Synthesis of a New Organic Linker Molecule for Protein Immobilization

Abstract

Recent biological and medicinal experimental techniques for diagnosis and drug discovery utilize microarrays of proteins immobilized on solid substrates. Such protein “chips” can have proteins on surface attached covalently or non-covalently between proteins and linker molecules frequently by chemisorption of the film components to the substrate from solution. For the well defined systems, organic monolayer films have formed versatile model study. The arrays of proteins attached to a solid surface have a great potential for detecting interactions of protein-ligand, proteinprotein, and antibody-antigen. Among several self-assembling systems, organosulfur compounds including dialkyl sulfides, dialkyl disulfides, and thiols have been investigated intensively on gold. It has been recognized that self-assembled monolayers (SAM) using organosufur compounds on gold are advantageous over other SAM systems because of higher structural order by densely packing long chains. In addition, versatile functionalization of terminal group at the monolayer surface suggests ideal model systems for immobilization and interfacial interaction. Several functional groups including anhydride, succinimidyl ester, maleimide, and phosphinothioester have been applied for the chemoselective binding of proteins. Here, we report a new potential substrate for the covalent and specific immobilization of nucleophilic molecules. The new organic linker, disulfide 1, has 4, 5-difluoro-2-nitroaniline amide moiety on the head and a long carbon chain with a triazole group. We envisioned that the compound would be prepared via 1,3-dipolar cycloaddition of azide 2 and alkyne 3, and hoped that a Meisenheimer complex of 4,5-difluoro-2-nitroaniline amide moiety would serve for a covalent attachment of amine or thiol moieties, and eventually proteins on the surface (Scheme 1). Prior to the synthesis of the molecule, we wanted to evaluate the reactivity of the substrate 1 toward nucleophilic aromatic substitution. Therefore, we first prepared a amide 4 from 4,5-difluoro-2-nitroaniline and 10-undecenoyl chloride and have tried the substitution reaction with various amines and a thiol as a model study (Table 1). The reaction was carried out in THF/water at room temperature. Primary and secondary amines reacted with the nitroaniline in minutes to afford good yields. Cyclic amines provided better than noncyclic secondary amines, and thiol provided the best yield in the shortest reaction time as expected. On the basis of the result, we anticipate the functional groups in proteins, amine or thiol, would show the similar reaction pattern toward the