Dissertation:
„Coordination properties of hydroxamic analogues of ferrichrome”
mgr Agnieszka Szebesczyk
Supervisor: prof. dr hab. Henryk Kozłowski
Co-supervisor: dr hab. Elżbieta Gumienna – Kontecka
The aim of the thesis was to determine the coordination characteristics of new analogues
of ferrichrome, one of siderophores. 16 compounds were analyzed, 9 of which were ferrichrome
analogues with a simplified structure. The hexapeptide ring, present in natural ferrichrome, was
replaced by tripodal anchoring template. Three arms, built of amino-acid and terminated with retro-hydroxamate moiety, were attached to the template. The arms differed in length, both by extending the amino acid as well as the template. Additionally, the arms were modified nearby the
hydroxamic groups by introducing methyl groups, amino groups at position α or β, or carboxylic
groups at β or γ position. The molecules were designed in order to identify the factors affecting
the stability of the complexes formed by analogues with FeIII ions, and to investigate the process of
recognition by receptors present in the bacterial cell membrane.
The remaining 7 compounds were monohydroxamates, with the same structure as single
arm of tripodal analogue. Analysis of such monomers aimed to determine the effect of introduced
modifications on the ability to form hydrogen bonds, which are important in the recognition process.
A number of physicochemical methods were used during the project. Mass spectrometry,
ESI-MS, was employed to identify the stoichiometry of formed complexes. Potentiometric titration
was used to determine the protonation constants of ligands. Stability constants of ferric complexes formed by studied ligands were determined by pH-metric titrations, both potentiometric and
spectrophotometric. Also the competitive spectrophotometric titration with EDTA was used for
determination of stability constants of trihydroxamate-ferric complexes of tripodal analogues.
Cyclic voltammetry was used to identify the reduction and oxidation potentials of complexes FeIIIanalog → FeII-analog → FeIII-analog. Investigations of bacterial growth stimulation were performed
in the laboratory of prof. Yitzhak Hadar in Rehovot, Israel.
The values of protonation constants, determined for the hydroxamic groups for both trihydroxamic and monhydroxamic ligands, fall in the range typical for such groups. The comparison of
the results for both types of compounds allowed to conclude, that the presence of carboxyl groups
in the trihydroxamic ligands, both in the β and γ position, results in the intramolecular interactions, such as van der Waals forces or hydrogen bonds. The intramolecular interactions caused the
increase of the protonation constants values of both carboxylic and hydroxamic groups. Such effect was not observed for monohydroxamates.
The results of ESI-MS studies indicates, that monohydroxamic ligands form ferric complexes with the metal-to-ligand stoichiometry 1:1, 1:2 and 1:3, while trihydroxamic analogues form
ferric complexes with stoichiometry 1:1.
The values of stability constants obtained for ferric complexes of monohydroxamates,
along with their spectroscopic parameters, are typical for this kind of compounds. Additionally, the
calculated pFe values (pFe = -log[FeIII]aq dla cFe = 1·10-6 M, Fe : ligand 1:10) lie in the range of 15.7 –
15.9, and are similar to e.g. pFe for acetohydroxamic acid, which is 15.7.
The values of stability constants obtained for ferric complexes of trihydroxamates reflect
the impact of the modifications of molecules on their coordination properties. The presence of
amino groups, which are charged in a wide range of pH, affects the formation of complexes with
FeIII ions not only by charge repulsion, but also by the introduction of steric hindrance. The effect
of amino group on the formation and stability of complexes is significantly greater, when it is in α
position with respect to the hydroxamic group.
The carboxylic groups demonstrate greater effect on the formation of ferric complexes.
Despite the introduction of desired hydrogen bonds, they disturb the process of formation of trihydroxamate-ferric complex by competition in binding FeIII ions. Only the trihydroxamic complexes
can be recognized by bacterial cell membrane receptors. In the case of compounds with carboxyl
groups, trihydroxamic complexes can be formed only after the deprotonation of the third hydroxamic group. Therefore, the trihydroxamate-ferric complexes of ligand, which has -COOH group at
position β relative to the hydroxamic groups, predominate the solution only above pH 7.3. As
a result, in the pH range in which bacteria exist, there still will be mixed complexes with two hydroxamates and one carboxyl involved in the metal ion coordination, and such complex cannot be
recognized by the receptor. Less significant impact on the stability constants was presented by the
modifications of the length of the arms. pFe values for ferric complexes of ligands with different
arm lengths are less than two orders of magnitude lower than those for natural ferrichrome. For
ligands with amino groups, these differences reach three orders of magnitude, and for carboxylate
analogues pFe values are even seven orders of magnitude (for ligand with –COOH in β position)
lower than for ferrichrome. However, even the pFe values which differ significantly from the one
for ferrichrome, fall within the range for natural siderophores, which indicates that the studied
compounds may be considered as biomimetic ferrichrome analogs.
The studies of bacterial growth stimulation demonstrated, that some of the analyzed compounds caused the growth of colonies of P. putida to the same extent as natural ferrichrome.
Moreover, ferric complexes of some analogues were recognized only by P. putida, and some also
by E. coli. Broad or narrow recognition is important for the further research and use of the new
biomimetic ferrichrome analogues, e.g. as a bacteria concentration factor, used to increase the
sensitivity of the tests detecting the presence of bacteria, or as a part of the system used to identify specific bacterial strain causing the infection.