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

Article abstract of DOI:10.1021/acs.bioconjchem.6b00482

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.

Full text of DOI:10.1021/acs.bioconjchem.6b00482

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Article
Diamagnetic imaging agents with a modular chemical
design for quantitative detection of #-galactosidase
and #-glucuronidase activities with catalyCEST MRI
Gabriela Fernandez-Cuervo, Kirsten A Tucker, Scott W Malm, Kyle Mitchell Jones, and Mark D Pagel
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00482 • Publication Date (Web): 22 Sep 2016
Downloaded from http://pubs.acs.org on September 23, 2016
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Bioconjugate Chemistry
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Diamagnetic imaging agents with a modular chemical design for quantitative detection
of βꢀgalactosidase and βꢀglucuronidase activities with catalyCEST MRI
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Gabriela FernándezꢀCuervo,† Kirsten A. Tucker,‡ Scott W. Malm,§ Kyle M. Jonesǁ and Mark D. Pagel*, ‡, ǁ,
†Department of Pharmaceutical Sciences, The University of Arizona, 1295 N Martin, P.O. Box 210202, Tucson AZ, 85721
‡Department of Chemistry and Biochemistry, The University of Arizona, 1306 E University Blvd, Tucson, AZ 85719
§Department of Pharmacology and Toxicology, The University of Arizona, 1295 N Martin, P.O. Box 210202, Tucson AZ, 85721
ǁ Department of Biomedical Engineering, The University of Arizona, 1127 E James E. Rogers Way, P.O. Box 210020, Tucson, AZ 85721
Department of Medical Imaging, The University of Arizona, 1515 N Campbell Avenue, Tucson AZ, 85724-5024
* E-mail: mpagel@u.arizona.edu
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ABSTRACT: Imaging agents for the nonꢀinvasive 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 βꢀgalactosidase and βꢀglucuronidase
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. MichaelisꢀMenten enzyme kinetics studies were performed by monitoring catalyCEST MRI signals, which were validated
with UVꢀvis assays.
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 when evaluating preclinical assessments of new
treatment strategies has proven to be crucial.2 βꢀgalactosidase (βꢀgal) and βꢀglucuronidase (βꢀgus) are glycoside hydrolase enzymes that
have been employed in prodrug monotherapy (PMT),3ꢀ11 antibody directed enzyme prodrug therapy (ADEPT)12ꢀ17 and gene directed
enzyme prodrug therapy (GDEPT).18ꢀ21 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 βꢀgal and βꢀgus activities in tissues. The
evaluation of βꢀgal and βꢀgus 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 βꢀgal and βꢀgus with
fluorescence,23ꢀ24 luminescence, 25 chemiluminiscence 26 and optoacoustic 27 imaging. However, these techniques suffer from poor spatial
resolution and/or depth of view in vivo. PET and SPECT agents have been designed that are retained in tissues after cleavage by βꢀgal or
βꢀgus.28ꢀ32 These radionuclide agents depend on the relative pharmacokinetics 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 19FꢀNMR is insensitive and equipment is lacking in the clinic.33 MRI contrast
agents have been developed that nonꢀinvasively detect βꢀgal and βꢀgus by changing their T1 relaxation time constant of the agent when
cleaved by these enzymes.34ꢀ35 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 βꢀgal and βꢀgus activities.
A new type of 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 exchanging 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.
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We have developed an imaging protocol, termed catalyCEST MRI, that can detect enzyme activity in vitro37ꢀ43 and in vivo.44ꢀ45
CatalyCEST MRI simultaneously detects enzymeꢀresponsive 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. Dualꢀsignal CEST MRI contrast
agents have been developed that are substrates for esterase,39 cathepsin B,42 sulfatase,43 urokinase Plasminogen Activator,44 and γꢀglutamyl
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 βꢀgal and βꢀgus suffer from this pitfall, as evidenced by the poor catalytic cleavage of a glycoside
substrates resulting in limited prodrug activation.47 In particular, the pKa of the alkyl alcohol leaving group of the substrate can influence
the activity of these enzymes.48ꢀ50 Therefore, we sought to expand this platform technology by incorporating a spacer between the
salicylate and glycoside moieties via a carbamate linkage, creating a “triꢀmodular” 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 galactosylꢀenzyme intermediate thus improving cleavage. A similar approach has been used to
develop other prodrugs51 suggesting that this approach is feasible for developing diamagnetic CEST agents.
