis Reveal
Conformation of N-methyl Enterobactin
Analog
•Since enterobactin
exhibits C3 symmetry,
the conformation of
the siderophore can
be elucidated by
analysis of the spinspin coupling of
alpha-methine (Hα)
and beta-methylene
protons (Hβ1 and Hβ2 )
on the seryl units by
1H NMR.
• The two possible conformations of enterobactin are the pseudoaxial
and pseudoequatarial forms and they have different splitting
patterns for the seryl protons
Specific Aim 3: NMR Analysis Reveal
Conformation of N-methyl Enterobactin
Analog
1H
NMR spectrum of Enterobactin backbone
Conclusions
• Any conformational changes in enterobactin can be
speculated by the spin-spin coupling constants of alphamethine(Hα) and beta-methylene protons (Hβ1 and Hβ2 ).
• 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 Hβ2 as a
pseudo triplet and the Hβ1 would display doublets of
doublets, the proton NMR pattern for pseudoequatorial
conformation.
Thank You!
Any questions?