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.

Graphical Abstract

INTRODUCTION

Understanding the functions of proteins in their innate in vivo tissue environment can provide useful biomarker information enabling appropriate and timely decisions with respect to drug development and the use of personalized therapy in routine clinical care.¹ For example, the detection of enzyme activity relative to detecting enzyme expression during the evaluation of preclinical assessments of new treatment strategies has proven to be crucial.² β-galactosidase (β-gal) and β-glucuronidase (β-gus) are glycoside hydrolase enzymes that have been employed in prodrug monotherapy (PMT),^3–11 antibody-directed enzyme prodrug therapy (ADEPT),^12–17 and gene-directed enzyme prodrug therapy (GDEPT).^18–21 Despite the appealing concept of targeted prodrug therapies, these treatment strategies have yielded inconsistent results throughout the past decade, potentially due to variable levels of β-gal and β-gus activities in tissues. The evaluation of β-gal and β-gus activities within in vivo tissues would improve our understanding of these treatment strategies and improve the paradigm of personalized medicine by identifying patients who could benefit from prodrugs that employ these enzymes.

Significant chemical efforts have been invested to develop new and improved biomedical imaging agents that detect enzyme activity within in vivo tissues.²² In particular, chemical agents have been developed that detect the activities of β-gal and β-gus with fluorescence,^23,24 luminescence,²⁵ chemilumi-niscence,²⁶ and optoacoustic²⁷ imaging. However, these techniques suffer from poor spatial resolution and depth of view in vivo. PET and SPECT agents have been designed that are retained in tissues after cleavage by β-gal or β-gus.^28–32 These radionuclide agents depend on the relative pharmaco-kinetics of the agent before and after cleavage, which may be influenced by many other physiological conditions, compromising the detection of enzyme activity. ¹⁹F NMR spectroscopy can show a change in chemical shift after enzyme cleavage; however, ¹⁹F-NMR is insensitive, and equipment is lacking in the clinic.³³ MRI contrast agents have been developed that noninvasively detect β-gal and β-gus by changing their T₁ relaxation time constant of the agent when cleaved by these enzymes.^34,35 Unfortunately, the concentration of the agent and other experimental conditions affect the T₁ time measured by MRI, which compromises the specificity for detecting these enzymes. Moreover, even though some agents rely on a shift in signal, most of these agents only count with one on–off signal, making it challenging to conduct controlled measurements. Therefore, a new imaging method and improved contrast agents are needed to detect β-gal and β-gus activities.

A new type of magnetic resonance imaging (MRI) contrast generated by chemical exchange saturation transfer (CEST) can be exploited for molecular imaging.³⁶ CEST MRI requires a labile proton on an agent with a relatively slow chemical exchange rate that can be selectively saturated while avoiding direct saturation of the MR frequency of water. This selective saturation greatly reduces the net magnetization of the agent’s proton pool, thus eliminating the detectable MR signal from these protons. These saturated protons exchange with bulk water and, in turn, decrease the water signal amplitude (Figure 1). Continuous selective saturation at the MR frequency of the agent’s labile proton can build a large population of saturated water protons, which greatly improves detection sensitivity.

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.

We have developed an imaging protocol, termed catalyCEST MRI, that can detect enzyme activity in vitro^37–43 and in vivo.^44,45 CatalyCEST MRI simultaneously detects enzyme-responsive and unresponsive “control” CEST signals and uses the ratio of the signals to detect enzyme activity with outstanding specificity relative to other conditions that affect CEST MRI. Dual-signal CEST MRI contrast agents have been developed that are substrates for esterase,³⁹ cathepsin B,⁴² sulfatase,⁴³ urokinase plasminogen activator,⁴⁴ and -glutamyl transferase⁴⁵ enzymes. Some of these agents are based on the chemical structure of salicylic acid, which provides an exchangeable proton source with an excellent chemical shift for selective saturation. Moreover, branching from the amino group of the amino salicylic acid analogue provides a second CEST proton source for the agent, which would furnish a single agent that simultaneously detects two CEST signals. Overall, these properties in addition to good biocompatibility make this scaffold an excellent potential candidate for clinical translation.⁴⁶ Derivatives of salicylic acid are relatively accessible, and enzyme responsive contrast agents can be easily synthesized by conjugating an enzyme’s substrate directly to the salicylate moiety.

