A catalyCEST MRI contrast agent that detects the enzyme-catalyzed creation of a covalent bond

Article abstract of DOI:10.1021/ja400254e

CatalyCEST MRI can detect enzyme activity by employing contrast agents that are detected through chemical exchange saturation transfer (CEST). A CEST agent, Tm-DO3A-cadaverine, has been designed to detect the catalytic activity of transglutaminase (TGase), which creates a covalent bond between the agent and the side chain of a glutamine amino acid residue. CEST appeared at -9.2 ppm after TGase conjugated Tm-DO3A-cadaverine to albumin, which also caused a decrease in CEST from albumin at +4.6 ppm. Studies with model peptides revealed similar appearances and decreases in detectable CEST effects following TGase-catalyzed conjugation of the contrast agent and peptide. The MR frequencies and amplitudes of these CEST effects were dependent on the peptide sequence, which demonstrated the sensitivity of CEST agents to ligand conformations that may be exploited to create more responsive molecular imaging agents. The chemical exchange rates of the substrates and conjugated products were measured by fitting modified Bloch equations to CEST spectra, which demonstrated that changes in exchange rates can also be used to detect the formation of a covalent bond by catalyCEST MRI.

Full text of DOI:10.1021/ja400254e

Communication
pubs.acs.org/JACS
A CatalyCEST MRI Contrast Agent That Detects the Enzyme-
Catalyzed Creation of a Covalent Bond
†
†
,†,‡,§,⊥
Dina V. Hingorani, Edward A. Randtke, and Mark D. Pagel*
†
‡
§
Department of Chemistry and Biochemistry, Department of Biomedical Engineering, Department of Medical Imaging, and
⊥
University of Arizona Cancer Center, University of Arizona, Tucson, Arizona 85724-5024, United States
*
S Supporting Information
ABSTRACT: CatalyCEST MRI can detect enzyme
activity by employing contrast agents that are detected
through chemical exchange saturation transfer (CEST). A
CEST agent, Tm-DO3A-cadaverine, has been designed to
detect the catalytic activity of transglutaminase (TGase),
which creates a covalent bond between the agent and the
side chain of a glutamine amino acid residue. CEST
appeared at −9.2 ppm after TGase conjugated Tm-DO3A-
cadaverine to albumin, which also caused a decrease in
CEST from albumin at +4.6 ppm. Studies with model
peptides revealed similar appearances and decreases in
detectable CEST effects following TGase-catalyzed con-
jugation of the contrast agent and peptide. The MR
frequencies and amplitudes of these CEST effects were
dependent on the peptide sequence, which demonstrated
the sensitivity of CEST agents to ligand conformations
that may be exploited to create more responsive molecular
imaging agents. The chemical exchange rates of the
substrates and conjugated products were measured by
fitting modified Bloch equations to CEST spectra, which
demonstrated that changes in exchange rates can also be
used to detect the formation of a covalent bond by
catalyCEST MRI.
Figure 1. CatalyCEST MRI. (A) MR saturation of the amide proton
of {Tm-DO3A-cadaverine}-protein (shown in red), followed by
chemical exchange with water, transfers the MR saturation to water.
(B) Conjugating the CEST agent to a protein’s glutamine side chain
by TGase converts an amine to an amide that generates CEST.
agent with Tm-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
acid (Tm-DO3A) would create a paramagnetic CEST agent
that can be similarly conjugated to glutamine via TGase (Figure
1
B). Tm-DO3A-cadaverine 1 was synthesized by alkylating
DO3A with a cadaverine derivative and using the product to
chelate Tm(III) (Schemes S1 and S2).
