As a result of limited therapeutic accumulation within mitochondria, targeting the mitochondria with antioxidants or therapeutics has been a major interest especially for cardiovascular disease and cancer. Small molecules can permeate through the mitochondrial outer membrane but fail to cross the inner membrane. Taking advantage of the high inner membrane potential gradient, lipophilic cations can easily accumulate within the mitochondria as well as permeate the phospholipid bilayers. Vitamin E conjugated to TPP + can accumulate into the mitochondria, where it decreases ROS more effectively than vitamin E alone, and is able to ameliorate oxidative stress-mediated disease. While conjugating vitamin E to TPP + has been previously described, our goal was to conjugate the vitamin E metabolite, a-CEHC, to TPP+ and to design a fast and efficient synthetic method using a lysine linker and solid phase synthesis. This method does not require isolation of synthetic intermediates, while reagents and by-products are washed away after each step. In addition, similar to trolox, a-CEHC contains the a-tocopherol ring structure but have a truncated side chain with one carbon longer than trolox. The chroman ring of vitamin E becomes redox active at the mitochondria, where it forms semiquinone after detoxifying a free radical via hydrogen donation. The semiquinone is further reduced by intramitochondrial ascorbic acid or by electron donation. The chroman ring is still intact in aCEHC when conjugated to TPP +. A lysine linker with two protecting groups was used, which enabled the conjugation of TPP + and a-CEHC. The masked lysine was coupled onto the Rink Amide MBHA resin. HBTU and HOBt were used to enhance the coupling rate. The Fmoc was then deprotected to allow for TPP + conjugation through its carboxylic acid group forming an amide bond. The Mtt protecting group was then removed. The removal of the protecting group enabled the carboxylic acid on a-CEHC side chain to form an amide bond with the lysine linker. The final product, TPP + -Lysine-a-CEHC, was then released from the resin via treatment with 95% TFA. The final product was characterized by MALDI-TOF mass spectrometry. The molecular weight peak was at 736.39, which corresponds to the expected peak for the MitoCEHC generated by ChemDraw software. The mass spectrometry data also shows virtually no trace of by-products, reagents or synthetic intermediates. The ability of final product to diminish oxidative stress was examined in vitro. Oxidative stress is defined as the overproduction of oxidizing chemical species and the failure to eradicate their excess by enzymatic or non-enzymatic antioxidants. Elevation in ROS production is a factor in the etiology of cardiovascular disease by modifying lipids, proteins, and nucleic acids. To further explore the antioxidant activity of the conjugated MitoCEHC, the oxidation of CM-H2DCFDA was measured. The H2DCFDA derivative with a thiol-reactive chloromethyl group was used due to its better retention in live cells than H2DCFDA. This derivative is retained better in cells because of its ability to bind covalently to intracellular components. BAEC were incubated with low and high glucose concentrations. The cells incubated under hyperglycemic conditions showed an increase in ROS production, which is mainly in the mitochondria. Flow cytometry data also showed decrease in ROS production in the hyperglycemic cells treated with MitoCEHC.

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