Unfortunately, many enzymes may be inactive or have sluggish activity when catalyzing the cleavage to a substrate that is directly linked to an imaging agent. Both β-gal and β-gus suffer from this pitfall, as evidenced by the poor catalytic cleavage of a glycoside substrates resulting in limited prodrug activation.⁴⁷ In particular, the pK_a of the alkyl alcohol leaving group of the substrate can influence the activity of these enzymes.^48–50 Therefore, we sought to expand this platform technology by incorporating a spacer between the salicylate and glycoside moieties via a carbamate linkage, creating a “tri-modular” scaffold that would extend salicylic acid from the enzyme substrate and avoid steric hindrance (Scheme 1a). Additionally, having an electron withdrawing substituent at the alkoxy leaving group provides further stabilization of the galactosyl-enzyme intermediate, thus improving cleavage. A similar approach has been used to develop other prodrugs,⁵¹ suggesting that this approach is feasible for developing diamagnetic CEST agents.

Scheme 1.

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

Our selection of a nitrobenzyloxy-carbamate moiety was inspired by the use of a similar spacer that was incorporated into a paramagnetic CEST agent that detected β-gal activity.^52,53 This type of spacer can spontaneously disassemble upon enzyme cleavage of the pyranose from the agent. This disassembly converted the carbamate to an amine on the paramagnetic agent, which caused the appearance of two CEST signals at −16.7 and −20.5 ppm that were used to detect enzyme activity. In our approach, the disassembly of the carbamate spacer is designed to cause a disappearance of the CEST signal from the carbamate, while the CEST signal from the salicylate moiety remains as an enzyme-unresponsive control signal (Scheme 1b). The comparison of enzyme-responsive and unresponsive CEST signals is an improvement in the design of agents for catalyCEST MRI. Furthermore, we used a carbamate spacer bearing a nitro group that more rapidly disassembles than does the spacer used in the paramagnetic agent, which can improve the temporal response to enzyme activity. Finally, our diamagnetic agent is designed to retain large chemical shifts without requiring a paramagnetic ion that is potentially toxic at high concentrations, which provides yet another advantage.

As an additional advantage, the selected spacer generates an optically active product after the spontaneous disassembly. Validation of molecular imaging probes is a major requirement for developing biochemical imaging agents, and the generation of an optical signal after β-gal or β-gus activity can provide this essential validation. Optical imaging is more rapid and economical relative to MRI, which facilitates straightforward validation studies in solution.

RESULTS AND DISCUSSION

Design of Enzyme-Responsive CEST MRI Agents for Detecting β-Gal or β-Gus Activity

To synthesize our agent, the spacer 1 was conjugated to the corresponding activated bromo-glycosides 2a and 2b, through a Koenigs–Knorr glycosylation reaction to furnish 3a and 3b (Scheme 2). Compounds 3a and 3b were then conjugated to 4 through a carbamate linkage between these moieties. In a two-step one-pot reaction, the amino group was converted into an isocyanate group in the presence of triphosgene, and subsequently reacted with the hydroxyl group of compounds 3. (Triphosgene is toxic and corrosive; MSDS must be consulted prior to use). The final step followed a suitable Zemplén deacetylation reaction to provide imaging agents 6a and 6b in good yields. More details about the synthesis and the studies described below can be found in the Supporting Information.

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).

Detection of Enzyme Catalysis

We detected the activity of β-gal or β-gus by acquiring CEST spectra from each agent before and after adding enzyme (Figure 2a,c). Before enzyme hydrolysis, we observed two CEST signals from each agent at 9.25 and 4.25 ppm, corresponding to the selective saturation of the exchangeable proton from the salicylate and the carbamate moieties, respectively. In the presence of enzyme, the signal at 4.25 ppm disappeared, corresponding to the conversion of the agent into 4-amino salicylic acid as a consequence of catalytic hydrolysis of the glycosidic ligand. The signal of the agent at 9.25 ppm did not change in amplitude. However, this signal showed a slight decrease in saturation frequency after enzyme cleavage due to the decoupling of the electron withdrawing carbamate from the salicylic acid moiety. Importantly, the ratio of the CEST peaks effectively detected enzyme activity in a concentration-independent manner. This result demonstrated the responsiveness of this agent for each enzyme.

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.

