Solid phase synthesis of mitochondrial triphenylphosphonium-vitamin E metabolite using a lysine linker for reversal of oxidative stress

Abstract

Mitochondrial targeting of antioxidants has been an area of interest due to the mitochondria's role in producing and metabolizing reactive oxygen species. Antioxidants, especially vitamin E (α-tocopherol), have been conjugated to lipophilic cations to increase their mitochondrial targeting. Synthetic vitamin E analogues have also been produced as an alternative to α-tocopherol. In this paper, we investigated the mitochondrial targeting of a vitamin E metabolite, 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (α-CEHC), which is similar in structure to vitamin E analogues. We report a fast and efficient method to conjugate the water-soluble metabolite, α-CEHC, to triphenylphosphonium cation via a lysine linker using solid phase synthesis. The efficacy of the final product (MitoCEHC) to lower oxidative stress was tested in bovine aortic endothelial cells. In addition the ability of MitoCEHC to target the mitochondria was examined in type 2 diabetes db/db mice. The results showed mitochondrial accumulation in vivo and oxidative stress decrease in vitro.

Figure 1. Solid phase synthesis of MitoCEHC (8).

Reagents and conditions: a) 20% piperidine, DMF. b) Fmoc-Lys[Mtt]-OH, HBTU, HOBt, DIPEA, DMF. c) 20% piperidine, DMF. d) (3-carboxyproppyl)TPP⁺, HBTU, HOBt, DIPEA, DMF. e) 94% DCM, 5% Tis, 1% TFA. f) α-CEHC, HBTU, HOBt, DIPEA, DMF. g) 95% TFA, 2.5% water, 2.5% Tis.

Figure 2. Mass Spectrometry and structure of MitoCEHC (8).

The MALDI-TOF Mass Spectrometry of the final product from resin cleavage shows a molecular weight peak at 736.39 m/z. In addition, the structure of MitoCEHC (8) was created using ChemDraw Ultra software, with a calculated m/z for C₄₄H₅₅N₃O₅P⁺ of 736.39 (100%), which corresponds to the Mass Spectrometry results.

Figure 3. Effect of MitoCEHC on lowering ROS.

ROS was measured via FACSCAN. The effect of 2 µM α-CEHC and 2 µM MitoCEHC on lowering ROS induced by high glucose in endothelial cells was tested 36 hours after treatment. MitoCEHC displays a higher significant effect on decreasing ROS than α-CEHC alone. Data are expressed as the percent of basal (5 mM glucose). Mean values were analyzed using one-way ANOVA with Tukey's posttest (*p<0.05, ***p<0.001).