Detection of DT-diaphorase Enzyme with a ParaCEST MRI Contrast Agent

Abstract

A responsive magnetic resonance (MRI) contrast agent has been developed that can detect the enzyme activity of DT-diaphorase. The agent produced different chemical exchange saturation transfer (CEST) MRI signals before and after incubation with the enzyme, NADH, and GSH at different pH values whereas it showed good stability in a reducing environment without enzyme.

Keywords: CEST MRI; NQO1; dt-diaphorase; molecular imaging; trimethyl lock.

Figure 1

Yb-DO3A-oAA-TML-Q was reacted with DT-diaphorase enzyme at pH 8.1 ± 0.1 and 37 °C for 15 h. CEST spectra were acquired with 6 μT, saturation power applied for 4 sec. (A) No reaction was detected when no enzyme, GSH, and NADH were added to the solution. (B) A new CEST signal at 9 ppm (solid red line) was detected when the CEST MRI agent was reacted with the enzyme, GSH, and NADH. The absence of NADH (C) or GSH (D) did not allow formation of the desired product whereas the CEST signal of unactivated CEST MRI agent (dashed lines in C and D) was clearly detected.

Figure 2

Yb-DO3A-oAA-TML-Q showed stability under anaerobic conditions (A) whereas the presence of DT-diaphorase enzyme and NADH in presence (B) or absence (C) of GSH activated the agent and produced a CEST signal at 9 ppm at pH 7.0 ± 0.25, 37 °C for 15 h, power level of 6 μT, and saturation time of 4 sec. The agent showed good stability in a reducing environment even in the presence of the enzyme and GSH in an anaerobic chamber (D).

Scheme 1

The quinone functional group in Yb-DO3A-oAA-TML-Q was reduced to hydroquinone in the presence of DT-diaphorase enzyme, NADH, and GSH. The newly formed hydroxy group attacked and dissociated the amide bond as a result of the steric effect between the methyl group on the benzene ring and the two adjacent methyl groups, consequently resulting in the release of the CEST agent.

Scheme 2

Synthesis of Yb-DO3A-oAA-TML-Q. The caboxylic acid form of quinone was prepared in 2 steps. Then it was reacted with tert-butyl protected DO3A-oAA that was prepared in 3 steps. The resulting product was de-protected and then used to chelate Yb^III.