We performed additional studies for both enzymes to further validate that the change in CEST signal was due to enzyme activation. Because the CEST effect at 4.25 ppm represents substrate before cleavage, we monitored this CEST signal over 4.5 h (Figure 2b,d). The CEST signal decreased in the presence of the corresponding enzyme for each agent. This CEST signal of agent 6a remained consistently high in the presence of inhibited β-gal, implying that this agent truly detects β-gal activity. The CEST signal of 6a also remained high in the absence of β-gal, demonstrating that this agent is stable in solution. The CEST signal was also consistently high for 6a treated with β-gus, indicating excellent specificity for detecting β-gal versus β-gus. The CEST signal for agent 6b, with no enzyme, showed a slow decrease, suggesting that the agent was slightly unstable in the buffer conditions that were tested. The addition of β-gal to the agent in these buffer conditions did not accelerate the rate of the signal decrease compared to that of the control, suggesting β-gal did not cleave the agent, thus indicating good specificity for 6b to detect β-gus against β-gal.

For the further verification of these results, liquid chromatography–mass spectrometry (LC–MS) analysis revealed that the glycosidic cleavage led to the anticipated 1,6-elimination and the release of both reporters, 4-hydroxy-3-nitrobenzyl alcohol spacer, and 4-amino salicylic acid (Figures S1 and S2). Qualitative confirmation of enzyme activity was noted as the solution became yellow after enzyme cleavage. As additional validation, the liberation of 4-hydroxy-3-nitrobenzyl alcohol (1) was detected by UV–vis absorbance at 425 nm (Figure S3). The 3.8- and 10.9-fold increase in optical absorbance after treatment with β-gal or β-gus, respectively, demonstrated that the agent had excellent detection sensitivity for confirming the activity of these two enzymes in solution.

Optimization of catalyCEST MRI Parameters

We used the HW-QUESP method to evaluate the dependence of CEST signal amplitude on saturation power.⁵⁴ A saturation power of 4 μ4T was shown to generate relatively strong CEST signals from the agent while still maintaining an acceptably low power below the SAR limit for in vivo studies (Figures S4 and S5). We used the RL-QUEST method to assess the effect of the saturation time on CEST signals.⁵⁵ A saturation time of 3 s was adequate for generating strong CEST signal amplitude from each agent while maintaining a relatively short acquisition time for eventual in vivo studies (Figures S6 and S7).

We evaluated the chemical exchange rates (k_ex) of 6a and 6b using a HW-QUESP CEST MRI analysis method (Figures S4 and S5). The carbamide proton had a k_ex of 701 and 693 Hz for 6a and 6b, respectively, and the salicylate proton’s k_ex was 972 and 1007 Hz for 6a and 6b. These k_ex values for the salicylate proton of the substrate agents were similar to the k_ex of the product, which was determined to be 934 Hz in a previous study.⁴⁵ These similar k_ex values of the substrates and product showed that the salicylate generated an enzyme-unresponsive “control” signal to improve the evaluation of enzyme activity. As expected, the k_ex of the salicylic acid proton and the carbamide proton were slower than the saturation frequency of each proton (2775 Hz at 9.25 ppm, and 1275 at 4.25 ppm, at 300 MHz magnetic field strength), which is a requirement for good CEST detection.

Michaelis–Menten Kinetics Analysis

We performed Michaelis–Menten enzyme kinetics studies to quantitatively demonstrate that our agents detect enzyme activity.³⁸ Samples of the agents ranging from 4 to 50 mM were treated with each enzyme, and 75 CEST spectra were acquired for 4.5 h (Figure 3a,d). The temporal disappearance of the CEST signal at 4.25 ppm was converted to the concentration of the uncleaved agent at each time point of the reaction using our previously determined HW-Conc CEST calibration method with an identical CEST MRI protocol (Figure 3b,e).⁵⁶ The initial velocity of each reaction, v_i, was determined from the rate of decrease of the uncleaved agent’s concentration during the first 2 h of the reaction. The good fitting of the Hanes–Woolf plot to the experimental results (R² = 0.80 for β-gal and 0.76 for β-gus) demonstrated that the CEST agent followed Michaelis–Menten kinetics and therefore was responsive to enzyme activity (Figure 3c,f). These plots were used to determine the reaction velocity, V_max, Michaelis constant, K_M, catalysis rate, k_cat, and the catalytic efficiency, k_cat/K_M, for each enzyme (Table 1).

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.

Table 1.

