Synthesis of a potential heterobifunctional cross-linking reagent with specificity for lysine residues

Abstract

Cross-linking reagents are used extensively in protein chemistry, and cleavable reagents which have two different reactive groups for attachment are of great interest (Ji, 1983). The use of such cleavable heterobifunctional compounds permits cleavage of the reagent after cross-linking, with individual protein amino acids retaining a label which facilitates their subsequent identification. In the current work, we have synthesized a cleavable heterobifunctional cross-linking reagent and evaluated its potential as a practical protein cross-linking reagent. A dione group was chosen as an essential part of the reagent on the basis of the reactivity of 2,4-pentanedione with proteins (Gilbert & O’Leary, 1975; El-Saleh et a[., 1984). Reagent design also included a cleavable phenacyl ester linkage (Sheehan & Daves, 1964), and an azido group for photo-induced non-specific labelling of amino acid residues (Bayley & Knowles, 1977). For synthesis of the reagent, ABAH (Fig. l), 20ml of 2 . 5 ~ N a O H and 6ml of 2,4-pentanedione were mixed at 25”C, and 2.5ml of propiolactone was then added over IOmin. After 2h , 20ml of ~ M H C I was added, and the solution was then extracted three times with ether. The ether extracts were combined and the product (4-acetyl-50x0hexanoic acid) was purified by vacuum distillation (Gresham et al., 1951). The purified acid (130mg) was then dissolved in 3 ml of acetone and 169 mg of p-azidophenacyl bromide and 0.097 ml of triethylamine were added (Moreland, 1956). After formation of a triethyl ammonium bromide precipitate, the product was recrystallized from methanol. Elemental analysis of the product (calculated C = 58.00%, H = 5.17%; experimental C = 57.98%, H = 5.20%) and ‘H-n.m.r. and I3C-n.m.r. spectroscopy (Fig. 1) confirmed the structure of the compound. ABAH was readily soluble in organic solvents, ethanol and methanol, soluble in 50% (v/v) ethanol, but only sparingly soluble in water ( < 0.5 mgiml). The cleavability of the phenacyl group in ABAH was tested at room temperature with sodium thiophenoxide in dimethylformamide. Cleavage of the ester occurred within 1 h as evidenced by the appearance of a singlet peak at 4.25 p.p.m. in the ‘H-n.m.r. spectrum (Griesbaum et al., 1963). The reactivity of ABAH with amines was tested using 40% (v/v) methylamine solution at room temperature. Enamine formation was confirmed by I3C-n.m.r. spectroscopy and by U.V. absorption spectroscopy (Gilbert & O’Leary, 1975). Reactivity with lysine ( 0 . 5 4 ~ in 40% aqueous methanol) was also confirmed by U.V. absorption spectroscopy. The reactivity of the dione group of ABAH with proteins was investigated using albumin, cytochrome c and tropomyosin. In each experiment, 5mg of reagent was predissolved in 1 ml ethanol and then added to 2 ml of a 9 mg/ml aqueous solution of the protein adjusted to pH 7.5. Reagent was found to precipitate out of solution on addition to the protein solution, and analysis of the protein solution by U.V.