Malonaldehyde, periodate oxidation

In theory, periodate oxidation could have given a clear-cut answer as to the composition of the isomeric mixture of deoxy ribose phosphates. The 4-phosphate (73), devoid of vicinal diol groups, should be resistant to periodate the 3-phosphate (74) should reduce one and only one molar equivalent of the oxidant and yield one molar equivalent of both formaldehyde and the phosphorylated dialdehyde (75), whereas the 5-phosphate (76) could be expected to reduce one molar equivalent of periodate relatively rapidly, followed by a slower overoxidation reaction owing to the oxidation of malonaldehyde, formed as a result of the glycol cleavage. 

Despite the above-mentioned short-comings, this approach to the estimation of those deoxy sugars which yield malonaldehyde when oxidized with periodate, seemed promising, since, as has been seen (58,59), the dye is formed quantitatively in the reaction of malonaldehyde with 2-thiobarbituric acid also, more recently, its constitution (49,57) and molar extinction coefficient (36) have been established. Thus, if conditions could be found in which malonaldehyde, while being formed quantitatively from the deoxy sugars, would be stable, an ideal method, independent of standard compounds, would be available for the quantitative determination of all of these sugars. 

Thus, if triose reductone is, in fact, the first intermediate in the periodate oxidation of malonaldehyde, the total consumption of periodate per mole of malonaldehyde should be four molar equivalents two moles of formic acid and one mole of carbon dioxide should be formed, in accordance with the sequence proposed by Fleury and his collaborators (22). As in the case of the periodate oxidation of malonic acid (32) the rate determining step should be the hydroxylation step. 

It is, however, more likely that the discrepancies observed in the periodate oxidation of malonaldehyde concern mainly the hydroxylation step. In the mechanism proposed (5) for this reaction, it is the enol form of malonaldehyde which is hydroxylated. However, titrations of a solution of malonaldehyde, prepared by hydrolysis of an aqueous solution (33) of carefully distilled 1, 3, 3-tri-ethoxypropene (46, 47), both with strong base and with iodine, indicate that only about 80% of the enol form is present in the equilibrium solution. On the other hand, the thio-barbituric acid test (58, 59) gave consistently higher values for the malonaldehyde content of the solution. The fact that only about 80% of the enol form is present in the equilibrium solution is all the more important as it can be shown (56) by titration with strong base that the enolization is slow, and moreover does not seem to go to completion. 

When 1, 3, 3-triethoxypropene was hydrolyzed with IN sulfuric acid, a solution of malonaldehyde whose optical density was perfectly stable at 350 m/x for at least one week was obtained. If the solution was made alkaline, the optical density at the same wavelength increased by a small value and then remained virtually constant for at least one week (56). It was also observed that in these solutions the extinction coefficient at 350 m/x was very low (observed 8.3, 61.5 and 69, for solutions of pH 0.4, 7.15 and 9.4 respectively) compared with previously reported values which varied from 200 ( 40) to 1000 ( 48). On the other hand, the absorption of solutions having a pH of 3 to 5, increased considerably with time (at pH 4.75, the extinction coefficient of malonaldehyde at 350 m/x was initially about 40 after four weeks a value of about 930 was recorded and the optical density of the solution was still increasing). This increase in absorption was accompanied by a marked decrease in the malonaldehyde content of the solution, as measured by the thiobarbituric acid method. As a corollary, it was found that aqueous solutions of malonaldehyde, prepared by autocatalyzed hydrolysis (33) of the same acetal and which had a pH of about 3.5, showed, at the completion of the hydrolysis, considerably higher extinction coefficient values at 350 m/x than did those malonaldehyde solutions which were prepared by hydrolysis with IN acid and subsequently adjusted to pH 4. It appears, therefore, that at pH values at which most of the periodate oxidations are carried out, malonaldehyde is unstable and undergoes a chemical reaction, the nature of which is not, as yet, known. 

These observations provide at least one explanation for the fact that variable results are obtained when malonaldehyde is oxidized with periodate. They also explain why widely differing values for the extinction coefficient of malonaldehyde at 350 m/x have been reported and make it unlikely that the absorption band at this wavelength is caused by the dialdehydo form of malonaldehyde. 

In the past, periodate titrations have been of limited value for establishing the structure of quercitols or cyclohexanetetrols. The former show overoxidation, because of the fact that malonaldehyde is formed, and this compound undergoes further oxidation. Some isomers of the tetrols... 

