The reaction conditions of galactose oxidase-catalyzed, targeted C-6 oxidation of galactose derivatives were optimized for aldehyde production and to minimize the formation of secondary products. Galactose oxidase, produced in transgenic Pichia pastoris carrying the galactose oxidase gene from Fusarium spp., was used as catalyst, methyl alpha-D-galactopyranoside as substrate, and reaction medium, temperature, concentration, and combinations of galactose oxidase, catalase, and horseradish peroxidase were used as variables. The reactions were followed by H-1 NMR spectroscopy and the main products isolated, characterized, and identified. An optimal combination of all the three enzymes gave aldehyde (methyl alpha-D-galacto-hexodialdo-1,5-pyranoside) in approximately 90% yield with a substrate concentration of 70 mM in water at 4 degrees C using air as oxygen source. Oxygen flushing of the reaction mixture was not necessary. The aldehyde existed as a hydrate in water. The main secondary products, a uronic acid (methyl alpha-D-galactopyranosiduronic acid) and an alpha,beta-unsaturated aldehyde (methyl 4-deoxy-alpha-D-threo-hex-4-enodialdo-1,5-pyranoside), were observed for the first time to form in parallel. Formation of uronic acid seemed to be the result of impurities in the galactose oxidase preparation. H-1 and C-13 NMR data of the products are reported for the alpha,beta-unsaturated aldehyde for the first time, and chemical shifts in DMSO-d(6) for all the products for the first time. Oxidation of D-raffinose (alpha-D-galactopyranosyl-(1-6)-alpha-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside) in the same optimum conditions also proceeded well, resulting in approximately 90% yield of the corresponding aldehyde. (C) 2008 Elsevier Ltd. All rights reserved.