Protonation of carboxyl groups in EuDOTA-tetraamide complexes results in catalytic prototropic exchange and quenching of the CEST signal.

The CEST properties of EuDOTA-tetraamide complexes bearing pendant carboxylate and carboxyl ethyl esters were measured as a function of pH. The CEST signal from the Eu3+-bound water molecule decreased in intensity between pH 8.5 and 4.5 while the proton exchange rates (kex) increased over this same pH range. In comparison, the CEST signal in the corresponding carboxyl ester derivatives was nearly constant. Both observations are consistent with stepwise protonation of the four carboxylic acid groups over this same pH range. This indicates that negative charges on the carboxyl groups above pH 6 facilitate the formation of a strong hydrogen-bonding network in the coordination second sphere above the single Eu3+-bound water molecule, thereby decreasing prototropic exchange of protons on the bound water molecule with bulk water protons. The percentage of square antiprismatic versus twisted square antiprism coordination isomers also decreased as the appended carboxylic acid groups were positioned further away from the amide. The net effect of lowering the pH was an overall increase in kex and a quenching of the CEST signal.This article is part of the themed issue 'Challenges for chemistry in molecular imaging'.

The presence of fast-exchanging proton species in aqueous solutions of paraCEST agents can impact rate constants measured for slower exchanging species when fitting CEST spectra to the Bloch equations.

LnDOTA-tetraamide complexes typically exist in solution as a mixture of square-antiprismatic (SAP) and twisted square-antiprismatic (TSAP) coordination isomers. In most cases, the SAP isomer, which is preferred for CEST imaging, predominates, and the presence of the minor TSAP isomer is assumed to have little influence on quantitative measures of the water-exchange rate constant for the SAP isomer. Here, we sought to confirm the validity of this assumption by mixing two chelates with different SAP and TSAP isomer populations while measuring the water-exchange rate constant of the SAP isomer. The results show that an increase in the population of the TSAP isomer in solution results in as much as a 30% overestimation of the water-exchange rate constant for the SAP isomer when CEST spectra are fit to the Bloch equations. This effect was shown to be significant only when the TSAP isomer population exceeded 50%.

The stereochemistry of amide side chains containing carboxyl groups influences water exchange rates in EuDOTA-tetraamide complexes.

Many Eu(III) complexes formed with DOTA-tetraamide ligands (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) have sufficiently slow water exchange kinetics to meet the slow-to-intermediate condition required to serve as chemical exchange saturation transfer (CEST) contrast agents for MRI. This class of MRI contrast agents offers an attractive platform for creating biological sensors because water exchange is exquisitely sensitive to subtle ligand stereochemistry and electronic effects. Introduction of carboxyl groups or carboxyl ethyl ester groups on the amide substituents has been shown to slow water exchange in these complexes, but less is known about the orientation or position of these side-chain groups relative to the inner-sphere Eu(III)-bound water molecule. In this study, a series of Eu(III) complexes having one or more carboxyl groups or carboxyl esters at the δ-position of the pendant amide side chains were prepared. Initial attempts to prepare optically pure EuDOTA-[(S)-Asp]4 resulted in a chemically pure ligand consisting of a mixture of stereochemical isomers. This was traced to racemization of (S)-aspartate diethyl ester during the synthetic procedure. Nevertheless, NMR studies of the Eu(III) complexes of this mixture revealed that each isomer had a different water exchange rate, differing by a factor of 2 or more. A second controlled synthesis and CEST study of EuDOTA-[(S)-Asp]4 and cis-EuDOTA-[(S)-Asp]2[(R)-Asp]2 confirmed that the water exchange rates in these diastereomeric complexes are controlled by the axial versus equatorial orientation of the carboxyl groups on the amide side chains. These observations provide new insights toward the development of even more slowly water exchanging systems which will be necessary for practical in vivo applications.

Nanoparticle-based PARACEST agents: the quenching effect of silica nanoparticles on the CEST signal from surface-conjugated chelates.

Silica nanoparticles of average diameter 53 ± 3 nm were prepared using standard water-in-oil microemulsion methods. After conversion of the surface Si-OH groups to amino groups for further conjugation, the PARACEST agent, EuDOTA-(gly)4 (-) was coupled to the amines via one or more side-chain carboxyl groups in an attempt to trap water molecules in the inner-sphere of the complex. Fluorescence and ICP analyses showed that approximately 1200 Eu(3+) complexes were attached to each silica nanoparticle, leaving behind excess protonated amino groups. CEST spectra of the modified silica nanoparticles showed that attachment of the EuDOTA-(gly)4 (-) to the surface of the nanoparticles did not result in a decrease in water exchange kinetics as anticipated, but rather resulted in a complete elimination of the normal Eu(3+) -bound water exchange peak and broadening of the bulk water signal. This observation was traced to catalysis of proton exchange from the Eu(3+) -bound water molecule by excess positively charged amino groups on the surface of the nanoparticles.

Amphiphilic EuDOTA-tetraamide complexes form micelles with enhanced CEST sensitivity.

The synthesis and characterization of four new DOTA-tetraamide ligands having variable alkyl chain lengths (C(1), C(12), C(14), and C(16)) and their respective europium (III) complexes are reported. The three EuL complexes having long alkyl chains spontaneously form micelles of variable size. The critical micelle concentration differed for each complex (lower for the C(16) complex than the C(12) complex) while micelle size increased with increasing alkyl chain length. Chemical exchange saturation transfer (CEST) experiments showed that all four Eu(III) complexes display slow-to-intermediate water exchange kinetics. As expected, the CEST signals in these complexes decreased with increasing temperatures due to faster water exchange but, interestingly, the CEST signals for the C(14) and C(16) complexes approached a maximum near 25°C consistent with exchange limited CEST at or near room temperature. The water residence lifetimes obtained by fitting the CEST spectra to the Bloch equations increased in parallel with an increase in alkyl carbon chain-length. By comparisons with the monomethylamide complex, which served as control, the data illustrate that micelle formation serves to slow the rate of water exchange in these systems. The complex having the largest CEST effect per unit Eu(III) concentration (the C(16) analog) had a detection limit of 5.3 µM. This represents an approximate 250-fold increase in sensitivity relative to the monomethylamide control (detection limit ~1.3 mM). These features highlight the potential of using micelle-based systems such as these as paramagnetic chemical exchange saturation transfer (PARACEST) agents for molecular imaging by MRI.