Diamagnetic Imaging Agents with a Modular Chemical Design for Quantitative Detection of β-Galactosidase and β-Glucuronidase Activities with CatalyCEST MRI

Abstract

Imaging agents for the noninvasive in vivo detection of enzyme activity in preclinical and clinical settings could have fundamental implications in the field of drug discovery. Furthermore, a new class of targeted prodrug treatments takes advantage of high enzyme activity to tailor therapy and improve treatment outcomes. Herein, we report the design and synthesis of new magnetic resonance imaging (MRI) agents that quantitatively detect β-galactosidase and β-glucuronidase activities by measuring changes in chemical exchange saturation transfer (CEST). Based on a modular approach, we incorporated the enzymes' respective substrates to a salicylate moiety with a chromogenic spacer via a carbamate linkage. This furnished highly selective diamagnetic CEST agents that detected and quantified enzyme activities of glycoside hydrolase enzymes. Michaelis-Menten enzyme kinetics studies were performed by monitoring catalyCEST MRI signals, which were validated with UV-vis assays.

Figure 1

CEST MRI mechanism. Radio frequency saturation of the carbamide hydrogen atom (green) results in the loss of the MRI signal (a saturated proton is shown in white). After the exchange of this saturated hydrogen atom with a hydrogen atom on water (blue), some of the MRI signal of water is lost, which can be measured with MRI. The MRI signal of the proton of the salicylic acid moiety (green) is unchanged because chemical exchange with saturated carbamide or saturated water protons is negligible. However, radio frequency saturation of the salicylic acid proton would cause a similar decrease in the water signal based the same mechanism.

Figure 2

catalyCEST MRI. (a) The experimental CEST spectra (blue circles), the Lorentzian line fitting of the experimental CEST spectra (blue lines), and the Lorentzian line shapes showed two CEST signals from the substrate 6a (solid red lines) and only one CEST signal for the product after β-gal catalysis (dashed red line). (b) The normalized CEST signal at 4.25 ppm decreased after treatment of 6a with β-gal (red). No change in CEST signal was observed after treatment with β-gus (green), with β-gal inhibited by PETG (purple), or in the absence of enzyme (blue). (c,d) Similar results were obtained before and after enzyme catalysis of 6b with β-gus.

Figure 3

Michaelis–Menten kinetics studies with catalyCEST MRI. (a) The decrease of the CEST signal at 4.25 ppm was monitored for 4.5 h with catalyCEST MRI after the addition of 0.25 units of β-gal to 25 mM of 6a. (b) The CEST signal amplitude was correlated with the concentration of 6a using the HW-Conc analysis method. The initial velocity, v_i, was determined by converting the CEST signal in panel a to concentration, using the calibration curve in panel b. (c) A Hanes–Woolf plot was used to determine Michaelis–Menten kinetics parameters. (d–f) This analysis was repeated for evaluating the kinetics of β-gus with its substrate 6b.

Figure 4

Michaelis–Menten kinetics studies with absorbance at 425 nm. (a) The absorbance at 425 nm was correlated with the concentration of 4-hydroxy-3-nitrobenzyl alcohol (1) using the Beer–Lambert law. (b) The initial velocity, v_i, was determined by monitoring the change in UV absorbance of 6a after the addition of β-gal enzyme and converting the absorbance at 425 nm to concentration using the calibration in panel a. (c) A Hanes–Woolf plot with initial velocities and substrate concentrations was used to determine Michaelis–Menten kinetics parameters. (d,e) This analysis was repeated for evaluating the kinetics of β-gus with its substrate 6b.

Scheme 1

(a) Modular Design of the CEST MRI Agent and (b) the Proposed Release Mechanism of the Enzyme Responsive Contrast Agent

Scheme 2. Agent Synthesis and Experimental Conditions

^a(i) Silver oxide, ACN, room temperature, 2 h, 86% (3a), 88% (3b). (ii) Triphosgene, 1:1 ACN/toluene, 60 to 80 °C, 2h. (iii) 3, 80 °C, 1 h, 13% (5a), 34% (5b). (iv) MeOH, NaOMe, room temperature, 30 min, 47% (6a), 83% (6b).