Alcohol, Aldehyde, and Ketone Liberation and Intracellular Cargo Release through Peroxide-Mediated α-Boryl Ether Fragmentation

Abstract

α-Boryl ethers, carbonates, and acetals, readily prepared from the corresponding alcohols that are accessed through ketone diboration, react rapidly with hydrogen peroxide to release alcohols, aldehydes, and ketones through the collapse of hemiacetal intermediates. Experiments with α-boryl acetals containing a latent fluorophore clearly demonstrate that cargo can be released inside cells in the presence of exogenous or endogenous hydrogen peroxide. These experiments show that this protocol can be used for drug activation in an oxidative environment without generating toxic byproducts.

Figure 1.

Oxidative breakdown of 7. (A) Reaction progress as determined by ¹H NMR. (B) Reaction progress as a function of pH.

Figure 2.

Structures 9 and 10 as potential origins for the slow breakdown of 8.

Figure 3.

Fluorophore release at low substrate and peroxide concentrations and pH stability studies.

Figure 4.

Comparison of fluorophore release by different oxidants. [33]₀ = 40 μM, [oxidant]₀ = 200 μM, pH 7.4.

Figure 5.

Fluorophore release in HeLa cells treated with exogenous H₂O₂. Cells were incubated with 33 (10 μM) in DPBS buffer for 45 min at 37 °C, followed by replacement with fresh DPBS containing (A) vehicle or (B) H₂O₂ (100 μM). After 30 min, fluorescence was imaged (Zeiss Axio Observer Z1, 20× objective, GFP filter (Set 38 HE; ex = 470 nm; em = 525 nm)). (C) Bright-field image of cells in panel B stained with Hoechst 33258 (1 μM) and imaged using a DAPI filter (Set 68; ex = 377 nm; em = 464 nm). (D) Mean fluorescence intensities were calculated from three individual HeLa cells and set relative to the mean fluorescence intensity prior to treatments (F/F_i). Error bars denote standard deviations; ***, P < 0.001.

Figure 6.

Fluorophore release in HEK293T cells through treatment with exogenous H₂O₂. Cells were treated with 33 (10 μM) in DPBS buffer for 45 min at 37 °C, followed by replacement with fresh DPBS containing (A) vehicle or (B) H₂O₂ (100 μM). After 30 min incubation, cellular fluorescence was imaged on a Zeiss Axio Observer Z1 microscope using a 20× objective and GFP (Set 38 HE) filter (ex = 470 nm, em = 525 nm). (C) Bright-field image of cells in panel B stained with Hoechst 33258 (1 μM) and imaged using a DAPI (Set 68) filter (ex = 377 nm, em = 464 nm). (D) Mean fluorescence intensities were calculated from individual ROIs (n = 3) and set relative to the mean fluorescence intensity prior to treatments (F/F_i). Error bars denote standard deviations.

Figure 7.

Cellular fluorophore release in HeLa cells by endogenous, PMA-stimulated H₂O₂ generation. Cells were pretreated in DMEM containing (A) DMSO or (B) PMA (1 uM) and incubated at 37 °C for 60 min. Media was replaced with fresh DPBS containing 33 (10 uM) and cells were incubated for an additional 60 min at 37 °C before fluorescence was imaged (Zeiss Axio Observer Z1, 20× objective, GFP filter (Set 38 HE; ex = 470 nm; em = 525 nm)). (C) Bright-field image of cells in panel B stained with Hoechst 33258 (1 μM) and imaged using a DAPI filter (Set 68; ex = 377 nm; em = 464 nm). (D) Mean fluorescence intensities were calculated from three individual HeLa cells and set relative to the mean fluorescence intensity prior to treatments (F/F_i). Error bars denote standard deviations; **, P < 0.01.

Scheme 1.

Alcohol Release through Boronate Oxidation

Scheme 2.

α-Boryl Alcohol Synthesis and Functionalization

Scheme 3.

Oxidative Alcohol Release

Scheme 4.

Etherification in the Absence of Strong Base

Scheme 5.

Synthesis of Cyclic Acetal Substrates

Scheme 6.

Aldehyde and Ketone Release through Oxidative Acetal Cleavage

Scheme 7.

Synthesis of a Latent Fluorophore