We report a novel ditopic Gd(III)-based probe selective to zwitterionic amino acid neurotransmitters (ZNTs) crafted for ratiometric MRI imaging. The probe displayed increased binding affinity to ZNTs and non-synchronized concentration-dependent changes of the r 1 -and r 2 -relaxivity. Through the application of a T 2 /T 1 weighted MRI strategy, we demonstrated signal enhancement for cooperatively bound glutamate and c-aminobutyric acid ZNTs over competitive hydrogencarbonate, which remained MR silent.A wide variety of neuronal diseases are characterized by the overexpression of zwitterionic amino acid neurotransmitters (ZNTs) into the synaptic cleft, which in abnormal amounts trigger a plethora of processes resulting in the partial impairment or death of nerve cells. 1 Excess secretion of the major excitatory glutamate (Glu) or inhibitory g-aminobutyric acid (GABA) ZNTs causes an imbalance in the excitation level of the brain, leading to various diseases, such as epileptic seizures that result in the degeneration of neuronal tissue. Thus, decoding the spatiotemporal patterns of brain chemodynamics could imply connection with related diseases. For this reason, ZNTs are recognized as irreplaceable biomarkers for monitoring neuronal activity. Currently, non-invasive imaging techniques based on magnetic resonance (MR), i.e. diamagnetic chemical exchange saturation transfer (diaCEST) or 13 C MR spectroscopy, are preferably used due to good spatio-temporal resolution and depth of penetration. However, they do not distinguish between intra-and extracellular concentration of ZNTs, and have insufficient chemical resolution and low sensitivity. 2,3 A promising way to address these issues involves the introduction of a paramagnetic sensor capable of altering image contrast upon interaction with the target metabolite. 4 Therefore, the development of MR imaging (MRI) probes responsive to extracellular ZNTs is of emerging importance.Molecular recognition between magnetic host sensors and guest molecules occurs via the formation of a ternary adduct, and is evaluated via its binding affinity. In the case of a 'turn-off' response, the interaction of biomarkers with the coordination cage of an MRI probe restricts inner-sphere water accessibility to the paramagnetic center resulting in a signal decrease. 5 So far, the direct sensing of NTs has been approached with two distinct designs, via genetically engineered metalloproteins and by small-molecule ditopic paramagnetic probes. 6-8 Although large molecule CAs tailored for monoamine neurotransmitters were shown to be capable of detecting target metabolites in the mM range, further improvement of small-sized probes is imperative due to issues associated with translation into biological systems. In terms of host-guest interaction, the low-molecular-weight sensors are bismacrocyclic Gd(III)-based complexes with a 1,4,7,10tetraazacyclododecane-1,7-dicarboxylic acid (DO2A) chelator, whose geometry allows for easy access to small anions. 9 In particular, the selectivity of the current sen...

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA - 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

A novel nano‐sized MRI probe sensitive to zwitterionic amino acid neurotransmitters (ZNTs) was prepared by the conjugation of bismacrocyclic ZNT‐responsive units to a generation 4 (G4) polyamidoamine (PAMAM) dendrimer. In vitro evaluation through 1H NMR relaxometric titrations provided binding profiles revealing a surface morphology‐dependent interaction with the ZNTs. The positively charged dendrimeric periphery gave higher initial relaxivities and a milder decrease upon interaction with the target molecules. To improve solubility under physiologically relevant conditions, polyethylene glycol (PEG) units were additionally conjugated to the dendrimeric surface. The PEGylated conjugate displayed discrimination towards acetylcholine and a slightly increased binding affinity towards glutamate over the massively abundant competitive metabolite hydrogencarbonate.

The efficacy of Gd-based low-molecular weight ditopic MRI probes on binding zwitterionic neurotransmitters (ZNTs) relies on their structural compatibility. ZNTs are challenging biomarkers for monitoring chemical neurotransmission due to their intrinsic complexity as target molecules. In this work, we focus on tuning the cyclen- and azacrown ether-based binding sites properties to increase the affinity toward ZNTs. Our approach consisted in performing structural modifications on the binding sites in terms of charge and size, followed by the affinity evaluation through T 1 -weighted relaxometric titrations. We prepared and investigated six Gd 3+ complexes with different structures and thus properties, which were found to be acetylcholine insensitive; moreover, two of them displayed considerably stronger affinity toward glutamate and glycine over hydrogencarbonate and other ZNTs. Complexes with small and non-charged or no substituents on the azacrown moiety displayed the highest affinities toward ZNTs, followed by strong decrease in longitudinal relaxivity r 1 of around 70%. In contrast, hosts with negatively charged substituents exhibited lower decrease in r 1 of nearly 30%. The thorough investigations involving relaxometric titrations, luminescence, and NMR diffusion experiments, as well as theoretical density functional theory calculations, revealed that the affinity of reported hosts toward ZNTs is greatly affected by the remote pendant on the azacrown derivative.