DBCO-amine

Formation of Chelate by DBCO-amine
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
(1) Metalation of Diethylenetriaminepentaacetic Acid (DTPA):​ Dissolve 10mM DTPA in 100mM MES buffer (pH=5.5) and heat to 75°C to promote dissolution. Add 10mM TbCl₃ and incubate at 75°C for 10 minutes to allow Tb³⁺ to chelate with DTPA, forming the DTPA:Tb complex.
(2) Click Reaction Coupling:​ Dissolve DBCO-amine in DMSO (final concentration 8.33%) and mix with 10mM DTPA:Tb in 100mM MES buffer (pH=5.5), ensuring the final concentration of DBCO-amine is 50mM. Add 50mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and react at room temperature under gentle shaking in the dark for 120 minutes. This facilitates the formation of an amide bond between the amino group of DBCO-amine and the central carboxyl group of DTPA:Tb, yielding the lanthanide chelator (CLC).
(3) Purification and Verification: Load the reaction mixture onto a pre-equilibrated Sephadex G10 size exclusion column and elute with 100mM MES buffer (pH=5.5). Collect fractions with a molecular weight ≥700Da (containing CLC). Verify the formation and purity of CLC using fluorescence spectroscopy (excitation at 350nm, detection at characteristic emission peaks such as 429nm and 544nm) and UPLC-MS (m/z = 806.2).
Precautions:
(1) DBCO-amine is light-sensitive. All steps should be performed in the dark to maintain reagent stability.
(2) For safety and health, wear a lab coat and disposable gloves during operation.

References:
[1] Bishop SC, Winefield R, Anbanandam A, et al. Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry. PLoS One. 2019 Mar 27;14(3):e0209726.