Richard L.M. Synge

Free and bound phenolic acids of lucerne (Medicago sativa cv europe)

Abstract The distribution of free and covalently-bound phenolic acids was studied in various fractions obtained from fresh lucerne shoots. p-Hydroxybenzoic, vanillic, p-coumaric and ferulic acids were present, both free and bound, in all the fractions. Salicylic and sinapic acids occurred only in a bound, alkali-labile state and were found almost entirely in the ‘aqueous phase’ fraction after treatment of methanol-chloroform-water extract according to Bligh and Dyer. Many other common phenolics were absent. Amounts of the phenolic acids much larger than those extracted by methanol-chloroform-water were extracted subsequently by phenol-acetic acid-water and passed into the ‘diffusate’ fraction on dialysis of this extract against 70% acetic acid. Small, though significant, quantities of phenolic acids remained with the bulk protein in the ‘bag contents’ fraction. The extent to which the phenolic acids in these last two fractions are held to protein by covalent bonds or by secondary-valence attractions is discussed, particularly in relation to the isolation of N-feruloylglycylphenylalanine after partial hydrolysis. Suggestions are made for improving analytical procedures.

Hydrogenation as an approach to study of reactions of oxidizing polyphenols with plant proteins

Abstract Coupling of oxidation products of o -diphenols with -NH 2 groups of plant proteins can damage nutritional availability of lysine residues. Relevant model coupling products (before or after reductive acetylation or permethylation) are unstable to acid hydrolysis. Hydrogenation over Rh/Al 2 O 3 , at room temperature and atmospheric pressure, gave cyclohexane derivatives stable to hydrolysis and retaining, with only partial hydrogenolysis, all groups originally attached to the aromatic nucleus. Plant bulk proteins were hydrogenated with substantial conversion of their aromatic amino acids; their S-containing amino acids were desulphurized. The technique is therefore promising for study of the fate of lysine residues in “enzymically browned” proteins.

Ascorbalamic acid: An ascorbigen-like plant constituent yielding in hot acid 3-(2-furoyl)alanine

Abstract Ascorbalamic acid (C 9 H 13 NO 8 ) was isolated from Brassica olerocea L. MS study of various methylated derivatives suggested a structure (Ia) derivable by CC coupling of C-3 of alanine with C-2 of ascorbic acid, followed by lactone → lactam rearrangement. Other derivatives provided supporting evidence, as did study of the reaction of L -3-chloroalanine with L -ascorbic acid in vitro . On treatment with hot 6 M HCl, ascorbalamic acid yielded L -aspartic acid and 3-(2-furoyl)alanine. For identification of the latter, DL -3-(2-furoyl)alanine and its N-2,4-dinitrophenyl and N-acetyl methyl ester derivatives were synthesized. Unlike ascorbigens, ascorbalamic acid is probably present in the living plant. It seemed to be present in all crucifers examined, but to have a capricious distribution in other orders. During permethylation, rearrangements of ester groups were observed, both with ascorbalamic acid and with pyrrolidonecarboxylic acid as a model.

Mass-spectrometric evidence for quinonoid-lysine coupling products in cigar protein.

Abstract On subjecting hydrolysates of hydrogenated cigar protein to derivatization and preparative GLC, two fractions were obtained which had volatilities and mass spectra consistent with an origin from oxidative-condensation products of chlorogenic acid with lysine residues. The mass-spectral evidence was chiefly from high-resolution mass spectrometry of the heaviest fragment ions; there was also high-resolution comparison of the lighter fragment ions with those from relevant model compounds. New compounds prepared were the hydantoin from 6-(1-trans-octahydroquinol-2-onyl)- DL -norleucine and the heptafluorobutyrylated n-propyl esters of four other less-common amino acids. Details of the mass spectra are provided in a Supplementary Publication § or have been sent to the Mass Spectrometry Data Centre.

Changes in bulk protein of tobacco leaves on aerobic autolysis: Hydrogenation studies and identification of bound quinic acid

Abstract During aerobic autolysis and in commercial curing, the bulk proteins of tobacco leaves become coupled with quinic acid, presumably in consequence of coupling of chlorogenic acid congeners with lysine e-NH 2 groups. Quinic acid derivatives, prepared from acid hydrolysates of such altered proteins, were identified by GC-MS. Such proteins were also hydrogenated over Rh/Al 2 O 3 with a view to stabilizing the hypothetical linkages. Difficulties in removing contaminant Al had to be overcome. Evidence was then obtained (by GLC of derivatives) for several components, in acid hydrolysates of hydrogenated altered proteins, which were neither normal hydrogenation products of the common amino acids nor derivatives of quinic acid. Details of the chromatograms and mass spectra of quinic acid derivatives are provided in a supplementary publications.