Synthesis of ¹³C-methyl-labeled amino acids and their incorporation into proteins in mammalian cells

Isotopic labeling of methyl-substituted proteinogenic amino acids with ¹³C has transformed applications of solution-based NMR spectroscopy and allowed the study of much larger and more complex proteins than previously possible with ¹⁵N labeling. Procedures are well-established for producing methyl-labeled proteins expressed in bacteria, with efficient incorporation of ¹³C-methyl labeled metabolic precursors to enable the isotopic labeling of Ile, Val, and Leu methyl groups. Recently, similar methodology has been applied to enable ¹³C-methyl labeling of Ile, Val, and Leu in yeast, extending the approach to proteins that do not readily fold when produced in bacteria. Mammalian or insect cells are nonetheless preferable for production of many human proteins, yet ¹³C-methyl labeling using similar metabolic precursors is not feasible as these cells lack the requisite biosynthetic machinery. Herein, we report versatile and high-yielding synthetic routes to ¹³C methyl-labeled amino acids based on palladium-catalyzed C(sp³)-H functionalization. We demonstrate the efficient incorporation of two of the synthesized amino acids, ¹³C-γ2-Ile and ¹³C-γ1,γ2-Val, into human receptor extracellular domains with multiple disulfides using suspension-cultured HEK293 cells. Production costs are reasonable, even at moderate expression levels of 2-3 mg purified protein per liter of medium, and the method can be extended to label other methyl groups, such as ¹³C-δ1-Ile and ¹³C-δ1,δ2-Leu. In summary, we demonstrate the cost-effective production of methyl-labeled proteins in mammalian cells by incorporation of ¹³C methyl-labeled amino acids generated de novo by a versatile synthetic route.