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Introduction Smart MR contrast agents (CA) exhibit modulation of their relaxivity by specific physiological or biochemical trigger-events. In an effort to develop novel smart MR contrast agents, the multifunctional chelating agent DO3A-EA has been synthesized. It serves as a valuable multipurpose precursor for smart contrast agents based on Gadolinium chelates in the design of relaxometric MR probes.
Synthesis 1,4,7-tris(carboxymethyl)10-(aminoethyl)-1,4,7,10-tetraazacyclododecane (DO3A-EA) was synthesized from cyclen by the reaction of tert-butylbromoacetate to get the tri-substituted product. It was further reacted with N-Boc-2-bromoethylamine to get 1,4,7-tris(carbobutoxymethyl)-10-(Boc-aminoethyl)-1,4,7,10 tetraazacyclododecane. The corresponding carboxylate derivative DO3A-EA was obtained by cleaving the tert-butyl groups by the treatment of DCM/TFA at RT. Yield was 85.
Results With DO3A-EA as precursor, following CAs were synthesized and tested:
Gd-DO3A-E-NCS: It forms stable macrocyclic complex with Gd(III) and can be used in Gd-preloading approach to avoid the binding of gadolinium with calcium binding chelates. MR relaxivity of Gd-DO3A-E-NCS (pH 7.5) was r1 = (3.29 ± 0.08) s-1mM-1.
Gd-DO3A-E-biotin: It can be used for targeted imaging in an antibody-avidin system. MR relaxivity of Gd-DO3A-E-biotin (pH 7.5) was r1 = (4.85 ± 0.08) s-1mM-1. Mixture of Gd-DO3A-E-biotin and avidin (4:1) showed 30 relaxivity enhancement for r1 and 311 for r2 relative to the unbound biotinylated Gd(III) complex.
DO3A-E-FITC: It can be used to track cellular binding and internalization. Additional loading with Gd3+ can provide MR contrast. DO3A-E-FITC and Eu-DO3A-E-FITC up to 50 μM did not show cytotoxicity after treatment for 24 hrs (NIH-3T3 cells, PI assay). Fluorescence microscopy of living cells displayed proper co-localization.
