Abstract
The creation of discrete, covalent bonds between a protein and a functional molecule like a drug, fluorophore, or radiolabeled complex is essential for making state-of-the-art tools that find applications in basic science and clinical medicine. Photochemistry offers a unique set of reactive groups that hold potential for the synthesis of protein conjugates. Previous studies have demonstrated that photoactivatable desferrioxamine B (DFO) derivatives featuring a para-substituted aryl azide (ArN3) can be used to produce viable zirconium-89-radiolabeled monoclonal antibodies (89Zr-mAbs) for applications in noninvasive diagnostic positron emission tomography (PET) imaging of cancers. Here, we report on the synthesis, 89Zr-radiochemistry, and light-triggered photoradiosynthesis of 89Zr-labeled human serum albumin (HSA) using a series of 14 different photoactivatable DFO derivatives. The photoactive groups explore a range of substituted, and isomeric ArN3 reagents, as well as derivatives of benzophenone, a para-substituted trifluoromethyl phenyl diazirine, and a tetrazole species. For the compounds studied, efficient photochemical activation occurs inside the UVA-to-visible region of the electromagnetic spectrum (∼365–450 nm) and the photochemical reactions with HSA in water were complete within 15 min under ambient conditions. Under standardized experimental conditions, photoradiosynthesis with compounds 1–14 produced the corresponding 89ZrDFO-PEG3-HSA conjugates with decay-corrected isolated radiochemical yields between 18.1 ± 1.8% and 62.3 ± 3.6%. Extensive density functional theory (DFT) calculations were used to explore the reaction mechanisms and chemoselectivity of the light-induced bimolecular conjugation of compounds 1–14 to protein. The photoactivatable DFO-derivatives operate by at least five distinct mechanisms, each producing a different type of bioconjugate bond. Overall, the experimental and computational work presented here confirms that photochemistry is a viable option for making diverse, functionalized protein conjugates.
Highlights
Chemical modification of proteins plays an essential role in the development of many state-of-the-art diagnostic and therapeutic agents used in fundamental science and clinical medicine.[1−3] For instance, the high affinity and specificity of monoclonal antibodies provides the basis of molecularly targeted antibody-drug conjugates (ADCs) via functionalization with a cargo molecule.[4,5] The cargo can be a cytotoxic drug for therapeutic intervention, a fluorophore for optical imaging, or a radioactive complex for applications in positron-emission tomography (PET) imaging or radioimmunotherapy
The photoactivatable desferrioxamine B (DFO)-derivatives operate by at least five distinct mechanisms, each producing a different type of bioconjugate bond
The synthetic route toward photoactivatable derivatives of the hexadentate chelate, DFO bearing a water-solubilizing tris-polyethylene glycol (PEG3) spacer is presented in Scheme 1.40 In general, DFO-PEG3 derivatives were synthesized in four linear steps starting from a carboxylic acid derivative of the photoactivatable unit
Summary
Chemical modification of proteins plays an essential role in the development of many state-of-the-art diagnostic and therapeutic agents used in fundamental science and clinical medicine.[1−3] For instance, the high affinity and specificity of monoclonal antibodies (mAbs) provides the basis of molecularly targeted antibody-drug conjugates (ADCs) via functionalization with a cargo molecule.[4,5] The cargo can be a cytotoxic drug for therapeutic intervention, a fluorophore for optical imaging, or a radioactive complex for applications in positron-emission tomography (PET) imaging or radioimmunotherapy. Formation of a chemically and metabolically stable covalent bond between the protein and the exogenous cargo molecule is central to many successful bioconjugation strategies. Traditional protein bioconjugation methods usually exploit the native reactivity of amino acid side chain groups to create new linkages. Used methods include the reaction of free sulfhydryl groups of cysteine (Cys) residues on proteins with reagents bearing Michael acceptors, or the use of electrophilic reagents including activated esters or isothiocyanates that react with the nucleophilic ε-NH2 group on lysine (Lys) residues to form biocompatible, and generally stable, amide or thiourea bonds.[5] Conjugation chemistries based on these routes are highly successful in the clinic, but there is growing appreciation that the nature of the bioconjugate bond can influence the pharmacokinetics and overall performance of the protein conjugate in vivo
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