Modular synthesis of cell-permeating 2-ketoglutarate esters

Abstract

Cell-permeating esters of 2-ketoglutarate (2-KG) have been synthesized through a convergent sequence from two modules in two and three steps, respectively. This route provides access to a full series of mono- and disubstituted 2-KG esters, enabling us to define the effect of regioisomeric masking on metabolite release and antihypoxic activity in cell-based assays. In addition to providing insight into the biological activity of cell permeable 2-KG esters, the straightforward and modular nature of this synthetic route may prove useful for the development of next-generation 2-KG analogues for diagnostic and therapeutic applications.

Figure 1.

(a) 2-Ketoglutarate and cell-permeable 2-KG esters. (b) Retrosynthetic analysis of 2-KG esters.

Figure 2.

(a) Schematic for cell-based assay of antihypoxic effects of 2-KG esters. A cell line stably expressing a HIF-luciferase gene fusion is treated with cell permeable 2-KG esters. Literature precedent suggests delivery of 2-KG can increase the activity of 2-KG dependent dioxygenases, including PHD2, the prolyl hydroxylase responsible for HIF degradation. (b) Quantitative analysis of cellular antihypoxic activity of 2-KG esters (1 mM). All values are normalized to the average HIF-Luciferase activity of untreated control cells (set at 100%). Compounds 1 and 3 reduce HIF-Luc luciferase activity, consistent with 2-KG release and dioxygenase activation. Values represent the average of three biological replicates, ± standard deviation.

Scheme 1.

Synthesis of Regiospecifically Protected KG Esters 1−3