Two different pathways have been proposed to explain the over-oxidation reaction of malonaldehyde. Huebner and his collaborators (32) based their conclusion on the observed behavior of digitoxose and suggested that malonaldehyde (7) was oxidized by three molar equivalents of periodate with the concomitant formation of three molar equivalents of formic acid ... 

However, when we oxidized malonaldehyde (56) in the conditions just described for triose reductone, although formic acid and carbon dioxide were produced in high yields, the periodate consumption was erratic. Similar results were obtained with deoxy sugars. This discrepancy may be caused by the incomplete enolization of the first intermediate, hydroxy malonaldehyde —i.e. tartronic dialdehyde (5,22,32), to triose reductone, or may concern the hydroxylation step itself. 

In an attempt to elucidate the reaction sequence by which the cyclohexanepentols are oxidized, we recorded simultaneously the time curves of periodate reduction and of malonaldehyde production during the oxidations. By this procedure, it is, in fact, possible to propose a reaction sequence for (1 d)-1, 2, 5/3, 4-cyclohexanepentol. 

Figure 4. Periodate uptake (10f) and malonaldehyde formation (MA) during the oxidation of (1 d)-1,2,5/3,4-cyclohexanepentol with sodium metaperiodate...

No hypotheses can be advanced for the sequences involved in the oxidation of cyclohexanepentols which react even more slowly with periodate than does (+)-quercitol. For instance, when the all-trans l 3, 5/2, 4-cyclohexanepentol (33) is oxidized (Figure 6), production of malonaldehyde starts very early and the time curve of periodate reduction indicates a very complex reaction. Nor is it possible to analyze satisfactorily the curves obtained with (1 l)-1, 2, 4/3, 5-cyclohexane-pentol (34) or with dl-1, 2, 3, 4/5-cyclohexanepentol (35) [this is the configuration predicted for the (1 D)-entantiomorph (41)]. 

Malonaldehyde (MA) is a major end product of oxidizing or rancid lipids and it accumulates in moist foodstuffs (6). Several MA-protein systems have been studied. Chio and Tappel combined RNAase and MA to demonstrate fluorescence attributed to a conjugated imine formed by crosslinking two e-amino groups with the dialdehyde (7). Shin studied the same reaction and found it to be dependent on pH and reactant concentrations (8). Crawford reported the reaction between MA and bovine plasma albumin (BPA) also to be pH dependent, and of first order kinetics with a maximum rate near pH 4.30. At room temperature 50-60% of the e-amino groups were modified—40% in the first eight hours, the remainder over a period of days (9). 

Malonaldehyde has been identified by Fleury and coworkers as a product of the rapid stage of the oxidation of (+)-proto-quercitol. 79 It is slowly oxidized, with the consumption of another four moles of periodate per mole, to formic acid and carbon dioxide. 

Analytical methods for the determination of malonaldehyde are particularly well developed. 2-Thiobarbituric acid in the presence of trifluoroacetic acid reacts with malonaldehyde to give a product whose absorption at 530 nm is proportional to the irradiation dose up to 400 rads. This reaction is proposed as a test for determining the absorbed dose, provided that the humidity of the sample, date of irradiation, and the temperature of storage are known.152 168 173 An alternative method is the reaction of malonaldehyde with 2-methylindole.174 The same reaction has also been used for the determination of deoxy sugars, but this requires oxidation with periodate to give malonaldehyde.175,176... 

The theoretical yield of carbon dioxide was obtained in about 10 hours from lactose by oxidation with 0.06 M sodium metaperiodate in acetate buffer at pH 5.0 and 50°. On the other hand, the use of phosphate buffer at pH 5.0 led to extensive over-oxidation of the products, giving carbon dioxide far in excess of one mole per mole. The concentration of periodate in the reaction mixture has a profound effect on the rate of oxidation of malonaldehyde derivatives and, in <0.01 M periodate, the reactions are very slow. ... 

This result is consistent with the hypothesis that malonaldehyde is formed in the reaction, since oxidation of this compound has been shown to be slow below 5 (Cantley, Hough and Pittet, 1963)- When this oxidation was run at room temperature it was found that additional periodate was consumed until a total of 8 moles/mole had been taken up in the course of 8 to 10 days. Formic acid and carbon dioxide could be detected as products of the reaction, but no malonaldehyde could be isolated or trapped (Schimbor, 1965)- Results of the carbon-by-carbon degradations of subunits are summarized in the following tables. 

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