Hydrolysis performic acid-oxidized

Finally, it is very common for methionine to be determined as part of the standard hydrochloric acid hydrolysis. Indeed, methionine is not nearly as labile to oxidation as cysteine is. While this is appropriate for many samples, there are studies (27,91) that indicate that seriously flawed recoveries (10-40% low) may result from methionine determination by standard acid hydrolysis if the samples contain high levels of carbohydrate. For these sorts of samples, it is recommended that determination of methionine as methionine sulfone (by performic acid oxidation) be pursued. 

That the hydroxyamino group is in all cases attached to the 8 carbon is evident from the fact that periodate oxidation yields nitrosodimer (rather than N2O, which would arise from an N -hydroxyamino acid (42, 89)), performic acid oxidation affords glutamic acid (136) and, finally, deacylation followed by catalytic hydrogenation, reaction with fluoro-dinitrobenzene and hydrolysis gives N8-dinitrophenylornithine (63). 

Figure 16. Hydrolysis rate of bovine ribonuclease relative to that of performic acid-oxidized ribonuclease as a function of temperature (155). The follotving enzymes were used O, aminopeptidase , carboxy-peptidase A A, trypsin and , chymotrypsin.

Cysteine analyses showed a continuing trend of successful use of disulfide exchange reagents. Performic acid oxidation, although still successfully used, overall fared as well as the simple direct analyses of cystine. Direct cystine analysis of the pre-hydrolysate was as successful as that of the protein sample, suggesting that chromatography and derivatization and not hydrolysis are the most important factors in successful analysis of cystine by this simple methodology. 

If appropriate precautions have been taken in the preparation of a protein, and if oxygen is completely removed before hydrolysis, methionine will usually be recovered from acid hydrolysates in yields greater than 95 %. However, in some proteins (particularly those that are chemically modified) and in many peptides the methionine may be at least partially oxidized to the sulfoxide or sulfone forms, and even though these may be analyzed with amino acid analyzers (see below), the total yield of methionine (and oxidized products) is usually somewhat low. A good check on total methionine content in a peptide or protein is obtained by analyzing for methionine sulfone after performic acid oxidation, since methionine and its sulfoxides are quantitatively converted to the sulfone by this procedure. 

The method described above gives direct analysis of methionine sulfoxide content in proteins. Another method makes use of carboxy-methylation of methionine at acid pH to give the carboxymethyl-sulfonium derivative ( 3.5). Methionine sulfoxide, which is not affected by the carboxymethylation reaction, is then oxidized to the sulfone which is stable to acid hydrolysis and can easily be quantitated. This is possible because methionine carboxymethyl-sulfonium salts are not affected by performic acid oxidation, although they are degraded by acid hydrolysis. Therefore, the methionine sulfone content is equal to the methionine sulfoxide content plus any sulfone that may have been initially present (shown by analysis before oxidation). 

Methionine carboxymethylsulfonium salts These derivatives of methionine (isomers) are prepared by treating proteins with iodoacetic acid the reaction is most specific for methionine at acid pH ( 3.5). These derivatives are not affected by performic acid oxidation (see under methionine sulfoxide), but are degraded by acid hydrolysis to give methionine, carboxymethyl-homocysteine, homoserine and homoserine lactone (Gundlach et al. 1959). 

Amino acids Acid hydrolysis (HCI 6N) followed by chromatography. There are many methods with variants in duration (24-48 hours) and temperature (110-I45°C). Methionine and cystine are obtained after performic acid oxidation and tryptophan after alkaline hydrolysis. 

The above sequence was extended to a preparation of 6-epicorynoline. Performic acid oxidation of deoxycorynolone, followed by treatment with alkali, afforded a mixture of diols 3, epimeric at C-5. Lithium aluminum hydride reduction of diols 3, followed by palladium hydrogenolysis of the benzylic C-5 hydroxyl, gave rise to 6-epicorynoline. It will be noted that in this instance performic acid oxidation, followed by hydrolysis, furnished C-6 beta hydroxyl derivatives. ... 

Finlayson 117) has reported that performic acid oxidation of lysozyme gives a higher conversion of tryptophan to kynurenine than that given by oxidation of free tryptophan. The tryptophan residues can also be converted into an oxidized form which does not give kynurenine on hydrolysis. 