Our selection of a nitrobenzyloxyꢀcarbamate moiety was inspired by the use of a similar spacer that was incorporated into a
paramagnetic CEST agent that detected βꢀgal activity.52ꢀ53 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 enzymeꢀunresponsive control signal (Scheme 1b). The comparison of enzymeꢀresponsive 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 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.
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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
βꢀgal or βꢀgus activity can provide this essential validation. Optical imaging is more rapid and economical relative to MRI, which
facilitates straightforward validation studies in solution.
Scheme 1. (a) Modular design of the CEST MRI agent. (b) The proposed release mechanism of the enzyme responsive contrast agent.
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Results and discussion
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Design of enzymeꢀresponsive CEST MRI agents for detecting βꢀgal or βꢀgus activity
To synthesize our agent, the spacer was conjugated to the corresponding activated bromoꢀglycosides 2a and 2b, through a Koenigsꢀ
Knorr glycosylation reaction to furnish 3a and 3b (Scheme 2). Compounds 3a and 3b were then conjugated to through a carbamate
linkage between these moieties. In a twoꢀstep oneꢀpot reaction, the amino group was converted into an isocyanate group in the presence of
triphosgene, and subsequently reacted with the hydroxyl group of compounds . (Triphosgene is toxic and corrosive; MSDS must be
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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.
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Scheme 2. (i) Silver oxide, ACN, room temperature, 2 h, 86% (3a), 88% (3b). (ii) Triphosgene, 1:1 ACN/toluene, 60 oC to 80 oC, 2h. (iii) 3, 80 oC, 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 βꢀgal or βꢀgus 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 ppm 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 4ꢀamino 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 βꢀgal catalysis (dashed red line). (b) The normalized CEST signal at 4.25 ppm
decreased after treatment of 6a with βꢀgal (red). No change in CEST signal was observed after treatment with βꢀgus (green), with βꢀgal inhibited by PETG (purple), or in the absence of enzyme
(blue). (c) and (d) Similar results were obtained before and after enzyme catalysis of 6b with βꢀgus.
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 hours (Figure 2b,d).
The CEST signal decreased in the presence of the corresponding enzyme for each agent. This CEST signal of agent 6a remained
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consistently high in the presence of inhibited βꢀgal, implying that this agent truly detects βꢀgal activity. The CEST signal of 6a also
remained high in the absence of βꢀgal, demonstrating that this agent is stable in solution. The CEST signal was also consistently high for
6a treated with βꢀgus, indicating excellent specificity for detecting βꢀgal versus βꢀgus. 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 βꢀgal to
the agent in these buffer conditions did not accelerate the rate signal decrease compared to the control, suggesting βꢀgal did not cleave the
agent, thus indicating good specificity for 6b to detect βꢀgus against βꢀgal.
To further verify these results, LCꢀMS analysis revealed that the glycosidic cleavage led to the anticipated 1,6ꢀelimination and the
release of both reporters, 4ꢀhydroxyꢀ3ꢀnitrobenzyl alcohol spacer, and 4ꢀamino salicylic acid (Figure S1, S2). Qualitative confirmation of
enzyme activity was noted as the solution became yellow after enzyme cleavage. As additional validation the liberation of 4ꢀhydroxyꢀ3ꢀ
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nitrobenzyl alcohol (1) was detected by UVꢀvis absorbance at 425 nm (Figure S3). The 3.8ꢀ and 10.9ꢀfold increase in optical absorbance
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after treatment with βꢀgal or βꢀgus, 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 HWꢀQUESP method to evaluate the dependence of CEST signal amplitude on saturation power.54 A saturation power of
4 µT 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 (Figure S4, S5). We used the RLꢀQUEST method to assess the effect of the saturation time on CEST
signals.55 A saturation time of 3 seconds was adequate for generating strong CEST signal amplitude from each agent while maintaining a
relatively short acquisition time for eventual in vivo studies (Figure S6, S7).
