Prochelators triggered by hydrogen peroxide provide hexadentate iron coordination to impede oxidative stress

Abstract

Prochelators are agents that have little affinity for metal ions until they undergo a chemical conversion. Three new aryl boronate prochelators are presented that are responsive to hydrogen peroxide to provide hexadentate ligands for chelating metal ions. TRENBSIM (tris[(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)-2-aminoethyl]amine), TRENBSAM (tris[(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl)-2-aminoethyl]amine), and TB (tris[(2-boronic acid-benzyl)2-aminoethyl]amine) convert to TRENSIM (tris[(salicylideneamino)ethyl]amine), TRENSAM (tris[(2-hydroxybenzoyl)-2-aminoethyl]amine), and TS (tris[2-hydroxybenzyl)2-aminoethyl]amine), respectively. The prochelators were characterized by (11)B NMR, and the structures of TRENBSAM, TRENBSIM, and the Fe(III) complex of TS were determined by X-ray crystallography. Of the three prochelator/chelator pairs, TB/TS was identified as the most promising for biological applications, as they prevent iron and copper-induced hydroxyl radical generation in an in vitro assay. TB has negligible interactions with metal ions, whereas TS has apparent binding constants (log K') at pH 7.4 of 15.87 for Cu(II), 9.67 Zn(II) and 14.42 for Fe(III). Up to 1 mMTB was nontoxic to retinal pigment epithelial cells, whereas 10 μM TS induced cell death. TS protected cells against H(2)O(2)-induced death, but only within a 1-10 μM range. TB, on the other hand, had a much broader window of protection, suggesting that it may be a useful agent for preventing metal-promoted oxidative damage.

Fig. 1

Hexadentate chelators (top) and prochelators (bottom).

Fig. 2

Side and top views of the crystal structures of TRENBSIM (A&B, respectively) and TRENBSAM (C&D) shown as stick diagrams. Hydrogen atoms have been omitted for clarity.

Fig. 3

¹¹B NMR pH titration of TB. Solutions of 10 mM TB with 25 mM HEPES in D₂O were probed by ¹¹B NMR. The red box on the left highlights peaks with a shift ~19 ppm. Peaks in the center blue box have a shift of ~8 ppm, while the green circled features shift to ~2 ppm. The broad feature at −5 ppm is an artifact of boron in the glass of the NMR probe.

Fig. 4

UV/Vis spectra showing the reaction of 100 μM TB (a) and 100 μM TRENBSAM (b) with 30 mM hydrogen peroxide; both samples are in 20 mM PBS pH 7.4. The conversion of TB to TS is complete within 4 min, whereas TRENBSAM takes over 30 min to convert to TRENSAM under these conditions.

Fig. 5

Plot of observed rate constants versus hydrogen peroxide concentration for 100 μM TB (green diamonds) and 100 μM TRENBSAM (red squares) in pH 7.4 phosphate buffered saline. The initial rates of oxidation differ by a factor of 10, as determined by measurement of spectrophotometric changes at 276 nm (TRENBSAM) and 277 nm (TB).

Fig. 6

UV/Vis spectra of copper binding by TS and TB. TB (green dashed line) does not bind copper (red solid line), while TS has a distinct absorbance at 420 nm (blue dotted line) upon copper binding. Conditions: 100 μM TB, 100 μM TS, and 100 μM Cu(ClO₄)₂ in 20 mM phosphate buffer, pH 7.4.

Fig. 7

Crystal structure of [FeHTS]ClO₄. Only one of the two Fe-TS molecules contained in the asymmetric unit is shown. Thermal ellipsoids are shown at 50% probability. Hydrogen atoms (including 2 H’s on N3) and the ClO₄ anion are not shown. Selected bond distances (Å) and angles (°): Fe–O1, 1.946(3); Fe–O2, 1.891(3); Fe–O3, 1.920(3); Fe–N1, 2.184(4); Fe–N2, 2.186(4); Fe–N4, 2.293(4); O1–Fe–O2, 99.5(1); O1–Fe–O3, 91.2(1); O2–Fe–O3, 103.6(5); O1–Fe–N1, 87.7(1); O1–Fe–N2, 172.8(1); O1–Fe–N4, 92.4(1); O2–Fe–N1, 90.6(1); O2–Fe–N2, 87.3(1); O2–Fe–N4, 162.9(1); O3–Fe–N1, 165.7(1); O3–Fe–N2, 89.1(1); O3–Fe–N4, 88.1(1); N1–Fe–N2, 90.2(1); N1–Fe–N4, 77.7(1); N2–Fe–N4, 80.4(1).

Fig. 8

Cyclic voltammograms of TS with Fe(NH₄)₂(SO₄)₂ (both 1 mM) in HEPES buffer at pH 7.4 at a hanging mercury drop electrode; scan rates as indicated.

Fig. 9

Effect of hexadentate prochelators and chelators on deoxyribose degradation by (a) iron or (b) copper-induced radical formation. Conditions: 400 μM H₂O₂, 10 μM FeCl₃ or 10 μM Cu(ClO₄)₂, 2 mM ascorbic acid, 15 mM 2-deoxyribose in pH 7.4 phosphate buffer. A and A_o are the absorbance readings at 490 nm with and without added chelator. Values of A/A_o <1 signify protection against radical generated deoxyribose degradation.

Fig. 10

Toxicity of TB and TS in ARPE-19 cells. The prochelator TB is not cytotoxic up to 1 mM, while the chelator TS induces cell death at concentrations as low as 1 μM.

Fig. 11

The effect of TB and TS on ARPE-19 cells treated with a cytotoxic dose of 200 μM hydrogen peroxide. The chelator TS is protective within a narrow concentration window of 1–10 μM, while the prochelator is protective at all concentrations tested over 1 μM.

Scheme 1

Synthesis of hexadentate chelators TRENSAM, TRENSIM and TS (R=OH) and prochelators TRENBSAM, TRENBSIM and TB (R=boronic ester or boronic acid).

Scheme 2

Boron environments of TB as a function of pH.