Chemical functionalization of proteins is a common approach toward their utilization in a plethora of functional assemblies. Availability of recombinant proteins that can be expressed and purified using standard methodologies makes essentially any protein with known amino acid sequence available through routine protocols of expression of recombinant DNA vectors. In contrast, due to presence of multiple residues of amino acids in any protein, conventional methods producing protein conjugates through reactions of side chains of cysteine or lysine rarely provide well-defined protein conjugates. To get selective derivatization of recombinant proteins our approach involves utilization of histidine tag as the site for covalent modification using the unique ability of the hexahistidine tag to form metal complexes that serve as a template for subsequent alkylation of a histidine residue in the tag with a Baylis-Hillman ester function.This approach was implemented using two step one-pot transformation. One the first step, hexahistidine tag of a recombinant protein is functionalized with an alkynyl group. This process proceeds in neutral aqueous solution under micromolar concentrations of reagents. It is specific to hexahistidine tag and no appreciable reaction of other amino acid residues on the protein takes place. On the second step, the alkynyl group is further derivatized through a conventional 'click' 1,3-dipolar cycloaddition with an azide functionalized counterpart. Due to presence of hexahistidine tag in the majority of recombinant proteins, selectivity of its derivatization, and mild reaction conditions the approach can be general method for functionalization of diverse recombinant providing versatile and highly predictable method for production of protein conjugates.

Specific derivatization of hexahistdine tag in recombinant proteins by Baylis-Hillman esters

248th National Meeting (2014)

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