We evaluated the chemical exchange rates (kex) of 6a and 6b, using a HWꢀQUESP CEST MRI analysis method (Figure S4, S5). The
carbamide proton had a kex of 701 Hz and 693 Hz for 6a and 6b, respectively, and the salicylate proton’s kex was 972 Hz and 1007 Hz for
6a and 6b. These kex values for the salicylate proton of the substrate agents were similar to the kex of the product, which was determined to
be 934 Hz in a previous study.45 These similar kex values of the substrates and product showed that the salicylate generated an enzymeꢀ
unresponsive “control” signal to improve the evaluation of enzyme activity. As expected, the kex of the salicylic acid proton and the
carbamide proton were slower than the saturation frequency of each proton (2,775 Hz at 9.25 ppm, and 1,275 at 4.25 ppm, at 300 MHz
magnetic field strength), which is a requirement for good CEST detection.
MichaelisꢀMenten kinetics analysis
We performed MichaelisꢀMenten enzyme kinetics studies to quantitatively demonstrate that our agents detect enzyme activity.38
Samples of the agents ranging from 4 mM to 50 mM were treated with each enzyme and 75 CEST spectra were acquired for 4.5 hours
(Figure 3a,d). The temporal disappearance of the CEST signal at 4.25 ppm was converted to concentration of the uncleaved agent at each
time point of the reaction, using our previously determined HWꢀConc CEST calibration method with an identical CEST MRI protocol
(Figure 3b,e).56 The initial velocity of each reaction, vi, was determined from the rate of decrease of the uncleaved agent’s concentration
during the first 2 hours of the reaction. The good fitting of the Hanesꢀ Woolf plot to the experimental results (R2 = 0.80 for βꢀgal and 0.76
for βꢀgus) demonstrated that the CEST agent followed MichaelisꢀMenten kinetics and therefore was responsive to enzyme activity (Figure
3c,f). These plots were used to determine the reaction velocity, Vmax, Michaelis constant, KM, catalysis rate, kcat, and the catalytic
efficiency, kcat/KM for each enzyme (Table 1).
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Figure. 3 MichaelisꢀMenten kinetics studies with catalyCEST MRI. (a) The decrease of the CEST signal at 4.25 ppm was monitored for 4.5 hours with catalyCEST MRI after
adding 0.25 units of βꢀgal to 25 mM of 6a. (b) The CEST signal amplitude was correlated with the concentration of 6a using the HWꢀConc analysis method. The initial velocity,
vi, was determined by converting the CEST signal in panel a to concentration, using the calibration curve in panel b. (c) A HanesꢀWoolf plot was used to determine Michaelisꢀ
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Menten kinetics parameters. (dꢀf) This analysis was repeated for evaluating the kinetics of βꢀgus with its substrate 6b
.
We repeated MichaelisꢀMenten kinetics studies of the agents 6a and 6b by monitoring the optical density of the spontaneously
disassembling spacer (Figure 4, Table 1). We converted the optical density to concentration using a BeerꢀLambert correlation with 4ꢀ
hydroxyꢀ3ꢀnitrobenzyl alcohol (1) as the standard for both agents. The initial velocity, vi, was determined from the increasing rate of 4ꢀ
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hydroxyꢀ3ꢀnitrobenzyl alcohol concentration produced from the liberation of free spacer during the first 2 and 1 hours of the cleavage of
6a and 6b by their respective enzymes. We used the HanesꢀWoolf plot to determine MichaelisꢀMenten kinetics constants to directly
compare results from the catalyCEST MRI experiment. The minor curvature in the HanesꢀWoolf plot for βꢀgus suggested a systematic
error in the kinetics analysis. This error was attributed to the competitive inhibition of the βꢀgus enzyme by glucuronic acid, which is a
product of the reaction.57
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Figure. 4 MichaelisꢀMenten kinetics studies with absorbance at 425 nm. (a) The absorbance at 425 nm was correlated with the concentration of 4ꢀhydroxyꢀ3ꢀnitrobenzyl alcohol (1) using the
BeerꢀLambert law. (b) The initial velocity, vi, was determined by monitoring the change in UV absorbance of 6a after adding βꢀgal enzyme, and converting the absorbance at 425 nm to
concentration using the calibration in panel a. (c) A HanesꢀWoolf plot with initial velocities and substrate concentrations was used to determine MichaelisꢀMenten kinetics parameters. (d) and
(e) This analysis was repeated for evaluating the kinetics of βꢀgus with its substrate 6b.
