A significant improvement of the efficacy of radical oxidant probes by the kinetic isotope effect.

The accurate and sensitive detection of radical oxidants is a central problem in the field of chemical biology. Radical oxidants can be detected in vitro with fluorescent leucodyes such as dihydroethidium (DHE), dihydrorhodamine (DHR), the hydrocyanines, redoxfluor-1, and their organelle-specific analogues. Although these probes are widely used in cell culture, their accuracy is compromised by their high levels of background fluorescence, which is caused by their spontaneous oxidation that is catalyzed by light or oxygen. Radical oxidants can be detected by using DHE, DHR, and the hydrocyanines, as they undergo an amine oxidation reaction with cellular oxidants, such as superoxide or hydroxyl radicals. However, the probes also generate background fluorescence by undergoing the same amine oxidation reaction with air and light; this reaction is often attributed to the effects of singlet oxygen (O2). [8] The mechanism of the amine oxidation differs significantly for reactions that involve either radical oxidants or singlet oxygen, and in particular, cleavage of the a-amine C H bond occurs at different points along the reaction coordinate for oxidation with these two oxidants. For example, amine oxidation by singlet oxygen proceeds via an exciplex intermediate, in which the C H bond cleavage occurs in the rate-determining step. This oxidation reaction therefore exhibits a relatively high kinetic isotope effect (KIE). For instance, the KIE for the oxidation of N,Ndimethyl([D2]benzyl)amine with singlet oxygen is 3.06 0.06. In contrast, a radical mechanism for amine oxidation proceeds through a sequence that involves either an electron transfer (ET)/proton transfer (PT) mechanism or a direct hydrogen atom transfer (HAT) mechanism, and generally has a lower KIE than oxidation with singlet oxygen. For example, the KIE for the oxidation of N,N-dimethyl([D2]benzyl)amine with the tert-butoxy radical (tBuOC) is only 1.4 0.7, and the KIE for the mechanistically similar oxidation of N,N-dimethyl([D2]benzyl)aniline with cytochrome P450 is 1.8 0.2. This difference in KIEs for the amine oxidation reaction between singlet oxygen and radical oxidants offers the possibility of selectively slowing down the aerial oxidation of radical oxidant probes while maintaining their reactivity with cellular radical oxidants. Herein we demonstrate that the efficacy of the commonly used radical oxidant probes, DHE (1), H-Cy3 (3), H-Cy5 (5), H-Cy7 (7), and DHR (9), can be dramatically improved by deuteration at their a-amine C H bond (Figure 1). Deuterated analogues of DHE (1), H-Cy3 (3), H-Cy5 (5), and H-Cy7 (7) have large KIEs (3.7–4.7) for aerial oxidation; however, their KIEs for oxidation with the superoxide radical anion are only between 2.5–2.8. This difference in KIEs causes the deuterated radical oxidant probes to generate less background fluorescence, but to still generate similar levels of fluorescence in cells that are stimulated to produce radical oxidants. Deuterated radical oxidant probes were significantly more accurate than their hydrogen analogues in the detection of radical oxidants in vitro, in cell culture, and in vivo. For example, the deuterated DHE analogue DDE had several advantages over its hydrogen analogue because of its lower background fluorescence. In particular, DDE had greater storage stability and higher accuracy than DHE, and was also used to detect radical oxidants produced in cell culture from angiotensin II (Ang II) stimulated rat aortic smooth muscle cells (RASM), whereas the oxidants were not detected by using DHE under identical experimental conditions. Similarly, D-Cy7 was also significantly better than its hydrogen analogue H-Cy7 (7) in the detection of radical oxidants in vivo because of its low background fluorescence. Based on these results, we anticipate numerous applications of deuterated radical oxidant probes in biology and medicine. Deuterated analogues of DHE (1), H-Cy3 (3), H-Cy5 (5), H-Cy7 (7), and DHR (9) were synthesized in excellent yields (> 93%) by reduction of commercially available ethidium bromide (11), Cy3 (12), Cy5 (13), Cy7 (14), and rhodamine (15) dyes, respectively, with sodium borodeuteride (see the Supporting Information). This reduction procedure specifically introduces a deuterium atom at the a-amine carbon atom of ethidium and the cyanines, and at the e-amine carbon atom of rhodamine. We investigated the stability of the deuterated probes 2, 4, 6, 8, and 10 to deuterium/hydrogen (D/H) exchange, and found them to be resistant to D/H [*] K. Kundu, S. Lee, Prof. W. R. Taylor, Prof. N. Murthy The Wallace H. Coulter Department of Biomedical Engineering and Parker H. Petit Institute of Bioengineering and Biosciences Georgia Institute of Technology Atlanta, GA 30332 (USA) Fax: (+1)404-894-4243 E-mail: [email protected]