Novel carboranyl amino acids and peptides: reagents for antibody modification and subsequent neutron-capture studies

Abstract

A new alpha-amino acid derivative incorporating the 1,2-dicarba-closo- dodecarborane(12) cage, namely 5-(2-methyl-1,2-dicarba-closo-dodecarborane(12)-1-yl)- 2-aminopentanoic acid (2), was synthesized by the alkylation of the benzophenone Schiff's base of glycine methyl ester with 3-(2-methyl-1,2-dicarba-closo-dodecaborane(12)-1-yl)pr opyl iodide (8). This amino acid was employed in the synthesis of peptide derivatives such as 19-21 using solid-phase Merrifield methods. Dipeptide 19 was converted to a water-soluble ionic derivative by the pyrrolidine-mediated carborane cage degradation reaction followed by cation exchange to afford sodium salt 22. Dansylation of 22 with dansyl chloride yielded fluorescence-labeled dipeptide 23. Undecapeptide 21 was dansylated while still anchored to the Merrifield resin. Following its cleavage from the resin with hydrogen fluoride, product 25 was acetylated to block the free amino group on the lysine residue and then converted to water-soluble derivative 27. Trial conjugations of dipeptide 23 and undecapeptide 27 to T84.66, an anti-CEA antibody, were carried out by means of carboxyl activation with N-hydroxysulfosuccinimide and N,N-diisopropylcarbodiimide. Studies of the chemical syntheses of these and other peptide derivatives and the conjugation of 23 and 27 to the antibody are described.

Homogeneous immunoconjugates for boron neutron-capture therapy: Design, synthesis, and preliminary characterization

“…The pentameric nido-carboranyl phosphodiesters (CB) 5 and (G4) 5 were synthesized on automated DNA-synthesis instruments as described (12,13) with no modification to the standard reagents or procedures and purified by HPLC. The purity of the oligomers was analyzed by PAGE (28.5% polyacrylamide gels͞3.5 M urea; ref.…”

“…Preparation and Characterization of (CB) 5 -and (G4) 5 -IgG3L309C Conjugates. IgG3L309C (300 g) at the concentration of 3 mg͞ml in phosphate buffer, pH 7.4, was treated with 100 l of 500 M cysteine͞50 mM ammonium citrate buffer, pH 7.5͞2 mM EDTA͞100 mM NaCl at 37°C for 4 h. The (CB) 5 and (G4) 5 oligomers were also dissolved in 50 mM ammonium citrate buffer, pH 7.5͞2 mM EDTA͞100 mM NaCl to a final concentration of 2 mM.…”

“…IgG3L309C (300 g) at the concentration of 3 mg͞ml in phosphate buffer, pH 7.4, was treated with 100 l of 500 M cysteine͞50 mM ammonium citrate buffer, pH 7.5͞2 mM EDTA͞100 mM NaCl at 37°C for 4 h. The (CB) 5 and (G4) 5 oligomers were also dissolved in 50 mM ammonium citrate buffer, pH 7.5͞2 mM EDTA͞100 mM NaCl to a final concentration of 2 mM. Bifunctional linker maleimidobenzoic acid N-hydroxysuccinimide ester (MBS; Pierce) was dissolved in ice-cold dimethylformamide to a final concentration of 200 mM.…”

“…Bifunctional linker maleimidobenzoic acid N-hydroxysuccinimide ester (MBS; Pierce) was dissolved in ice-cold dimethylformamide to a final concentration of 200 mM. MBS solution (100 l) and 100 l of either (CB) 5 solution or (G4) 5 solution were added to a 1-ml screw-cap vial. The mixture was incubated at room temperature for 4 h with constant stirring.…”

“…The excess unreacted MBS was removed by ten 300-l extractions with water-saturated ethyl acetate. The aqueous phase, containing MBS-activated (CB) 5 or (G4) 5 , was added to the cysteine-reduced antibody. The mixture was incubated at 37°C for 4 h. The antibody conjugates were purified by CENTRI-SEP column (Princeton Separations, Adelphia, NJ) and analyzed by SDS͞PAGE (5% sodium phosphate-buffered gels for intact proteins and 12% Tris͞glycine-buffered gels for proteins treated to break the interchain disulfide bonds).…”