Michaelis–Menten Kinetics Constants

Michaelis–Menten enzyme kinetics	β-galactosidase-responsive agent (6a)			β-glucuronidase-responsive agent (6b)
	catalyCEST MRI	UV–vis	MRI/UV–vis	catalyCEST MRI	UV–vis	MRI/UV–vis
Vmax	58.9 × 10−8 M s−1	5.16 × 10−8 M s−1	11.4	143 × 10−8 M s−1	1.26 × 10−8 M s−1	113
kcat	12.3 × 101 s−1	2.16 × 101 s−1	5.7	128 × 10−1 s−1	1.13 × 10−1 s−1	113
KM	54.9 × 10−4 M	2.60 × 10−4 M	21.1	131 × 10−4 M	2.16 × 10−4 M	60.6
kcat/KM	2.24 × 104 M−1 s−1	8.32 × 104 M−1 s−1	0.27	9.80 × 102 M−1 s−1	5.21 × 102 M−1 s−1	1.88

We repeated Michaelis–Menten kinetics studies of the agents 6a and 6b by monitoring the optical density of the spontaneously disassembling spacer (Figure 4 and Table 1). We converted the optical density to concentration using a Beer–Lambert correlation with 4-hydroxy-3-nitrobenzyl alcohol (1) as the standard for both agents. The initial velocity, v_i, was determined from the increasing rate of 4-hydroxy-3-nitrobenzyl alcohol concentration produced from the liberation of free spacer during the first 2 and 1 h of the cleavage of 6a and 6b by their respective enzymes. We used the Hanes–Woolf plot to determine Michaelis–Menten kinetics constants to directly compare results from the catalyCEST MRI experiment. The minor curvature in the Hanes–Woolf plot for β-gus suggested a systematic error in the kinetics analysis. This error was attributed to the competitive inhibition of the β-gus enzyme by glucuronic acid, which is a product of the reaction.⁵⁷

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.

For agent 6a, responsive to β-gal, the K_M, k_cat, and catalytic efficiency constants were comparable within an order of magnitude between the analyses performed with catalyCEST MRI and UV–vis studies. This order of magnitude comparison is reasonable considering that MRI techniques are less precise than optical techniques. This result provided confidence that catalyCEST MRI can quantitatively evaluate enzyme activity. For agent 6b, responsive to β-gus, the K_M and k_cat values differed by about 2 orders of magnitude between each analysis method. This difference was attributed to the minor systematic error caused by the competitive inhibition of β-gus by free glucuronic acid. This competitive inhibition has a stronger influence on the UV–vis analysis than catalyCEST MRI that sustains saturation of the enzyme with a substrate for a longer time. Furthermore, the systematic error was canceled by the Hanes–Woolf analysis method when the catalytic efficiency was determined, which explains why the catalytic efficiencies, k_cat/K_M, determined by the catalyCEST MRI and UV–vis analyses are similar despite differences in K_M and k_cat values.

Based on the Michaelis–Menten kinetics analysis, it was determined that β-gal and β-gus had similar K_M constants for the respective contrast agent, demonstrating similar binding affinities for their ligands. β-gal has a faster k_cat value than β-gus. The rate of the spontaneous disassembly of the spacer should be identical for 6a and 6b after cleavage of the glycosidic ligand, so the faster k_cat of β-gal indicates faster glycosidic cleavage of 6a. Coupled with the evidence that glucuronic acid can be a competitive inhibitor for the β-gus enzyme, agent 6a and β-gal appear to be the better choice for subsequent application of in vitro and in vivo imaging with catalyCEST MRI.

In conclusion, we have shown that diamagnetic CEST agents can detect and quantify the enzyme activities of glycoside hydrolase enzymes. The implementation of a modular design facilitated agent activation and, thus, detection of both enzymes. Incorporating the spontaneously disassembling spacer provided a trimodular design for detecting enzyme catalysis. Additionally, we took advantage of this spacer to incorporate a chromophore moiety that validated our MRI studies with UV–vis studies. These CEST MRI agents were also used to test substrate specificity and assess enzyme inhibition, which expands the utility of catalyCEST MRI as a platform technology for enzyme studies. Based on our kinetics results, it was determined that β-gal has superior catalysis properties relative to β-gus for this class of modular catalyCEST MRI contrast agents.

These findings demonstrate that the newly synthesized modular agents 6a and 6b have the potential to become a reliable catalyCEST MRI imaging probe for the noninvasive quantitative detection of enzyme activities. In addition, our results suggest that studies involving in vitro validation and in vivo translation are warranted. Such techniques may facilitate the identification and validation of new prodrugs that are activated by β-gal or β-gus. Moreover, the modular design of these agents facilitates the conjugation of other enzyme substrates to the carbamate spacer, such that this approach constitutes a platform technology for the detection of enzyme activity.

