Synthesis of aminobenzyltriethylenetetraamino-hexaacetic acid: Conjugationof the chelator to protein by an alkylamine linkage

The conjugation of a chelating agent to an antibody as an anchoring site fo r a radionuclide is the first step in the successful preparation of a radio labeled antibody for a diagnostic and therapeutic application. The high aff inity of the protein bound chelator towards radionuclide ensures a higher s electivity in the delivery of the radionuclide to the targeted tissue. 4-Am inobenzylderivativetriethlenetetraaminohexaacetic acid (TTHA), a hexadentat e chelating agent has been now prepared for conjugation with proteins in vi ew of the higher affinity of TTHA metal ions as compared to DTPA. The laten t crosslinking potential of alpha-hydroxy aldehydes has been used to conjug ate the new chelating agent to proteins through an alkylamine linkage. On i ncubation of amino benzyl TTHA with glycoladehyde at neutral pH and room te mperature, the reagent is converted to oxoethyl amino benzyl TTHA. On addit ion of albumin to this reaction mixture, the oxo ethylamino benzyl TTHA gen erates reversible schiff base adducts with the amino groups of albumin. The reduction of the Schiff base adducts of the chelator with the protein by s odium cyanoborohydride stabilizes the schiff base adducts as stable alkylam ine linkages. 4-Thiocyanatobenzyl TTHA has also been prepared and conjugate d to albumin through a thiocarbamoyl linkage. Both preparations of TTHA con jugated albumin complexed with Tc-99m and In-111, with high affinity and no decomposition of the complex was noticed for at least up to 6 hrs after th e preparation. The radiolabels complexed with these TTHA -albumin conjugate s could not be 'chased' out by free DTPA. A comparison of the biodistributi on of In-111, bound to the TTHA conjugated through an alkylamine and a thio carbamoyl linkage showed that In-111 complexed with alkylamine linked TTHA was retained in blood to a level nearly 17% higher compared to that seen wi th thicarbamoyl linked TTHA, one hr after the injection into mice. Thus, th e alkylamine linkage appears to be more stable under the in vivo conditions . The glycolaldehyde mediated alkylation procedure offers a mild, simple an d rapid method for preparation of drug-protein (antibody) conjugates.