title: Efficient site-specific radiolabeling of a modified C2A domain of synaptotagmin I with [99mTc(CO)3]+: a new radiopharmaceutical for imaging cell death.
abstract: 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 microg of C2AcH with [99mTc(CO)3(OH2)3]+ at 37 degrees 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 degrees C. Serum stability results indicated that >95% of radiolabeled C2AcH remained stable for at least 18 h at 37 degrees 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.
authors.affiliation: Division of Imaging Sciences, King's College London, St. Thomas' Hospital, London SE17EH, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Tavaré
authors.forename: Rafael
authors.initials: R
authors.lastname: Torres Martin De Rosales
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Blower
authors.forename: Gregory E D
authors.initials: GE
authors.lastname: Mullen
mesh_terms: D000818:Animals; D001665:Binding Sites; D016923:Cell Death; D003952:Diagnostic Imaging / Q000379:methods; D015195:Drug Design; D004355:Drug Stability; D007553:Isotope Labeling / Q000379:methods*; D010718:Phosphatidylserines / Q000737:chemistry; D019275:Radiopharmaceuticals / Q000138:chemical synthesis* / Q000737:chemistry; D051381:Rats; D050863:Synaptotagmin I / Q000737:chemistry*; D013667:Technetium / Q000737:chemistry
chemical_list: D010718:Phosphatidylserines; D019275:Radiopharmaceuticals; D050863:Synaptotagmin I; D013667:Technetium