EXPERIMENTAL PROCEDURES

Synthesis of Enzyme-Responsive CEST Agents

The CEST agents were synthesized as described in the Supporting Information. Compounds were analyzed with ¹H and ¹³C NMR spectroscopic methods using a Bruker Avance-III 400 MHz NMR spectrometer. Final products were confirmed with high-resolution electrospray ionization (HRMS (ESI negative mode)) mass spectroscopy analysis recorded with a Bruker 9.4 T Apex-Qh hybrid Fourier transfer ion-cyclotron resonance (FT-ICR) instrument. All compounds were purified by flash column chromatography when needed. Full characterization is provided in the Supporting Information.

Enzyme Reactions

β-gal and β-gus from Escherichia coli were purchased from Sigma-Aldrich as lyophilized. Samples of the agents were prepared as shown in Table S1. The substrate concentrations were varied between 4–50 mM and 0.050–3.125 mM, depending on the experiment and detection method. The temperature was adjusted to 37.0 ± 0.2 °C for enzyme incubation and for image acquisition in MRI and UV–vis experiments. Samples were analyzed after enzyme reactions with reverse-phase LC–MS (Shimadzu Corporation) to verify enzyme hydrolysis.

CEST MRI Acquisition Protocol for the Detection of Enzyme Catalysis

MRI studies were performed with a preclinical Bruker Biospec MRI scanner operating at 7.05 T (300 MHz) magnetic field strength with a 72 mm quadrature transceiver coil. A CEST–fast imaging with steady-state free precession (CEST–FISP) acquisition protocol was used for all CEST MRI studies.⁴¹ We acquired a series of 107 images after selective saturation was applied using a continuous wave pulse at 3 μT power for 4 s with frequencies ranging from 15 to −15 ppm. FISP acquisition parameters included TR: 3.196 ms; TE: 1.598 ms; excitation flip angle: 30°; centric encoding; number of averages: 1; matrix: 128 × 128; field of view: 6.4 × 6.4 cm; in-plane spatial resolution: 500 × 500 mm; slice thickness: 1 mm. The temporal resolution of acquiring one image with one selective saturation frequency was 5.441 s. The total time to acquire 107 images for a full CEST spectrum was 9:42 min.

CEST MRI Acquisition Protocol for Michaelis–Menten Kinetics Studies

A total of 75 image sets for the generation of a 62 point CEST spectrum were collected after selective saturation was applied using a continuous wave pulse at 3 μT power for 4 s with frequencies ranging from 15 to −15 ppm. The same FISP parameters were used for consistency. The total acquisition time was 4.5 h.

Analysis of CEST Spectra

A region of interest in the image of each sample was selected from the acquired image to generate a CEST spectrum for the sample. To measure CEST signal amplitudes, the spectrum was analyzed by fitting three Lorentzian line shapes to account for the direct saturation of water and the CEST signals at 4.25 and 9.25 ppm.⁴¹ The center, width and amplitude of each Lorentzian line shape were allowed to change to optimize the fit. The Lorentzian line shape fitting automatically referenced the bulk water chemical shift at 0 ppm, negating the effect of B₀ inhomogeneities in the CEST MR images.

UV–Vis Enzymatic Microplate Assays

Serial dilutions of appropriate substrate were incubated with 0.125 U of β-gal or 250 U of β-gus enzyme at 37.0 °C for 16 h. Formation of product was detected in a Synergy HTX multimode plate reader (BioTek, Winooski, VT) at 425 nm every 2 min.

ABBREVIATIONS

β-gal

β-galactosidase

β-gus

β-glucoronidase

CEST

chemical exchange saturation transfer

FISP

fast imaging with steady-state precession

LC–MS

liquid chromatography–mass spectrometry

MeOH

methanol

MRI

magnetic resonance imaging

NaOMe

sodium methoxide

NMR

nuclear magnetic resonance

PET

positron emission tomography

PETG

phenylethyl β-d-thiogalactoside

SPECT

single photon emission computed tomography

UV

ultraviolet

HW-QUESP

Hanes–Woolf quantifying exchange using saturation power

RL-QUEST

reverse linear quantifying exchange using saturation time