Synthetic studies of azulenyl and pseudoazulenyl nitrones

Free radicals have been implicated in various pathological conditions such as, stroke, aging and ischemic heart disease (IHD), as well as neurodegenerative diseases like Alzheimer’s, Parkinson’s, and Huntington’s disease. The role of antioxidants in protection from the harmful effects of free radicals has long been recognized. Trapping extremely reactive free radicals and eliminating them from circulation has been shown to be effective in animal models. Nitrone-based free radical traps have been extensively explored in biological systems. Examples include nitrones such as PBN, NXY-059, MDL-101,002, DMPO and EMPO. However, these nitrones have extremely high oxidation potentials as compared to natural antioxidants such as Vitamin E (α-tocopherol), and glutathione. Becker et al. (1995) synthesized novel azulenyl nitrones, which were shown to have oxidation potentials much lower than that of any of the previously reported nitrone based spin traps. Another azulenyl nitrone derivative, stilbazulenyl nitrone (STAZN), was shown to have an even lower oxidation potential within the range of natural antioxidants. STAZN, a second generation free radical trap, was found to be markedly superior than the two most studied nitrones, PBN and NXY-059, in animal models of cerebral ischemia and in an in vitro assay of lipid peroxidation. In this study, a third generation azulenyl nitrone was synthesized with an electron donating group on the previously synthesized STAZN derivative with the aim to lower the oxidation potential even more. Pseudoazulenes, because of the presence of an annular heteroatom, have been reported to possess even lower oxidation potential than that of the azulenyl counterpart. Therefore, pseudoazulenyl nitrones were synthesized for the first time by extracting and elaborating valtrate from the roots of Centranthus ruber (Red valerian or Jupiter’s beard). Several pseudoazulenyl nitrones were synthesized by using a facile experimental protocol. The physical and biological properties of these pseudoazulenyl nitrones can be easily modified by simply changing the substituent on the heteroatom. Cyclic voltammetry experiments have shown that these pseudoazulenyl nitrones do indeed have low oxidation potentials. The oxidation potential of these nitrones was lowered even more by preparing derivatives bearing an electron donating group at the 3-position of the five membered ring of the pseudoazulenyl nitrone.

Oxidative DNA Damage Modulates Trinucleotide Repeat Instability Via DNA Base Excision Repair

Trinucleotide repeat (TNR) expansion is the cause of more than 40 types of human neurodegenerative diseases such as Huntington’s disease. Recent studies have linked TNR expansion with oxidative DNA damage and base excision repair (BER). In this research, we provided the first evidence that oxidative DNA damage can induce CAG repeat deletion/contraction via BER. We found that BER of an oxidized DNA base lesion, 8-oxoguanine in a CAG repeat tract, resulted in the formation of a CTG hairpin at the template strand. DNA polymerase β (pol b) then skipped over the hairpin creating a 5’-flap that was cleaved by flap endonuclease 1 (FEN1) leading to CAG repeat deletion. To further investigate whether BER may help to shorten an expanded TNR tract, we examined BER in a CAG repeat hairpin loop. We found that 8-oxoguanine DNA glycosylase removed the oxidized base located in the loop region of the hairpin leaving an abasic site. Apurinic/apyrimidinic (AP) endonuclease 1 then incised the 5’-end of the abasic site leaving a nick in the loop. This further converted the hairpin into an intermediate with a 3’-flap and a 5’-flap. As a 5’-3’ endonuclease, FEN1 cleaved the 5’-flap, whereas a 3’-5’ endonuclease, Mus81/Eme1, removed the 3’-flap. The coordination between FEN1 and Mus81/Eme1 ultimately resulted in removal of a CAG repeat hairpin attenuating or preventing TNR expansion. To further explore if pol β bypass of an oxidized base lesion, 5’,8-cyclodeoxyadenosine, may affect TNR instability, we examined pol β DNA synthesis in bypassing this base lesion and found that the lesion preferentially induced TNR deletion during BER and Okazaki fragment maturation. The repeat deletion was mediated by the formation of a loop in the template strand induced specifically by the damage. Pol β then skipped over the loop structure creating a 5’-flap that was efficiently removed by FEN1 leading to repeat deletion. Our study demonstrates that pol β-mediated BER plays an important role in mediating TNR deletion and removing a TNR hairpin to prevent TNR expansion. Our research provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.

Synthetic Studies of Azulenyl and Pseudoazulenyl Nitrones

Free radicals have been implicated in various pathological conditions such as, stroke, aging and ischemic heart disease (IHD), as well as neurodegenerative diseases like Alzheimer’s, Parkinson’s, and Huntington’s disease. The role of antioxidants in protection from the harmful effects of free radicals has long been recognized. Trapping extremely reactive free radicals and eliminating them from circulation has been shown to be effective in animal models. Nitrone-based free radical traps have been extensively explored in biological systems. Examples include nitrones such as PBN, NXY-059, MDL-101,002, DMPO and EMPO. However, these nitrones have extremely high oxidation potentials as compared to natural antioxidants such as Vitamin E (á-tocopherol), and glutathione. Becker et al. (1995) synthesized novel azulenyl nitrones, which were shown to have oxidation potentials much lower than that of any of the previously reported nitrone based spin traps. Another azulenyl nitrone derivative, stilbazulenyl nitrone (STAZN), was shown to have an even lower oxidation potential within the range of natural antioxidants. STAZN, a second generation free radical trap, was found to be markedly superior than the two most studied nitrones, PBN and NXY-059, in animal models of cerebral ischemia and in an in vitro assay of lipid peroxidation. In this study, a third generation azulenyl nitrone was synthesized with an electron donating group on the previously synthesized STAZN derivative with the aim to lower the oxidation potential even more. Pseudoazulenes, because of the presence of an annular heteroatom, have been reported to possess even lower oxidation potential than that of the azulenyl counterpart. Therefore, pseudoazulenyl nitrones were synthesized for the first time by extracting and elaborating valtrate from the roots of Centranthus ruber (Red valerian or Jupiter’s beard). Several pseudoazulenyl nitrones were synthesized by using a facile experimental protocol. The physical and biological properties of these pseudoazulenyl nitrones can be easily modified by simply changing the substituent on the heteroatom. Cyclic voltammetry experiments have shown that these pseudoazulenyl nitrones do indeed have low oxidation potentials. The oxidation potential of these nitrones was lowered even more by preparing derivatives bearing an electron donating group at the 3-position of the five membered ring of the pseudoazulenyl nitrone.