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Open Access Publications from the University of California

Streptavidin as a Host for Copper(II) Complexes

  • Author(s): Paretsky, Jonathan Daniel
  • Advisor(s): Borovik, Andrew S
  • et al.

In nature, metal ions are utilized in many proteins and enzymes in order to perform a variety of chemical transformations that are essential to maintaining human life. The range of selectivity and functionality can be attributed to the unique environment surrounding each active site, which allow for control over not only the primary coordination sphere—containing atoms covalently bound to the metal ion, but also the secondary coordination sphere (microenvironment)—made up of non-covalent interactions such as hydrogen bonds (H-bonds). Although nature is very efficient at these chemical processes, achieving the same level of selectivity is difficult in synthetic systems. In order to reproduce the functions found in metalloproteins it is necessary to maintain control over both the primary and secondary coordination spheres of synthetic systems as seen within active sites. To accomplish this, typically a rigid organic framework is constructed that will coordinate to a metal ion and additionally provide a supporting network of non-covalent interactions: intramolecular H-bonds.

The Borovik group has performed extensive research the area of secondary coordination sphere effects around transition metal complexes and has developed a multitude of ligand frameworks that bind metal ions and incorporate intramolecular H-bond donors or acceptors that can stabilize exogenous ligands bound to the transition metal (TM) complexes. While these systems have been extremely effective and have been used to prepare unique high-valent metal-oxo/hydroxo species, they fall short in being able to provide extensive networks of H-bonds and long-range interactions found in microenvironments of metalloproteins. To address these challenges and gain further control over the secondary coordination sphere, this dissertation presents the development of new research program within the Borovik group in collaboration with the Ward lab: the development of artificial metalloproteins using biotin-Streptavidin (Sav) technology. Although Sav does not naturally contain metal ions, TM complexes of interest can be attached to biotin and inserted into the protein. This is an attractive feature, because of Sav’s exceptionally high affinity for biotin , which can be exploited for the use of installing biotinylated metal complexes. Furthermore, the selective binding of biotin provides a reproducible anchor for TM complexes within the active site of protein. In addition to being able to chemically modify a ligand, the protein is amenable to site directed mutagenesis, which allows for alterations of the microenvironment around the TM complexes. This combined chemogenetic approach allows for enhanced control over primary and secondary coordination spheres of TM complexes.

The first portion of this document describes the study of biotinylated copper(II) complexes with varied linker lengths between the Cu(II) complex and biotin. Utilizing different linker lengths was proposed to allow for: 1) to design discrete monomeric species confined within separate subunits or 2) to create a bimetallic systems at the dimer interface. Additionally, different mutants of Sav varying the amino acid residue at a site proximal to the Cu(II) complex were investigated to observe the protein effects on the secondary coordination sphere of the TM complex. Changing the serine at the112 position to either lysine, aspartate, or cysteine had drastic effects on the microenvironment of the Cu(II) complex. This was studied through Uv-Vis and electronic paramagnetic resonance (EPR) spectroscopies, and X-ray diffraction studies (XRD). XRD studies explicitly showed the molecular structures of the Cu(II) complexes bound within Sav and the effects of linker length changes and site directed mutagenesis. This research has shown proof of concept that Cu(II) complexes can be prepared within Sav and different microenvironments can be reproducibly attained with different linker lengths and protein mutants, and my work has established this methodology as a promising new research program within the group.

Another way of harnessing the dimer-of-dimers nature of Sav would be to prepare a TM complex linked to two biotin molecules to potentially bridge across two subunits of the protein. As reproducibility and rigidity of a binding environment is essential to selectivity in chemical transformations, this was envisioned as a method of firmly anchoring a TM complex within the protein to be studied further. To probe this hypothesis a bis-biotinylated Schiff-base was prepared and its binding to Sav studied using HABA titrations and gel electrophoresis. It was shown that instead of bridging two subunits in a homotetramer, the ligand and its complexes bridged separate Sav molecules to form oligomers of proteins.

Prior to developing the biotin-Sav program, I investigated the use of well-established tripodal, tetradentate ligands within the Borovik lab. Previous research within the lab had shown that the Fe(II) complexes of these ligands were capable of activating aryl azides to form Fe(III)-amido compounds via a putative Fe(IV)-imido intermediates. It was proposed that this would be amenable to a catalytic system wherein a C¬–H bond within an aryl azide substrate could be activated by an Fe(IV)-imido and I anticipated subsequent radical rebound would form a new C–N bond. However, the system did not demonstrate the ability to perform C–H amination.

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