Conjugation of p-Aminophenyl Glycosides with Squaric Acid Diester to a Carrier Protein and the Use of Neoglycoprotein in the Histochemical Detection of Lectins

Abstract

The coupling of p-aminophenyl 2-acetamido-2-deoxy-3-0-β-d-galactopyranosyl-β-d-galactopyranoside (gal-β 1, 3-galNAc) to bovine serum albumin (BSA) was achieved by using 1, 2-diethoxycyclobutene-3, 4-dione (squaric acid diester) as a new coupling reagent. Two selective consequential steps afforded the desired neoglycoprotein: reaction of the p-aminophenyl group of gal-β 1, 3-galNAc with squaric acid diester gave the corresponding squaric acid amide ester, which was transformed into the BSA conjugate by coupling with the lysyl ∈-amino groups of BSA through formation of a squaric acid 1, 2-bisamide. The experimental conditions for the reactions and the optimization of average were performed by using p-anisidine as model substance, the methyl group substituting for the carbohydrate part of a p-aminophenyl glycoside. Neoglycoproteins have proven to be valuable tools for lectin detection. To evaluate the properties of this type of probe, the obtained neoglycoprotein with the histochemically crucial T-antigen structure was used for glycocytological and glycohistochemical studies. Three cultured human tumor cell lines and tissue sections from human breast carcinomas were chosen. Its efficiency was similar in comparison to measurements with a probe, derived by diazotization with the p-aminophenyl glycosides of gal-β 1, 3-galNAc and already shown to be a reliable marker for lectin localization in tissue sections and cultured cells. © 1991, American Chemical Society. All rights reserved.