Formation of protein conjugates of phosphorothioate nucleoside diphosphate beta-S.

Abstract

The present study shows that the sulfur of the phosphorothioate 5'-nucleoside diphosphate beta-S adds across the olefmic double bond of the maleimide moiety of the thiol-sensitive probe, N-1 pyrene maleimide. The reaction of phosphorothioate with the maleimide moiety provided a basis for coupling the phosphorothioate nucleoside diphosphate beta-S to maleimidederivatized bovine serum albumin. Formation of proteinnucleotide conjugates employing the thiol-reactivity of the phosphorothioate group has not hitherto been reported. Proteinnucleotide conjugates crosslinked by reaction of phosphorothioate sulfur with maleimide-derivatized protein have significant potential for ligand-mediated delivery of nucleotides. Currently available methods for forming protein-nucleotide conjugates include ultraviolet irradiation (1), bifunctional chemicals (2) and photochemical cross-linking (3). These crosslinking methods induce changes in the bases of the nucleotide, thereby interfering with the function of the nucleotide. Also, water-soluble carbodiimides such as ethyl(dimethylaminopropyl) carbodiimide (EDC) are agents for crosslinking nucleotides to proteins (4). However, EDC lacks specificity and reacts with Ni of guanosine and also attacks thymidine (5). Moreover, coupling of proteins to nucleotides with EDC leads to protein-protein conjugates that would reduce yield of ligand-nucleotide conjugates, complicate conjugate purification and would compete with ligand-nucleotide conjugates for binding sites on the target cell membrane. The purpose of this work was to develop a practicable covalent crosslinking system between proteins and nucleotides that leaves the nucleotide bases intact and that has the potential to avoid the other aforementioned problems. Although the sulfur of phosphorothioate nucleotides is known to possess considerable thiol character (6-11), this thiol-like reactivity has never been applied, to the best knowledge of the authors, as the basis for preparation of protein-nucleotide conjugates. In this paper we first determine the reactivity of phosphorothioate adenosine diphosphate beta-S towards maleimide with a fluorescent maleimide probe and then utilize this reactivity to form covalent conjugates of the phosphorothioate nucleoside diphosphate and a maleimide-derivatized protein. N-1 pyrene maleimide (PM), a sulfhydryl-specific reporter molecule (12) was reacted with 5'-adenosine diphosphate beta-S (ADPS). 0.01 M ADPS (Boehringer-Mannheim) was reacted with 0.1 mM PM (Sigma Chemical Co) in 0.1 M sodium phosphate buffer (pH 8.8) with 5% acetone, at ambient temperature for 17 hours in the dark. Fluorescence emission intensity was recorded from 350-450 nm upon excitation at 330