hemical exchange saturation transfer (CEST) is a MRI
contrast mechanism that reduces the detectable water
C
To show the practical application of 1 to detect the catalytic
activity of TGase, 25 mM 1 was added to 0.75 mM bovine
serum albumin and 10 mM glutathione in Tris-HCl buffer at
pH 7 (glutathione was included to maintain a reducing
environment that is required for TGase activity). A CEST
spectrum was recorded using a 600 MHz NMR spectrometer
by applying selective radio frequency saturation from 30 to −30
ppm in 1 ppm increments (Figure 2A). A function of
Lorentzian line shapes was fit to the CEST spectrum to
accurately measure the chemical shifts that generated maximum
CEST (Figure 2B). A CEST effect was detected at +4.6 ppm,
which was assigned to the diamagnetic amide and amine
protons of albumin. After this sample was incubated with 0.5
unit (0.327 μM) of microbial TGase from S. mobaraensis at 37
signal after selective saturation of an exchangeable proton of a
1
MRI contrast agent (Figure 1A). CEST agents have been
designed that detect the catalytic activity of enzymes, termed
“
catalyCEST MRI”, including CEST agents that are catalyzed
2
−4
5,6
7
8
by protease,
galactosidase,
esterase, and deaminase
enzymes. In these examples, the enzyme-catalyzed cleavage of
a covalent bond within the agent caused a change in CEST
produced by the agent. CEST agents that can only be cleaved
by enzymes limits catalyCEST to the detection of a subset of
enzyme activities. CEST agents that create a covalent bond
through enzyme catalysis are needed to expand this technology.
We have designed a CEST agent to detect transglutaminase
11
(TGase) catalysis. The entire family of TGases catalyzes the
°
C for 24 h, the CEST effect at +4.6 ppm dramatically
decreased, and a new CEST effect appeared at −9.2 ppm. This
formation of an amide bond between the side chains of lysine
and glutamine amino acids, which cross-links the extracellular
9
reaction was repeated three times to ensure that the appearance
matrix in tissues, especially in tumors. Previous reports have
shown that a fluorescent imaging agent with a generic aliphatic
amine ligand can be conjugated to a glutamine side chain via
TGase activity. We hypothesized that replacing a fluorescent
Received: January 15, 2013
1
0
©
XXXX American Chemical Society
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dx.doi.org/10.1021/ja400254e | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Figure 2. CEST before and after TGase catalysis. (A) The CEST
spectrum and (B) Lorentzian line shape spectrum of a mixture of
albumin, Tm-DO3A-cadaverine and L-glutathione before (black) and
after (red) incubation with TGase showed a decrease in CEST at +4.6
ppm and the appearance of CEST at −9.2 ppm following the creation
of a covalent bond by TGase catalysis.
of the CEST effect at −9.2 ppm was reproducible. This change
was attributed to the conjugation of 1 to the glutamine side
chains of albumin, causing a decrease in diamagnetic amines
and an increase in paramagnetic amides within the sample.
More specifically, the newly formed amide was expected to
form a dative bond with the Tm(III) ion, causing the amide
proton to experience a hyperfine contact shift from the
paramagnetic Tm(III) ion and resonate at a unique value of
Figure 4. Lorentzian line shape spectra of peptides and Tm-DO3A-
cadaverine. (A) GQR, (B) QR, (C) ZQG, and (D) Boc-Gln-OH
before (black) and after TGase catalysis (red).
and albumin, the CEST effect at −10.8 ppm was assigned to the
paramagnetic amide created by TGase activity. The appearance
of CEST at +5.8 ppm after conjugation to GQR or QR was
attributed to a change in the chemical exchange rate of the QR
amide proton or the guanidinium group of arginine after
conjugation to 1. CEST at +5.8 ppm was unexpected, and
further demonstrated the sensitivity of CEST agents to ligand
conformations that cause changes in chemical exchange rates,
which may be exploited to create additional responsive
molecular imaging agents.
−
9.2 ppm, as shown in similar reports of CEST with Tm(III)
chelates.
5,12
The CEST spectrum of 1 alone showed a weak CEST effect
at −5.2 ppm (Figure 3). This paramagnetic CEST effect was
We investigated the TGase-catalyzed creation of amide
bonds between 1 and other peptides. The conjugation of 1 and
CBZ-protected QG generated a three CEST effects at +4.6,
+
9.8, and +22.5 ppm (Figure 4C). These positive CEST effects
were attributed to heterogeneous ligand conformations of the
hydrophobic CBZ-QG peptide. The conjugation of 1 and Boc-
protected glutamine converted a CEST effect at +7.2 ppm to a
broad CEST effect between −10 and −20 ppm (Figure 4D).
This result was attributed to the hydrophobicity of Boc-Gln-
OH, which may cause conformational heterogeneity that would
create a broad CEST effect. These studies were performed once
for the ZQG system and twice for Boc-Gln-OH. These results
demonstrate that a full CEST spectrum should be acquired
during in vivo studies to ensure the detection of CEST effects
that arise from 1 conjugated to hydrophilic or hydrophobic
proteins.