For agent 6a responsive to βꢀgal, the KM, kcat and catalytic efficiency constants were comparable within an order of magnitude
between the analyses performed with catalyCEST MRI and UVꢀvis 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 βꢀgus, the KM and kcat values differed by about two orders of magnitude between each
analysis method. This difference was attributed to the minor systematic error caused by the competitive inhibition of βꢀgus by free
glucuronic acid. This competitive inhibition has a stronger influence on the UVꢀvis analysis than catalyCEST MRI that sustains saturation
of the enzyme with a substrate for a longer time. Furthermore, the systematic error was cancelled by the HanesꢀWoolf analysis method
when determining the catalytic efficiency, which explains why the catalytic efficiencies, kcat/KM, determined by the catalyCEST MRI and
UVꢀvis analyses are similar, despite differences in KM and kcat values.
Table 1. MichaelisꢀMenten kinetics constants.
βꢀgalactosidaseꢀresponsive agent (6a
)
βꢀglucuronidaseꢀresponsive agent (6b)
MichaelisꢀMenten
Enzyme Kinetics
catalyCEST MRI
58.9 x 10ꢀ8 M sꢀ1
12.3 x 101 sꢀ1
54.9 x 10ꢀ4 M
2.24 x 104 Mꢀ1 sꢀ1
UVꢀvis
MRI/UVꢀvis
11.4
catalyCEST MRI
143 x 10ꢀ8 M sꢀ1
128 x 10ꢀ1 sꢀ1
131 x 10ꢀ4 M
9.80 x 102 Mꢀ1 sꢀ1
UVꢀvis
MRI/UVꢀvis
113
Vmax
kcat
5.16 x 10ꢀ8 M sꢀ1
2.16 x 101 sꢀ1
2.60 x 10ꢀ4 M
8.32 x 104 Mꢀ1 sꢀ1
1.26 x 10ꢀ8 M sꢀ1
1.13 x 10ꢀ1 sꢀ1
2.16 x 10ꢀ4 M
5.21 x 102 Mꢀ1 sꢀ1
5.7
113
KM
21.1
60.6
kcat/KM
0.27
1.88
Based on the MichaelisꢀMenten kinetics analysis, βꢀgal and βꢀgus had similar KM constants for the respective contrast agent,
demonstrating similar binding affinities for their ligands. βꢀgal has a faster kcat value than βꢀgus. 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 kcat of βꢀgal indicates faster glycosidic
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cleavage of 6a. Coupled with the evidence that glucuronic acid can be a competitive inhibitor for the βꢀgus enzyme, agent 6a and βꢀgal
appear to be the better choice for subsequent application of in vitro and in vivo imaging with catalyCEST MRI.
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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 triꢀmodular design for detecting enzyme catalysis. Additionally, we took advantage of this
spacer to incorporate a chromophore moiety that validated our MRI studies with UVꢀvis 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, βꢀgal has superior catalysis properties relative to βꢀgus 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 nonꢀinvasive 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 βꢀgal or βꢀgus. Moreover, the modular design of these agents facilitates the conjugation of other enzyme substrates to
the carbamate spacer, so that this approach constitutes a platform technology for the detection of enzyme activity.
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Experimental Procedures
Synthesis of enzymeꢀresponsive CEST agents. The CEST agents were synthesized as described in the Supporting Information.
1
Compounds were analyzed with H and 13C NMR spectroscopic methods using a Bruker AvanceꢀIII 400 MHz NMR spectrometer. Final
products were confirmed with highꢀresolution electrospray ionization (HRMS (ESI negative mode)) mass spectroscopy analysis recorded
with a Bruker 9.4 T ApexꢀQh hybrid Fourier transfer ionꢀcyclotron resonance (FTꢀICR) instrument. All compounds where purified by
flash column chromatography when needed. Full characterization is provided in the Supporting Information.
Enzyme reactions. βꢀgal and βꢀgus from E. coli were purchased from Sigma Aldrich as lyophilized. Samples of the agents were prepared
as shown in Table S1 of the Supporting Information. The substrate concentrations were varied between 4 – 50 mM and 0.050 – 3.125 mM,
o
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 UVꢀvis experiments. Samples were analyzed after enzyme reactions with reverse phase LCꢀMS (Shimadzu Corp.)
