Esterification of an Unnatural Amino Acid Structurally Deviating from Canonical Amino Acids Promotes Its Uptake and Incorporation into Proteins in Mammalian Cells

Abstract

Genetically encoded unnatural amino acids (UAAs) enable novel chemical and physical properties to be selectively introduced into proteins directly in live cells, which provides great potential for addressing biological questions at the molecular and cellular level in native settings.[1-3] UAAs have been genetically incorporated into proteins in mammalian cells using orthogonal tRNA-codon-synthetase sets,[4-6] yet the current low incorporation efficiency hinders their effective application. Efforts to improve efficiency have focused on optimizing the expression and activity of the orthogonal tRNA and synthetase,[5-7] whereas the bioavailability of the UAA inside mammalian cells, a prerequisite for incorporation, has not been addressed. In addition, there are many UAAs that have not been genetically incorporated into proteins successfully, such as glycosylated and phosphorylated amino acids. These amino acids can be invaluable for studying the contribution of posttranslational modifications to protein function and the role of a target protein in cellular signal transduction. Among many reasons, the inability of the UAA to enter cells prevents evolving a mutant synthetase specific for the UAA using cell-based selections or screens.[2] Here we show that an UAA structurally deviating from the canonical amino acids in side chain could not be efficiently transported into mammalian cells, but masking the carboxyl group of the UAA as an ester greatly increased the rate of cellular uptake and intracellular concentration of the UAA. This resulted in a significant increase in the incorporation of this UAA into proteins in mammalian cells. Among three esters tested, acetoxymethyl ester (AME) yielded the highest UAA incorporation efficiency with a concomitant reduction of the UAA required in the growth media.