Evelyn Kersten

Mechanistic Studies on the Formation of Pyrroles and Pyridines from [1-13C]-D-Glucose and [1-13C]-D-Arabinose

The Maillard reaction of [1- 13 C]-D-glucose and [1- 13 C]-D-arabinose with 4-aminobutyric acid (representing peptide bound lysine as well as a Strecker inactive amino acid) and L-isoleucine (representing a Strecker active amino acid) was investigated to get more insight into the reaction pathways involved. The extent and position of the labeling were determined by MS data. The results support 3-deoxyaldoketose as intermediate of N-alkyl-2-formyl-5-hydroxymethyl- and N-alkyl-2-formyl-5-methylpyrroles (1, 3, 7, 9) and disqualify 4-deoxy- and 1-deoxydiketose routes to N-alkyl-2-hydroxyacetyl- and N-alkyl-2-acetyl-pyrroles (2, 4, 8, 10), respectively. The 1, 3-dideoxy-1-amino-2, 4-diketose C is postulated as a new key intermediate in the formation route to 2, 4, 8, and 10 from D-glucose. In addition, this β-dicarbonyl route is correlated to the 3-deoxy-aldoketose route by keto-enol tautomerism as demonstrated by Maillard experiments in deuterium-oxide. The D-exchange ratios of compounds 1, 3, 7, 9, 13, and 14 (which were examined by MS- and 1 H NMR spectroscopy) indicated incorporation of D-atoms at C4 of glucose. The β-dicarbonyl pathway of [1- 13 C]-D-glucose/L-isoleucine (glycine) Maillard experiments generates 2-acetyl-[5- 13 C]-pyrrole and 2-methyl-3-[6- 13 C]pyridinol via a Strecker active intermediate. Based on the results of these labeling experiments and on the results of Maillard experiments in deuteriumoxide a revised scheme of the Maillard reaction of D-glucose with amines and α-amino acids is presented.

Mechanistic Studies on the Formation of Maillard Products from [1-13C]-D-Fructose

Ketoses are known to react with amino compounds via ketimines to form the so-called Heyns compounds (2-aminoaldoses), which are assumed to undergo subsequent transformations parallel to those observed with the corresponding Amadori compounds. Until now, this assumption was not established by separate mechanistic studies. Therefore, we prepared [1- 13 C]-D-fructose from [1- 13 C]-D-glucose by enzymatic methods. In a series of model experiments [1- 13 C]-D-fructose was heated with 4-aminobutyric acid (Strecker inactive), L-isoleucine (Strecker active), and L-proline (secondary amine type), respectively. The labeled products were analyzed by capillary GC/MS and NMR spectroscopy and the labeling characteristics were examined from MS data. Compared to corresponding experiments with [1- 13 C]-D-glucose, the significant results are: (1) With 4-aminobutyric acid only trace amounts of 3-deoxyaldoketose products are formed in the D-fructose system, whereas 1-deoxydiketose products were generated in comparable amounts from D-glucose and D-fructose; and (2) With L-isoleucine both D-glucose and D-fructose form 3-deoxyaldoketose- and 1-deoxydiketose products in comparable amounts; but with D-fructose the most effective reaction is the formation of pyrazines initiated by a retro aldol cleavage into C 3 +C 3 fragments. This cleavage is also responsible for the formation of mixtures of isotopomeric products in D-fructose systems.

Fragmentation of Sugar Skeletons and Formation of Maillard Polymers

Labelling experiments involving the reaction of 13 C-labelled hexoses, pentoses, and D-lactose with 4-aminobutyric acid (GABA) are described. The distribution of the label was investigated by MS and gave an insight into the formation pathways leading to complementary labelled compounds from hexoses and pentoses with intact carbon skeletons and indicated distinct fragmentations of the sugar skeletons into C 5 -and C 4 -compounds (furans, N-alkylpyrrolemethanols, N-alkyl-2-formylpyrroles and N-alkylpyrroles). These compounds undergo polycondensations to melanoidin-like macromolecules under mild reaction conditions. In a series of model experiments, different types of polymers were investigated, and individual oligomers were characterized by 1 H/ 13 C-NMR spectroscopy and FAB-/MALDI-TOF-MS. We postulate that these polycondensation reactions represent the most important driving force in the Maillard reaction.