Electrophilic chelating agents for irreversible binding of metal chelates to engineered antibodies

Chmura, Albert J.; Schmidt, Brian D.; Corson, Donald T; Traviglia, Stacey; Meares, Claude F.

doi:10.1016/s0168-3659(01)00485-0

Cited by 14 publications

(15 citation statements)

References 19 publications

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“…The starting metal-chelating compounds used for the electrochemical functionalization of carbon electrode surfaces, i.e., 4-aminophenyl-IDA ( 3 ) and 4-aminophenyl-NTA ( 6 ), were synthesized as outlined in Scheme . The synthesis of 3 was based upon a literature method used to prepare 1-(4-aminobenzyl)ethylenediamine- N , N , N ‘, N ‘-tetraacetic acid 2 Synthesis of IDA- and NTA-Terminated Aniline Derivatives …”

Section: Resultsmentioning

confidence: 99%

Dense Monolayers of Metal-Chelating Ligands Covalently Attached to Carbon Electrodes Electrochemically and Their Useful Application in Affinity Binding of Histidine-Tagged Proteins

et al. 2005

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In this work, monolayers of metal complexes were covalently attached to the surface of carbon electrodes with the goal of binding monolayers of histidine-tagged proteins with a controlled molecular orientation and a maintained biological activity. In this novel method, which is simple, versatile, and efficient, the covalent attachment was accomplished in a single step by the electrochemical reduction of aryl diazonium ions that were substituted with a nitrilotriacetic (NTA) or an imminodiacetic (IDA) ligand at the para position. The transient aryl radicals that were generated in the reduction were grafted to the surfaces of glassy carbon, highly oriented pyrolitic graphite, and graphite-based screen-printed electrodes, producing dense monolayers of the ligands. The NTA- and IDA-modified electrodes were shown to efficiently chelate Cu(II) and Ni(II) ions. The presence of the metal was established using X-ray photoelectron spectroscopy and electrochemistry. Surface coverages of the ligands were indirectly determined from the electroactivity of the copper(II) complex formed on the electrode surface. Studies on the effect of electrodeposition time and potential showed that, at sufficiently negative potentials, the surface coverage reached a saturating value in less than 2 min of electrodeposition time, which corresponds to the formation of a close-packed monolayer of ligand on the electrode surface. Once loaded with a metal ion, the modified electrode was able to bind specifically to histidine-tagged proteins such as the horseradish peroxidase (His-HRP) or to an enhanced, recombinant green-fluorescent protein via its N-terminal hexahistidine tail. In the case of His-HRP, the amount of active enzyme specifically immobilized by metal-chelating binding was determined from the analysis of electrocatalytic currents using cyclic voltammetry. The electrochemical grafting makes it possible to accurately controlled and electronically address the amount of deposited ligand on the conductive surfaces of carbon electrodes with any size and shape.

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Section: Resultsmentioning

confidence: 99%

Dense Monolayers of Metal-Chelating Ligands Covalently Attached to Carbon Electrodes Electrochemically and Their Useful Application in Affinity Binding of Histidine-Tagged Proteins

et al. 2005

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“…We also designed and synthesized several bifunctional chelating agents bearing reactive groups to form covalent bonds with the engineered site on the antibody (see examples in Fig. 2); (Chmura et al, 2002).…”

Section: Methodology For Infinite Affinitymentioning

confidence: 99%

“…To confirm our design of weakly electrophilic chelates we tested them in vivo (Chmura et al, 2002). Para substituted derivatives of benzyl-EDTA were sythesized by alkylation of tert-butyl aminobenzyl-EDTA with the acid chlorides of chloroacetic acid, chloropropionic acid, acryloyl chloride and bromoacetyl bromide.…”

Section: Methodology For Infinite Affinitymentioning

confidence: 99%

Molecular tools for targeted imaging and therapy of cancer

Meares

Chmura

Orton

et al. 2003

J of Molecular Recognition

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Here we review an approach to the design and production of antibody/ligand pairs for use in cell targeting procedures, to achieve functional affinity far greater than avidin/biotin. Using fundamental chemical principles, we have developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. By eliminating the dissociation of the ligand from the antibody, we have made the affinity functionally infinite. This methodology is applicable to other biological binding pairs.

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“…A S95C mutation was introduced into the sequence of CHA255 by site-directed mutagenesis, and a chimeric Fab incorporating the engineered mutant was expressed and evaluated for permanent binding activity: the CHA255 S95C mutant readily formed a covalent bridge to In-AABE. The acrylamide-based Michael acceptor displayed minimal cross-reactivity with biologically relevant nucleophiles, such as albumin and glutathione, relative to a non-electrophilic control in vitro and in vivo [75,91]. Requirements for a successful infinite affinity system involving a synthetic probe molecule and a genetically engineered receptor include no covalent bond formation with molecules other than the binding partner, and no significant affinity for molecules other than the binding partner.…”

Section: Irreversibly Binding Anti-metal Chelate Antibodies: Artificimentioning

confidence: 99%

“…The cysteine nucleophile is designed to lie close to the electrophilic domain of a 2D12.5 bound Y-DOTA ligand such as Y-AABD, which incorporates a relatively unreactive acrylamide. A weakly electrophilic moiety such as an acrylamide-based Michael acceptor is useful because it displays minimal cross-reactivity with biologically relevant nucleophiles, such as albumin and glutathione [75,87,91]. The mutual reactivities of the cysteine and acrylamide groups on the protein and ligand are significantly enhanced by the increase in effective local concentration that results from the high affinity binding interaction, yielding a permanent covalent bond between the receptor and ligand.…”

Section: Irreversibly Binding Anti-metal Chelate Antibodies: Artificimentioning

confidence: 99%

Irreversibly binding anti-metal chelate antibodies: Artificial receptors for pretargeting

Corneillie¹,

Whetstone²,

Meares³

2006

Journal of Inorganic Biochemistry

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Electrophilic chelating agents for irreversible binding of metal chelates to engineered antibodies

Cited by 14 publications

References 19 publications

Dense Monolayers of Metal-Chelating Ligands Covalently Attached to Carbon Electrodes Electrochemically and Their Useful Application in Affinity Binding of Histidine-Tagged Proteins

Dense Monolayers of Metal-Chelating Ligands Covalently Attached to Carbon Electrodes Electrochemically and Their Useful Application in Affinity Binding of Histidine-Tagged Proteins

Molecular tools for targeted imaging and therapy of cancer

Irreversibly binding anti-metal chelate antibodies: Artificial receptors for pretargeting

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