Efficient Site-Specific Radiolabeling of a Modified C2A Domain of Synaptotagmin I with [99mTc(CO)3]+: A New Radiopharmaceutical for Imaging Cell Death

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• 作者：Richard Tavar ; Rafael Torres Martin De Rosales ; Philip J. Blower ; Gregory E. D. Mullen
• 刊名：Bioconjugate Chemistry
• 出版年：2009
• 出版时间：November 18, 2009
• 年：2009
• 卷：20
• 期：11
• 页码：2071-2081
• 全文大小：350K
• 年卷期：v.20,no.11(November 18, 2009)
• ISSN：1520-4812

文摘

We describe the design and synthesis of a new Tc-99m labeled bioconjugate for cell-death imaging, based on C2A, the phosphatidylserine (PS)-binding domain of rat synaptotagmin I. Since several lysine residues in this protein are critical for PS binding, we engineered a new protein, C2AcH, to include the C-terminal sequence CKLAAALEHHHHHH, incorporating a free cysteine (for site-specific covalent modification) and a hexahistidine tag (for site-specific radiolabeling with [^99mTc(CO)₃(OH₂)₃]⁺). We also engineered a second derivative, C2Ac, in which the C-terminal sequence included only the C-terminal cysteine. These proteins were characterized by electrospray mass spectrometry, SDS/PAGE, and size exclusion chromatography and radiolabeled with [^99mTc(CO)₃(OH₂)₃]⁺. Conjugates of the proteins with the rhenium analogue [Re(CO)₃(OH₂)₃]⁺ were also synthesized. Site-specific labeling was confirmed by performing a tryptic digest of rhenium tricarbonyl-labeled C2AcH, and only peptides containing the His-tag contained the [Re(CO)₃]⁺. The labeled proteins were tested for binding to red blood cells (RBC) with exposed PS in a calcium dependent manner. Labeling 100 μg of C2AcH with [^99mTc(CO)₃(OH₂)₃]⁺ at 37 °C for 30 min gave a radiochemical yield of >96%. However, C2AcH that had first been conjugated with fluorescein maleimide or iodoacetamide via the Cys residue gave only 50% and 83% radiochemical yield, respectively, after incubation for 30 min at 37 °C. Serum stability results indicated that >95% of radiolabeled C2AcH remained stable for at least 18 h at 37 °C. Site-specifically labeled C2AcH exhibited calcium-dependent binding to the PS on the RBC, whereas a nonspecifically modified derivative, C2AcH-B, in which lysines had been modified with benzyloxycarbonyloxy, did not. We conclude that (i) the combination of Cys and a His-tag greatly enhances the rate and efficiency of labeling with [^99mTc(CO)₃(OH₂)₃]⁺ compared to either the His-tag or the Cys alone, and this sequence deserves further evaluation as a radiolabeling tag; (ii) non-site-specific modification of C2A via lysine residues impairs target binding affinity; (iii) ^99mTc-C2AcH has excellent radiolabeling, stability and PS binding characteristics and warrants in vivo evaluation as a cell-death imaging agent.