Figure 3. CEST of individual reactants. (A) The CEST spectrum and
(
B) Lorentzian line shape spectrum of an individual sample of albumin
showed CEST at +5.6 ppm (purple circles and purple line), L-
glutathione showed CEST at +5.4 ppm (green circles and green line),
and Tm-DO3A-cadaverine showed CEST at −5.2 ppm (blue circles
and blue line).
attributed to a non-covalent supra-molecular adduct between
the aliphatic amines and the carboxylate groups of DO3A.
Similar non-covalent interactions have been observed with
1
3
lanthanide chelates. This observation demonstrated the
sensitivity of CEST agents to ligand interactions that may be
further exploited to create responsive molecular imaging agents.
As expected, the CEST spectra of albumin or glutathione alone
A responsive CEST effect can also arise from changes in
chemical exchange rates. The Bloch-McConnell equations
modified for chemical exchange were fit to CEST spectra to
1
4,15
16,17
showed a CEST effect at +4.6 ppm.
These control samples
measure chemical exchange rates (Table S1).
The average
were only analyzed once because no reaction was involved.
To further validate the creation of a covalent bond between 1
and a side chain of glutamine, CEST spectra were recorded
before and after the addition of 0.5 unit of TGase to a solution
of 25 mM 1, 25 mM GQR peptide, and 10 mM glutathione in
the same buffer (Figure 4A). Before addition of TGase, a weak
CEST effect was detected at −9.0 ppm from the supra-
molecular adduct of 1. After addition of TGase, the CEST
effect shifted to −10.8 ppm and dramatically increased, and a
new CEST effect was observed at +5.8 ppm. Similar results
were obtained before and after conjugating 1 with a QR peptide
using TGase (Figure 4B). These reactions with the two
peptides were reapeated three times to ensure that the changes
in CEST effects were reproducible. As with the reaction of 1
chemical exchange rate of 1 that generated weak CEST at
−12.7 ppm was determined to be 2533 Hz. This rate is slower
than expected for an amine group at 37 °C and pH 7.0, which
suggested that the aliphatic amine had an intra- or
18
intermolecular interaction that reduced the exchange rate.
After 1 was conjugated to albumin, the strong CEST at −11.6
ppm had an average chemical exchange rate of 1,260 Hz. This
rate is much faster than expected for a diamagnetic amide group
at 37 °C and pH 7.0, which suggested that the amide of the
14
product was proximal to the lanthanide ion. Furthermore, this
measured change in exchange rate from 2533 Hz before TGase
catalysis to 1,260 Hz after TGase catalysis provided an
additional method for detecting the creation of a covalent
bond by TGase enzyme activity.
B
dx.doi.org/10.1021/ja400254e | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
The chemical exchange rates of 1 conjugated to CBZ-
protected QG were determined to be 3323 Hz at 22.5 ppm and
Arizona Cancer Center, and the Community Foundation of
Southern Arizona.
6
739 Hz at 9.8 ppm. These chemical exchange rates are faster
than expected for diamagnetic amide and carbamate groups,
which suggests that the amide or carbamate protons in this
product are proximal to the lanthanide ion.
Detecting TGase catalysis in biological systems has been
accomplished by using a T1 relaxation agent attached to a short
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ASSOCIATED CONTENT
■
(
23) Yoo, B.; Sheth, V. R.; Howison, C. M.; Douglas, M.; Pineda, C.;
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Synthesis methods, characterization of synthesis products,
CEST MR methods, CEST spectra, and the fittings of
Lorentzian line shapes and Bloch equations to CEST spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
24) Li, A. X.; Hudson, R. H. E.; Barrett, J. W.; Jones, C. K.;
AUTHOR INFORMATION
■
Corresponding Author
mpagel@u.arizona.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Dr. Nabila Brabez for advice regarding
synthesis, and Dr. Julio Car
discussions. This work was supported by NIH grants R01
CA169774-01 and P50 CA95060, the Phoenix Friends of the
́
́
denas-Rodriguez for helpful
C
dx.doi.org/10.1021/ja400254e | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX