How Lipid Unsaturation,
Peroxyl Radical Partitioning,
and Chromanol Lipophilic Tail Affect the Antioxidant Activity of α-Tocopherol:
Direct Visualization via High-Throughput Fluorescence Studies Conducted
with Fluorogenic α-Tocopherol Analogues
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Abstract
The preparation of two highly sensitive fluorogenic α-tocopherol
(TOH) analogues which undergo >30-fold fluorescence intensity enhancement
upon reaction with peroxyl radicals is reported. The probes consist
of a chromanol moiety coupled to the <i>meso</i> position
of a BODIPY fluorophore, where the use of a methylene linker (BODIPY-2,2,5,7,8-pentamethyl-6-hydroxy-chroman
adduct, H<sub>2</sub>B-PMHC) vs an ester linker (<i>meso</i>-methanoyl BODIPY-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
acid, H<sub>2</sub>B-TOH) enables tuning their reactivity toward H-atom
abstraction by peroxyl radicals. The development of a high-throughput
fluorescence assay for monitoring kinetics of peroxyl radical reactions
in liposomes is subsequently described where the evolution of the
fluorescence intensity over time provides a rapid, facile method to
conduct competitive kinetic studies in the presence of TOH and its
analogues. A quantitative treatment is formulated for the temporal
evolution of the intensity in terms of relative rate constants of
H-atom abstraction (<i>k</i><sub>inh</sub>) from the various
tocopherol analogues. Combined, the new probes, the fluorescence assay,
and the data analysis provide a new method to obtain, in a rapid,
parallel format, relative antioxidant activities in phospholipid membranes.
The method is exemplified with four chromanol-based antioxidant compounds
differing in their aliphatic tails (TOH, PMHC, H<sub>2</sub>B-PMHC,
and H<sub>2</sub>B-TOH). Studies were conducted in six different liposome
solutions prepared from poly- and mono-unsaturated and saturated (fluid
vs gel phase) lipids in the presence of either hydrophilic or lipophilic
peroxyl radicals. A number of key insights into the chemistry of the
TOH antioxidants in lipid membranes are provided: (1) The relative
antioxidant activities of chromanols in homogeneous solution, arising
from their inherent chemical reactivity, readily translate to the
microheterogeneous environment at the water/lipid interface; thus
similar values for <i>k</i><sub>inh</sub><sup>H<sub>2</sub>B‑PMHC</sup>/<i>k</i><sub>inh</sub><sup>H<sub>2</sub>B‑TOH</sup> in the range of 2–3 are recorded both in homogeneous solution
and in liposome suspensions with hydrophilic or lipophilic peroxyl
radicals. (2) The relative antioxidant activity between tocopherol
analogues with the same inherent chemical reactivity but bearing short
(PMHC) or long (TOH) aliphatic tails, <i>k</i><sub>inh</sub><sup>PMHC</sup>/<i>k</i><sub>inh</sub><sup>TOH</sup>, is
∼8 in the presence of hydrophilic peroxyl radicals, regardless
of the nature of the lipid membrane into which they are embedded.
(3) Antioxidants embedded in saturated lipids do not efficiently scavenge
hydrophilic peroxyl radicals; under these conditions wastage reactions
among peroxyl radicals become important, and this translates into
larger times for antioxidant consumption. (4) Lipophilic peroxyl radicals
show reduced discrimination between antioxidants bearing long and
short aliphatic tails, with <i>k</i><sub>inh</sub><sup>PMHC</sup>/<i>k</i><sub>inh</sub><sup>TOH</sup> in the range
of 3–4 for most lipid membranes. (5) Lipophilic peroxyl radicals
are scavenged with the same efficiency by all four antioxidants studied,
regardless of the nature of their aliphatic tail or the lipid membrane
into which they are embedded. These data underpin the key role the
lipid environment plays in modulating the rate of reaction of antioxidants
characterized by similar inherent chemical reactivity (arising from
a conserved chromanol moiety) but differing in their membrane mobility
(structural differences in the lipophilic tail). Altogether, a novel,
facile method of study, new insights, and a quantitative understanding
on the critical role of lipid diversity in modulating antioxidant
activity in the lipid milieu are reported