NMR Studies of the Metal-Loading Kinetics and Acid−Base Chemistry of DOTA and Butylamide-DOTA

• Published in: Bioconjugate chemistry 1999-05, Vol.10 (3), p.454-463
• Main Authors: Keire, David A, Kobayashi, Mitsuo
• Format: Article
• Language: English
• Subjects: Heterocyclic Compounds, 1-Ring - chemistry; Hydrogen-Ion Concentration; Kinetics; Magnetic Resonance Spectroscopy; Titrimetry; Yttrium - chemistry
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc980128u

The conjugation of a chelating agent to a protein via a covalent linkage has been previously reported to change the metal-binding characteristics of the chelator. A fundamental understanding of these binding changes would enable design of a new generation of metal-chelating agents for biological applications. To assess the effect of conjugation on the commonly used chelating agent 1 4,7,10-tetraaazacyclododecane-N,N‘,N‘ ‘,N‘ ‘‘-tetraaacetic acid (DOTA), we synthesized a model protein conjugate, 1,4,7-tris(carboxymethyl)-10-(butylaminocarboxymethyl)-1,4,7,10-tetraaazacyclododecane (BD) and explored the metal-binding characteristics via NMR. The extent of ionization of the carboxylic acid groups and the two protonated macrocycle nitrogens of DOTA and BD were determined as a function of pH by chemical shift changes in proximal carbon-bonded protons. In addition to the expected sensitivity of the chemical shifts to titration of proximate acidic groups, BD resonances from carbon-bonded protons 10−17 bonds distant from the deprotonation site also shifted significantly, indicating the presence of conformational changes. Furthermore, increased shielding of the amide and alkyl proton signals upon deprotonation of the carboxylic acid groups indicates the presence of pH-dependent hydrogen-bonded BD isoforms. On the basis of these NMR data, we propose new structures for the doubly protonated forms of DOTA and BD. To measure metal loading, the yttrium-loading rates (type I to type II) of DOTA and BD were determined by following the intensity of type I and type II proton signals as a function of time. The yttrium-loading rates of BD are approximately one-half those of DOTA at pHs between 4.6 and 6.5 and 37 °C. The loading rates measured as a function of pH indicate that while both the doubly protonated and singly protonated forms of DOTA are reactive to metal loading, only the singly protonated form of BD is reactive.