A similar type of linkage between coenzyme and protein in which position 8a of the flavin is at the oxidation level of carbonyl was found in thiamine dehydrogenase (from soil bacterial 138) and in P-cyclo-piazonate oxidocyclase from Penicillium cyclopium 88, 155). In these cases, however, it is probable that N(l) of a histidine residue constitutes the bridge to the protein backbone. The oxidation level of position 8a was deduced from decay data similar to those mentioned above for the 8a-thiohemiacetals. Of particular interest is the fluorescence profile of the flavin peptide obtained by trypsin-chymotrypsin hydrolysis of p-cyclopiazonate oxidocyclase. Fluorescence quenching similar to that observed with SD-flavin (9) is observed, but in this case the pK attributed to protonation of the histidine imidazole is shifted from 4.7 to 5.4, and the maximal fluorescence obtained is only 20% of that of FMN 164). Upon performic acid oxidation of the peptide the emission intensity is... 

Na/NH3, -30°, 3 min, 1(X)% yield. This protective group is stable to acidic hydrolysis (4.5 N HBr/HOAc 1 N HCV, CF3CO2H, reflux). There is no evidence of S N acyl migration in 5-(A-ethylcarbamates) (RS = cysteinyl). Oxidation of 5-(A-ethylcarbamoyl)cysteine with performic acid yields cysteic acid. ... 

Under these reducing conditions of hydrolysis of tryptophan peptides, cystine is reduced to cysteine and its coelution with proline using standard buffer gradients, makes quantitation difficult. Thus, cysteine and cystine are generally derivatized prior to acid hydrolysis by oxidation to cysteic acid with performic acid 21 or alkylation, upon reduction in the case of cystine, with iodoacetic acid 21 or, more appropriately, with 4-vmylpyridine)22 23 50 Conversion of cysteine into 5- 3-(4-pyridylethyl)cysteine bears the additional advantage of suppressing epimerization via the thiazoline intermediate, thus allowing for standardization of the acid-hydrolysis dependent racemization of cysteine in synthetic peptides)24 ... 

Treatment of cm- and fraws-cycloundeoene and cyclododecene with perform acid likewise gives complex mixtures of products derivable by 1 abnormal formolysis of the corresponding epoxides.1401-14o By contrast, mothylenecyclooctane oxide yields no diole on treatment with formic acid and subsequent alkaline hydrolysis, the main product being formykycJooctame.1 2 ... 

Oxidize methionine and cysteine to methionine sulfone and cysteic acid using performic acid prior to acid hydrolysis. 

The analysis of methionine and cysteine is problematic. The sulfur containing side chains of these amino acids are prone to oxidation. The standard hydrochloric acid hydrolysis will cause the partial conversion of these amino acids into cystine, cysteine, cysteine sulfinic acid, cysteic acid, methionine, methionine sulfoxide, and methionine sulfone. The classic strategy (79) for dealing with this problem is simply to drive the oxidative process to completion (i.e., convert all the cyst(e)ine to cysteic acid) and then to analyze chromatographically for cysteic acid and/or methionine sulfone. This is traditionally accomplished by a prehydrolysis treatment of the sample with performic acid. While this method has sufficed over the years, the typical recovery (85 -90%) and precision (4% intra- and 15% interlaboratory) have been poor (80). 

An interesting study (85) explores the use of sodium azide as an oxidative agent instead of performic acid. The big advantage offered here is that the oxidation of cysteine to cysteic acid is effected concurrent with the hydrochloric acid hydrolysis. The authors claim that the presence of 0.2% (w/v) NaN3 in the HC1 digestion does not represent an explosion risk. Recoveries of cysteine as cysteic acid were typically —90% for pure proteins. 

M Spindler, R Stadler, H Tanner. Amino acid analysis of feedstuffs determination of methionine and cystine after oxidation with performic acid and hydrolysis. J Agric Food Chem 32 1366-1371, 1984. 

Oxidation of deferrialbomycin S2 with performic acid cleaves the hydroxamic acid linkages and converts the bound NS-hydroxyomithine to glutamic acid, the latter still in peptide combination. Partial acid hydrolysis and identification of fragments affords the structure cyclo-(seryl)s-(glutamyl)3 (135). The oxygen of the side chain of the first serine is linked via sulfur to N1 of 3-methylcytosine, which in turn is acylated with an unknown fragment. 

TMS ethers of primary alcohols and most secondary alcohols do not survive even the simplest synthetic manipulations — especially if protic solvents are involved. For example, Swern oxidation or Collins oxidation conditions will cleave a primary TMS ether and perform the oxidation in the presence of a secondary TMS ether.3 Owing to the sensitivity of TMS ethers., deprotection can usually be achieved under very mild conditions (e,g., acetic acid or potassium carbonate in methanol). The rate of hydrolysis depends on both steric and electronic effects with hindered environments decreasing the rate and electron-withdrawing substituents on the hydroxyl function increasing the rate. In a synthesis of Zaragozic Acid A. the... 

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