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 CESTꢀFISP 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: 128x128; field of view: 6.4x6.4 cm;
inꢀplane spatial resolution: 500x500 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 MichaelisꢀMenten Kinetics studies. 75 image sets to generate a 62ꢀpoint 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 hours.
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 ppm 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.
UVꢀvis enzymatic microplate assays. Serial dilutions of appropriate substrate were incubated with 0.125 U of βꢀgal or 250 U of βꢀgus
enzyme at 37.0 °C for 16 hours. Formation of product was detected in a Synergy HTX multiꢀmode plate reader (BioTek, Winooski, VT)
at 425 nm every 2 minutes.
Associated Content
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
Author Information
Corresponding Author
* E-mail: mpagel@u.arizona.edu. Phone: +1 520-404-7049
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Acknowledgements
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The authors thank Edward A. Randtke, John Jewett, Kristen Keck and Walter Klimecki for helpful interactions. Gabriela Fernándezꢀ
Cuervo is a Howard Hughes Medical Institute Gilliam Fellow also supported by the Biological Chemistry Program Training Grant: T32
GM008804. This research study was also funded by the NIH through grant R01 CA169774 and P01 CA95060.
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Abbreviations
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9
βꢀgal, βꢀgalactosidase; βꢀgus, βꢀglucoronidase; CEST, chemical exchange saturation transfer; FISP, fast imaging with steadyꢀstate precession;
LCꢀMS, liquid chromatographyꢀmass spectrometry; MeOH, methanol; MRI, magnetic resonance imaging; NaOMe, sodium methoxide; NMR,
nuclear magnetic resonance; PET, positron emission tomography; PETG, phenylethyl βꢀdꢀthiogalactoside; SPECT, single photon emission computed
tomography; UV, ultraviolet
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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 exchanging 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.
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Scheme 1. (a) Modular design of the CEST MRI agent. (b) The proposed release mechanism of the enzyme
responsive contrast agent.
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Scheme 2. (i) Silver oxide, ACN, room temperature, 2 h, 86% (3a), 88% (3b). (ii) Triphosgene, 1:1
ACN/toluene, 60 oC to 80 oC, 2h. (iii) 3, 80 oC, 1 h, 13% (5a), 34% (5b). (iv) MeOH, NaOMe, room
temperature, 30 min, 47% (6a), 83% (6b).
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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 βꢀgal catalysis (dashed red
line). (b) The normalized CEST signal at 4.25 ppm decreased after treatment of 6a with βꢀgal (red). No
change in CEST signal was observed after treatment with βꢀgus (green), with βꢀgal inhibited by PETG
(purple), or in the absence of enzyme (blue). (c) and (d) Similar results were obtained before and after
enzyme catalysis of 6b with βꢀgus.
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Figure. 3 MichaelisꢀMenten kinetics studies with catalyCEST MRI. (a) The decrease of the CEST signal at
4.25 ppm was monitored for 4.5 hours with catalyCEST MRI after adding 0.25 units of βꢀgal to 25 mM of 6a.
(b) The CEST signal amplitude was correlated with the concentration of 6a using the HWꢀConc analysis
method. The initial velocity, vi, was determined by converting the CEST signal in panel a to concentration,
using the calibration curve in panel b. (c) A HanesꢀWoolf plot was used to determine MichaelisꢀMenten
kinetics parameters. (dꢀf) This analysis was repeated for evaluating the kinetics of βꢀgus with its substrate
6b.
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Page 15 of 15
Bioconjugate Chemistry
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Figure. 4 MichaelisꢀMenten kinetics studies with absorbance at 425 nm. (a) The absorbance at 425 nm was
correlated with the concentration of 4ꢀhydroxyꢀ3ꢀnitrobenzyl alcohol (1) using the BeerꢀLambert law. (b)
The initial velocity, vi, was determined by monitoring the change in UV absorbance of 6a after adding βꢀgal
enzyme, and converting the absorbance at 425 nm to concentration using the calibration in panel a. (c) A
HanesꢀWoolf plot with initial velocities and substrate concentrations was used to determine MichaelisꢀMenten
kinetics parameters. (d) and (e) This analysis was repeated for evaluating the kinetics of βꢀgus with its
substrate 6b.
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