BZM does not generate ROS, but directly reacts with EGCG to form a new boronate product. (A) RPMI/8226 cells were treated with 20 nM BZM, 5 mM H₂O₂, or exposed to hypoxia for 18 hours in the presence or absence of 20 μM EGCG. Shown is the fold increase of intracellular reactive oxygen species (ROS) levels (mean ± SE; n ≥ 3) from 1 representative experiment; ** indicates statistically significant (P = .005) differences between hypoxia-treated or H₂O₂-treated cells versus untreated (control) cells; ns, not statistically significantly different from untreated control. The entire experiment was repeated in different variations and with different incubation times; yet, BZM consistently failed to increase the levels of ROS. The generation of intracellular ROS was measured via labeling with 2′,7′-dichlorofluorescein diacetate (H₂DCFDA) and subsequent analysis by flow cytometry.²³  In some of the repetitions, total ROS production was determined by ultraviolet spectrometry after incubating cells with H₂DCFDA. (B) The direct binding of EGCG to BZM was investigated with the alizarin red S (ARS) reporter assay. In the absence of EGCG, there is increasing binding between ARS and rising (0-150 μM) concentrations of BZM. However, the addition of EGCG (250 μM) effectively reduces this interaction via direct competitive binding of EGCG to BZM. (C) Results from the ARS assay with MG-132 (65 μM) and MG-262 (65 μM) in the presence or absence of EGCG (250 μM). Note that MG-132 does not bind to ARS and there is no interference by EGCG, but that MG-262 strongly binds to ARS and this binding is effectively prevented by EGCG. All results are presented as mean ± SD (arbitrary units) of relative binding increase. These measurements were also repeated with TEAVIGO-EGCG and yielded very similar outcomes. (D) ¹H NMR spectra (400 MHz; left) and ¹³C NMR spectra (100 MHz; right) of combinations of BZM and EGCG in 20% D₂O in CD₃CN. (i) Pure BZM, (ii) pure EGCG, (iii) a 2:1 mixture of BZM and EGCG, and (iv) a 1:1 mixture of BZM and EGCG. Selected peaks from the NMR spectra indicate the presence of the new adduct from the reaction of a 1,2-diol unit of EGCG with the boronic acid group of BZM. By increasing the amount of EGCG relative to BZM, from 2:1 in panel iii to 1:1 in panel iv, the product peaks (red arrows in the ¹H NMR and red dots in the ¹³C NMR) increase relative to the BZM peaks (black arrows in the ¹H NMR and black dots in the ¹³C NMR).