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

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 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.

Fig. 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.

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.

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).

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).