Radioiodination of cyanocobalamin conjugates containing hydrophilic linkers: preparation of a radioiodinated cyanocobalamin monomer and two dimers, and assessment of their binding with transcobalamin II.
This report describes an investigation aimed at preparation of radioiodinated cyanocobalamin (CN-Cbl) monomers and dimers with improved water solubility and decreased nonspecific binding. In the investigation, synthesis and radioiodination reactions of one monomeric and two dimeric CN-Cbl derivatives were conducted. The initial step in the synthesis of the CN-Cbl derivatives was mild acid hydrolysis of CN-Cbl, 1, followed by separation of the resultant corrin ring b-, d-, and e-monocarboxylate isomers. The investigation was limited to preparation of conjugates of CN-Cbl-e-carboxylate, 2, as earlier studies had shown binding of that isomer with recombinant human transcobalamin II (rhTCII) was similar to CN-Cbl. In a second synthetic step, the hydrophilic linker moiety, 4,7,10-trioxa-1,13-tridecandiamine, 3, was conjugated with 2 to form the adduct, 4. The synthesis of a monomeric CN-Cbl derivative, 6a, which can be used for radioiodination, was accomplished by reaction of 4 with p-tri-n-butylstannylbenzoate tetrafluorophenyl (TFP) ester, 5a. Two CN-Cbl dimers containing the arylstannane radioiodination moiety were also synthesized. The first dimer, 8a, was synthesized by cross-linking 4 with a stannylbenzoyl-aminoisophthalate di-TFP ester, 7a. The second dimer, 11a, was synthesized by reacting benzene tricarboxylate tri-TFP ester, 10, in a stepwise manner with 1 equiv of the adduct of 5a and 3 (forming 9a), followed by 2 equiv of 4. Iodobenzoate HPLC standards, 6b, 8b, and 11b, used in the radioiodination studies, were prepared in a manner similar to that of the stannylbenzoate derivatives. Radioiodinations were performed by reacting 6a, 8a, or 11a with N-chlorosuccinimide and Na[(125)I]I in methanol under neutral conditions. Radiochemical yields of 17-42% were obtained. Evaluation of the binding properties of radiolabeled CN-Cbl conjugates with rhTCII showed that the dimer of CN-Cbl, 11b, bound more avidly than the monomer, 6b, and that the binding affinity of the dimer is essentially equivalent to that of unmodified CN-Cbl. Incubation of radioiodinated monomer, [(125)I]6b, and dimer, [(125)I]11b, with rhTCII followed by size-exclusion chromatographic analysis provided data that the monomer bound one rhTCII molecule whereas two rhTCII molecules were bound to approximately 30% of the dimer.
Wilbur
D S
DS
Department of Radiation Oncology, University of Washington, Seattle, Washington 98195, USA. dswilbur@u.washington.edu
Pathare
P M
PM
Hamlin
D K
DK
Rothenberg
S P
SP
Quadros
E V
EV
D001667:Binding, Competitive; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods; D011485:Protein Binding; D019275:Radiopharmaceuticals / Q000138:chemical synthesis* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D014155:Transcobalamins / Q000378:metabolism*; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D014867:Water / Q000737:chemistry
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D019275:Radiopharmaceuticals; D011994:Recombinant Proteins; D014155:Transcobalamins; D014867:Water; D014805:Vitamin B 12