Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry

Abstract

The lanthanides (Ln 3+ ), or rare earth elements, have proven to be useful tools for biomolecular NMR, X-ray crystallographic, and fluorescence analyses due to their unique 4 f orbitals. However, their utility in biological applications has been limited because site-specific incorporation of a chelating element is required to ensure efficient binding of the free Ln 3+ ion. Additionally, current Ln 3+ chelator syntheses complicate efforts to directly incorporate Ln 3+ chelators into proteins as the multi-step processes and a reliance on organic solvents promote protein denaturation and aggregation which are generally incompatible with direct incorporation into the protein of interest. To overcome these limitations, herein we describe a two-step aqueous synthesis of a small molecule lanthanide chelating agent amenable to site-specific incorporation into a protein using copper-free click chemistry with unnatural amino acids. The bioconjugate combines a diethylenetriaminepentaacetic acid (DTPA) chelating moiety with a clickable dibenzylcyclooctyne-amine (DBCO-amine) to facilitate the reaction with an azide containing unnatural amino acid. Incorporating the DBCO-amine avoids the use of the cytotoxic Cu 2+ ion as a catalyst. The clickable lanthanide chelator (CLC) reagent reacted readily with p -azidophenylalanine (paF) without the need of a copper catalyst, thereby demonstrating proof-of-concept. Implementation of the orthogonal click chemistry reaction has the added advantage that the chelator can be used directly in a protein labeling reaction, without the need of extensive purification. Given the inherent advantages of Cu 2+ -free click chemistry, aqueous synthesis, and facile labeling, we believe that the CLC will find abundant use in both structural and biophysical studies of proteins and their complexes.

Synthesis and Characterization of Carboxymethylated Polyethylenimines

“…Additionally, the functionalization degree indicates that >30 mol % of the primary/secondary amino groups are still not fully functionalized even in CM-PEI_4. Also, the chemical shift value of 3.85 ppm is completely consistent with a methylene adjacent to a di­(carboxymethyl)­amino group, the same value was found for DTPA in a very large pH range between 2 and 10. , …”