Emissive Alkylated Guanine Analogs as Probes for Monitoring O 6-Alkylguanine-DNA-transferase Activity

Abstract

Human O ⁶-alkylguanine-DNA-transferase (hAGT) is a repair protein that provides protection from mutagenic events caused by O ⁶-alkylguanine lesions. As this stoichiometric activity is tissue-specific, indicative of tumor status, and correlated to chemotherapeutic success, tracking the activity of hAGT could prove to be informative for disease diagnosis and therapy. Herein, we explore two families of emissive O ⁶-methyl- and O ⁶-benzylguanine analogs based on our previously described ^th G _N and ^tz G _N , thieno- and isothiazolo-guanine surrogates, respectively, as potential reporters. We establish that O ⁶ -Bn ^th G _N and O ⁶ -Bn ^tz G _N provide a spectral window to optically monitor hAGT activity, can be used as substrates for the widely used SNAP-Tag delivery system, and are sufficiently bright to be visualized in mammalian cells using fluorescence microscopy.

Figure 1

(a) Structures of guanine (displaying the conventional purine numbering system), as well as ^thG_N and ^tzG_N. (b) Potential conversion of O⁶-alkylguanine analogs (O⁶-Bn^thG_N and O⁶-Me^thG_N or O⁶-Bn^tzG_N and O⁶-Me^tzG_N) to the respective guanine analogs (^thG_N or ^tzG_N) by hAGT.

Scheme 1. Synthesis of O⁶-Benzyl Thieno- and Isothiazolo-guanine Analogs

Figure 2

(a) Structures of ^thG_N-H1, ^thG_N-H3, O⁶-Bn^thG_N, O⁶-Me^thG_N, ^tzG_N, O⁶-Bn^tzG_N, and O⁶-Me^tzG_N nucleobase analogs. (b) Absorption (dashed lines) and emission (solid lines) of ^thG_N (blue), O⁶-Bn^thG_N (dark green), and O⁶-Me^thG_N (lime). (c) Absorption (dashed lines) and emission (solid lines) of ^tzG_N (red), O⁶-Bn^tzG_N (orange), and O⁶-Me^tzG_N (yellow).

Figure 3

Data and curve fits of normalized fluorescence changes upon dealkylation of O⁶-Bn^thG_N to ^thG_N at 400 nm (blue) and O⁶-Bn^tzG_N to ^tzG_N at 450 nm (red) by (a) hAGT and (b) SNAP-Tag. Data presented are the averages of three independent experiments. For normalized data with error bars, see Figure S5. Insets: Before and after emission spectra for O⁶-Bn^thG_N (blue) and O⁶-Bn^tzG_N (red) reactions. Arrows show the observed change at the monitored wavelength.

Figure 4

(a) Live-cell imaging of O⁶-Bn^thG_N, O⁶-Me^thG_N, O⁶-Bn^tzG_N, and O⁶-Me^tzG_N in HEK293T cells after 1 h of incubation versus untreated cells. (b) Fluorescence images of HEK293T cells and CHO-K1 cells incubated with 500 μM O⁶-Me^tzG_N and (c) 100 μM O⁶-Bn^thG_N at indicated times. Cells’ nuclei were visualized using NucRed.

Figure 5

Real-time fluorescence monitoring of O⁶-Bn^tzG_N (a) and O⁶-Bn^thG_N (b) in live HEK293T (black) and CHO-K1 (red) cells. Data points are averages of fluorescence intensities measured over time from at least 10 ROIs and normalized.