Here we have shown that ONOO− reacts directly with boronic compounds yielding the corresponding hydroxyl derivatives (phenols or alcohols) as final products. As compared to reactivity between ONOO− and other small organic molecules, the bimolecular rate constant (k = 106 M−1 s−1) measured for the reaction between ONOO− and boronates is very high. This high reactivity of boronic compounds toward ONOO−, as compared with other oxidants, makes them attractive candidates as potential traps and fluorescent probes for cellular imaging of ONOO−. From product analyses and substrate consumption studies, we conclude that boronates react with ONOO− at a 1:1 stoichiometry, yielding the corresponding phenol as a major product (80-85%) and possibly free radical intermediates and radical-derived products as minor products (<20%). We propose the reaction scheme in which the initial reaction occurs with the formation of the ONOO− adduct to the boronic compound that subsequently decomposes predominantly via a nonradical pathway forming the phenolic product (). HOCl and H2O2 react stoichiometrically with boronates yielding the corresponding phenol (), although at much slower rates as compared to ONOO− ().
Proposed mechanism of oxidation of boronates by ONOO−, HOCl, and H2O2
Studies with X/XO and NONO-ates indicate that neither O •2− nor •NO react with boronates. We verified that nitrogen dioxide alone doesn’t convert the boronic compounds into phenols. ONOO−-mediated oxidation of PBE to 4′-hydroxyacetophenone was enhanced in the presence of low concentrations of GSH, and moderately attenuated at high concentrations of GSH (). The GSH-mediated increase in 4′-hydroxyacetophenone formation was oxygen-dependent and CAT-independent. This suggests that the reactive species derived from glutathionyl radical and molecular oxygen, but not glutathionyl radical per se, can oxidize boronic compounds to the corresponding phenols. The lack of the CAT effect also excludes the possible involvement of H2O2 that could be produced via the reaction of glutathiyl radical (GS•) radical with glutathiolate anion (GS−), with formation of glutathione disulfide radical anion (GSSG•−) and subsequent oxygen reduction leading to O •2− radical anion that dismutates to H2O2. Enhanced production of 4′-hydroxyacetophenone from PBE was also observed in incubations containing HCO −and GSH, possibly due to formation of GS• 3 radical.
Boronates react with ONOO−
nearly a million times faster than with H2
. The boronic acid group, wherein the boron atom is sp2
-hybridized, is a very strong electrophile, and its reaction with a powerful nucleophile, ONOO−
, is energetically favored. Nearly four decades ago, Keith and Powell reported that the decomposition of ONOO−
was increased in borate buffer, which was attributed to a transperoxidation reaction between ONOO−
and borate, forming a peroxyborate intermediate [28
]. Recently, it was shown that peroxymonophosphate reacts with boronates much faster than does H2
]. In the case of oxidants studied in this paper, reaction timescales ranged from milliseconds (for ONOO−
), seconds (for OCl−
), and hours (for hydroperoxide anion, HOO−
) (, ). At physiological pHs (=7.4), the percentage of HOO−
is 0.005% as compared to ONOO−
(83%) and OCl−
(46%). This is consistent with the pKa
’s of H2
(11.7), ONOOH (6.7), and HOCl (7.47). This may partially explain the differences in the observed second-order rate constants for these species with boronates. Although HOCl reacts with boronates a hundred to a thousand times slower than ONOO−
, this reaction may still be viable, because of its increased chemical stability. However, due to more rapid electrophilic reactions of HOCl with endogenous amines and thiols, it is rather unlikely that the boronic compounds can effectively compete for HOCl in the cellular systems. Moreover, the specific scavengers of HOCl (e.g., taurine) can be used to identify the nature of intracellular oxidant responsible for boronate oxidation.
The conversion of phenylboronic esters by ONOO−
, HOCl, and H2
to the corresponding phenolic product is shown in . The initial step of the reaction involves a bimolecular collision between the electrophilic boronate and the nucleophilic anion (ONOO−
). With H2
and HOCl, nearly a 100% conversion of the boronate to the corresponding phenolic product occurs (). Additional products were not formed with H2
; however, as shown in , higher concentrations of HOCl caused a rapid decrease in the levels of phenolic product. This is attributed to formation of the corresponding chlorinated phenol. In the case of ONOO−
, the yield of the phenolic product was about 85% (). The published reports on the reaction between ONOO−
and carbonyl compounds and carbon dioxide indicate that the adducts formed decompose by a nonradical and radical pathway [13
]. It is likely that a similar radical-mediated minor decomposition pathway occurs for the boronate/ONOO−
adduct. Formation of dityrosyl type products strongly implicates a role for radical-mediated decomposition. We are currently investigating the radical-mediated minor pathway in detail. We believe that the proposed reaction pathway between boronates and ONOO−
, and HOO−
is quite general, and should be applicable to many other boronates. The rapid direct reaction between arylboronates and ONOO−
, as compared to H2
, coupled with a nearly stoichiometric conversion of the adduct into phenolic product, make boronate-based fluorescent probes ideal candidates for cellular imaging of ONOO−
. Potential boronate-based fluorescence probes, some of which have already been reported in the literature [20
], are shown in Figure S1
. It is likely that fluorescein-based boronates will be eminently suitable for detection and imaging of cellular ONOO−
due to their high fluorescent quantum yields and suitable excitation/emission characteristics.
In conclusion, we have shown that several oxidants (ONOO−, HOCl, H2O2) react stoichiometrically at different rates with boronates, a class of organic compounds, which can be incorporated into several fluorescent probes, to generate the corresponding phenols. The present study opens up new possibilities for quantitative detection and inhibition of ONOO− in cells by using boronate-containing probes in biological systems.