Selective Functionalization of a Genetically Encoded Alkene-Containing Protein via “Photoclick Chemistry” in Bacterial Cells

Abstract

We report a tetrazole-based, photoclick chemistry that can be employed to selectively functionalize an alkene genetically encoded in a protein inside E. coli cells. The reaction involved the treatment of E. coli cells with cell-permeable tetrazoles followed by a brief photo irradiation at 302 nm (4 min) and an overnight incubation at 4 degrees C. This in vivo alkene functionalization procedure was simple, straightforward, and nontoxic to E. coli cells. Additionally, fluorescent adducts were formed, facilitating the monitoring of the reaction in vivo. This reaction should offer a new tool for the study of alkene-containing proteins in living systems.

Enzyme immobilization: an update

“…Another related family of reactions is "photoclick chemistry," which relies on photoirradiation to trigger pyrazoline formation between tetrazoles and alkenes [33]. An added advantage of this method is that the newly formed heterocycle is fluorescent, enabling the monitoring of the reaction progress.…”

Selective chemical protein modification

“…Their generation requires irradiation with ultraviolet light (this is termed a 'photo-click'). The reaction has been used to modify a number of alkenyl-UAAs site specifically, such as homoallylglycine 112 and cyclopropenes 113 , while the genetic incorporation of tetrazoles has also been achieved as a reactive handle for undertaking 'photo-click' reactions 114 . While the rates of reaction are now approaching levels seen for norbornene-tetrazine conjugations, the reaction is still somewhat slower than cyclooctene-tetrazine reactions.…”

Protein Engineering with Non-Natural Amino Acids

“…However, the reaction requires an alkene (such as trans-cyclooctene or norbornene) with much more strain than NNAAs that have been incorporated in vivo (such as homoallylglycine). One solution has been to use a tetrazole that can be photoactivated to produce a nitrile imine intermediate that then reacts with unactivated alkenes (Song et al, 2008). This has been used successfully with a Zdomain protein containing O-allyl-Tyr expressed from E. coli.…”