Ozone reactions with amino acids

Distinct changes in several properties of lysozyme occur after reaction with ozone. The lytic activity of the ozonized lysozyme shows the same trend at various pHs as the native enz3mie (Fig. 2) this may suggest that the pK values of the ionizable groups involved in catalysis have not been altered by ozonplysis. The amino acid composition of ozonized lysozyme differs from that of the native enz3mie in three residues — methionine, tryptophan and t3H osine. None of the other amino acids is affected by ozone. The extensive loss of enz5miic activity must be ascribed to the oxidative modification of these three amino acid residues in the lysozyme. 

Mudd, J. B., R. Leavitt, A. Ongun, and T. T. McManus. Reaction of ozone with amino acids and proteins. Atmos. Environ. 3 669-681, 1%9. 

Ozone causes both quantitative and qualitative changes in carbon dioxide fixation patterns. Wilkinson and Bames, using carbon dioxide-found a reduction in radioactivity in soluble sugars and increases in free amino acids and sugar phosphates in white pine after a 10-min exposure to ozone at 0.10 ppm. Miller observed a decrease in carbon dioxide-fixation in ponderosa pines that correlated with loss of chlorophyll, after exposure to ozone at 0.30-0.35 ppm. The Hill reaction rates of chloroplasts isolated from healthy and ozone-injured ponderosa pine indicated that both light and dark reactions of the chloroplasts from ozone-injured plants were depressed. Barnes found depressed photosynthesis and stimulated respiration in seedlings of four pine species of the southeastern United States after exposure to ozone at 0.15 ppm. 

There is some information concerning the reaction of ozone with chemicals under aqueous conditions. The information available suggests that double-bond cleavage takes place, just as it does under nonaqueous conditions, except that ozonides are not formed. Instead, the zwitterionk intermediate reacts with water, producing an aldehyde and hydrogen peroxide. In addition to double-bond cleavage, a number of other oxidations are possible. Mudd et showed that the susceptibility of amino acids is in the order cysteine, tryptophan, methionine. 

A previous study of the reaction of ozone with lysoz3mie dissolved in anhydrous formic acid gave rise to the conclusion that the only amino acid residues affected in the early stages of the reaction were the tryptophan residues 108 and 111 ( ). Conversion of these residues to N -formyl-kynurenine did not cause loss in enzyme activity. Imoto et al. (7) have pointed out that this result is anomalous since modifications of tryptophan 108 (e.g. with iodine) normally causes inactivation. 

One approach to unnatural amino acids is to use a readily available amino acid, such as L-phenyl-alanine, as the starting material. The Birch reduction of L-phenylalanine (1) was carried out with lithium in ammonia, followed by acylation of the amino group to produce compound 2, which was further esterihed to produce the cyclohexa-l,4-dienyl-L-alanine derivative 3 (Scheme 11.1). The ozonolysis step of the reaction was carried out at -78°C in a dichloromethane solution presaturated with ozone to reduce the extent of oxidation of the diene 3 to produce 4. Cyclization was then carried out by the introduction of either hydroxylamine hydrochloride to produce the isoxazol-5-ylalanine derivative 5 or phenylhydrazine to give a 1 1 mixture of (l-phenylpyrazol-3-yl)alanine derivative 6 and the (l-phenylpyrazol-2-yl)alanine derivative 7.4,5... 

Experiments in vitro are consistent with some of the chemical investigations. Enzymes are readily inactivated by ozone, and the inactivation can be traced to the more susceptible amino acid residues (Table XI). Reactions with unsaturated fatty acids have been examined, and the production of malonaldehyde and hydrogen peroxide has been detected (52-54). The lipid products have not been analyzed, and the toxicity of such products is yet to be determined. 

Amino Acids and Peptides. - Wasserman s method of one-carbon homologation of carboxylic acids to give a-ketocarboxylates involves reaction with cyanomethylenetriphenyl-phosphorane followed by ozone (Scheme 24) and has been used as a key step in a chemo-enzymatic synthesis of isotopically labelled L-valine, L-isoleucine, and o/fo-isoleucine. Alkylation of the carbanion derived from the imino-substituted methylphosphonate diphenyl ester (186) with indol-3-ylmethyl bromide followed by appropriate deprotection has been used to prepare the phosphonate analogue (187) of tryptophan (Scheme 25). The deprotected analogue (188) and derived peptides show activity as inhibitors of chymotrypsin. Two approaches to solid phase Wadsworth-Enunons reactions which have applications in combinatorial chemistry have been reported. In one diethylphosphonoacetamide is bound to PEG-PAL resin via a peptide link, while... 

In the decades following Miller s experiment, many scientists repHcat-ed it, varied reactants, conditions, and energy sources, and have successfully produced amino acids and other important precursors. The energy sources in Miller s experiment (electric arcs and boiling water) were far too high to be realistic for the prebiotic Earth but were employed to make reactions occur in days rather than months or years. Eollowing decades of research, it is now understood that the great volcanic activity of the early planet would produce far more CO2, CO, and N2 than NH3, and CH4, Miller s precursors. Moreover, with no O2 and therefore no ozone (O3) in the atmosphere, intense UV radiation would decompose H2O to form radicals that destroy NH3 and convert CH4 to CO2 and... 

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