Templated alkylation of hexahistidine function in recombinant proteins

Chemical functionalization of proteins is a common approach toward their utilization in a plethora of functional assemblies. It is extensively used in the study of protein structure, labelling of proteins, their immobilization, drug targeting, modification of immunological properties, bioanalytics, and other applications. 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. 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 and lysine result rarely provide well-defined protein conjugates. To overcome this problem other approaches involving site-specific incorporation of unnatural amino acids, introduction of aldehyde tags into proteins by the formylglycine generating enzyme, and native chemical ligation were tried. To increase versatility, protein bioconjugates are prepared in two stages through attachment of a reactive functionality that is capable of highly selective subsequent transformations such as biotinylation. Alternatively, proteins are functionalized with groups capable of highly specific bioorthogonal reactions such as cycloaddition or “click reactions” with complementary components attached to a small molecule or another protein. However, preparation of well-defined conjugates from an arbitrary protein remains a considerable challenge.

Approach for site selective derivatization of recombinant proteins

Our approach involves tethering of NTA functionality to Baylis-Hillman group through the ester group using a flexible linker. In that case formation of Ni²⁺ ternary complex between hexahistidine fragment and NTA would bring the reactive double bond of Baylis-Hillman ester group into the vicinity of one of imidazole rings that do not form the coordination bond with Ni²⁺ cation. The resultant addition-elimination process would form a C-N covalent bond while breaking linkage with NTA function leaving the ArCH=CH(CO₂Me)CH₂- moiety attached to hexahistidine residue.

The reaction of alkylation can be used for introduction of a "sticky end" group such as alkynyl which can be a versatile substrate for a subsequent bioorthogonal reaction to introduce variety of functionalities. To prove this approach a sample of recombinant Protein A possessing a hexahistidine tag was alkylated with alkynyl carrying ester 7b to perform two stage derivatization with a fluorescent functionality. On the first stage Protein A (16 M) was treated with 2 eq. of ester 7b, 6 eq. of N-methylimidazole, and 2 eq. of either Cu2+ or Zn2+ cations to produce alkynyl functionalized protein 13. To determine the rate of possible intermolecular alkylation of Protein A of the hexahistidine sequence, or of any other reaction site, a control experiment in the absence of these metal cations was also performed.

Derivatizations of Protein A 12 with an alkynyl function through the alkylation with ester 7b followed by a "click" cycloaddition with fluorescein azide 14 to produce fluorescent protein 15. Left bottom: scan of fluorescent image of gel electrophoresis of modified Protein A.

The hexahistidine group can be alkylated in aqueous solutions at micromolar concentrations of reagents assembled from Baylis-Hillman esters tethered to nitrolotriacetate function through a flexible linker. The reaction is templated by formation of quaternary complex composed from these reactants, a monodentate ligand such as N-methylimidazole, and a metal cation such as Ni²⁺, Cu²⁺, or Zn²⁺. While currently achieved alkylation time are currently higher than in the existing bioconjugation methods, this reaction can be a promising way for specific derivatization of recombinant proteins possessing a hexahistidine tail through introduction of an alkynyl function that can be used for subsequent bioorthogonal cycloaddition reactions. Studies of this versatile approach are currently being extended to development of new alkylating agents to perform derivatizations of recombinant proteins with quantitative yield using stoichiometric amounts of reagents under low micromolar concentrations in vitro and in vivo.

Department of Chemistry & Biomolecular Science

Templated alkylation of hexahistidine function in recombinant proteins

Clarkson University