Succinic acid labelled with

Table 5.1 Example of organic acids produced commercially by micro-organisms organic adds considered in this chapter are labelled with. A related add, a-oxoglutaric acid, is easy to produce microbiologically but has no current end use succinic acid is produced chemically. Amino adds are beyond the scope of this chapter.

Sprinson and coworkers [30] conducted the methylmalonyl-CoA mutase reaction in deuterium oxide using a crude mitochondrial preparation. The presence of methylmalonyl-CoA epimerase insured that (1) all substrate molecules incorporated one atom of deuterium into position 2, and (2) in the course of the reaction the (2R)-epimer of methylmalonyl-CoA was continuously supplied by epimerization of the (25)-epimer, which was in turn generated by the enzymic carboxylation of propionyl-CoA. Alkaline hydrolysis of the product and subsequent purification furnished succinic acid which was mainly monodeuterated (70% 2H,-, 15% 2H2-labelled and 13% unlabelled species). A positive ORD curve revealed its (5) configuration indicating stereochemical retention for the AdoCbl-dependent rearrangement (Fig. 22). No plausible explanation could be offered for the formation of doubly deuterated and unlabelled species. Essentially the same results were later obtained with a highly purified mutase preparation from Propionibacterium sher-manii (J. Retey, unpublished). 

Though the pertinent experiment in the corrin series has not yet been reported, a record of the analogous experiment in the porphyrin series has appeared [84], In contrast to corrins, in haems all four acetate side chains of the parent urogen III have been decarboxylated to methyl groups. In principle, these could be carved out as acetic acid and analysed for chirality if the starting succinate had been stereospecifically labelled with deuterium as well as with tritium. The synthesis of (2/ )-[2-2H,2-3 H]succinic acid was achieved by incubating (37 ,S)-[3-3 H]-... 

It has been established that nicotinic acid is the precursor of the pyridine ring in anabasine 140) and that carbons 2 and 3 are derived from the methylene carbons of succinate 127). It has also been shown that glycerol serves as a precursor for C-4, C-5, and C-6 119). Lysine serves as the precursor of the piperidine ring of anabasine 140, 141), C-2 of lysine becoming C-2 of anabasine. Tracer experiments with lysine-2- labeled with on the a- or e-nitrogen indicate that the piperidine nitrogen is derived from the e-amino group 142). The mechanism by which nicotinic acid is incorporated into nicotine must involve C-6 since nicotinic acid-G- H was incorporated with loss of the label, whereas nicotinic acid-2-3H was incorporated with retention of the label 140). A... 

Radioactive acetyl CoA can be generated by direct synthesis from C-acetate or from (3 oxidation of radioactive fatty acids, such as uniformly labeled palmitate. Examination of the reactions of the citric acid cycle reveals that neither of the two carbons that enter citrate horn acetate is removed as carbon dioxide during the first pass through the cycle. Labeled carbon from C-methyl-labeled acetate appears in C-2 and C-3 of oxaloacetate, because succinate is symmetrical, with either methylene carbon in that molecule labeling C-2 or C-3 of oxaloacetate. The conversion of oxaloacetate to phosphoenolpyruvate yields PEP labeled at C-2 or C-3 as well. Formation of glyceraldehyde 3-phosphate and its isomer dihydroxyacetone phosphate gives molecules, both labeled at carbons 2 and... 

In 1960 it was demonstrated that aseptically grown plants of Peganum har-mala are capable of incorporating [carboxy- C]anthranilic acid into vasicine (28). With Adhatoda vasica plants it was shown that the incorporation of an-thranilic acid involves retention of the carboxyl group (765). The origin of the non-anthranilic acid moiety is still controversial. It was found that in Adhatoda vasica C-1, C-2, and N-10 of (28) may be derived from aspartic acid and C-3 and C-3a from a Cj unit (161,166) (Scheme 9). This was supported experimentally by the incorporation of [S- Claspartic acid, [3- C]malic acid, and [2,3- " C] succinic acid into 28 with localization of the label at C-1 and C-2. The... 

All the enzymes contain ferrous iron. Ferrous ions are essential for the catalytic activity of butyrobetaine hydroxylase (259), proline-4- 254) and 3- 242, 243), hydroxylases, thymidine hydroxylase 260), lysine hydroxylase 261), trimethyllysine hydroxylase 247), thymine oxygenase 262—264), cephalosporin hydroxylase 249), and hydroxy-phenylpyruvate 265—267). In addition, the enzymes show a non-stoichio-metric requirement for ascorbic acid which is supposed to maintain the ion co-factor in the ferrous oxidation state (2, 242, 243, 254). Labelling studies with butyrobetaine hydroxylase 268), proline-4-hydroxylase 269), thymine oxygenase (270), cephalosporin hydroxylase (257) and hydroxy-phenylpyruvate hydroxylase (277) all indicate a common pattern of oxygen incorporation. One atom of oxygen becomes the new hydroxyl group while the other is located in one of the carboxylic acid groups of succinic acid. 

In addition to these standard methods, a number of specialized reactions of limited scope have been used to prepare some biologically important S-labelled thiols. For instance, 2-thiouracil- S has been prepared by the condensation of thiourea- S with Na0CH=CHC02Et . a-Amino- -mercaptobutyric acid- S was prepared by the acid hydrolysis of 4-carboxy-5-methyl-2-phenylthiazoline- S 2,3-Dimercaptosuccinic acid- S2 was obtained by the hydrolysis of 2,3-bis(acetylthio)succinic acid- S. D,L-Cysteine- S was obtained by the acid hydrolysis of PhCOS-CH2CH(HNCOC0H5)CO2Me... 

Succinic acid is incorporated into the 2-, 3- and 7-carbon atoms of ricinine (3-cyano-4-methoxy-l-methyl-2-pyridone). Carbon 1 of succinic acid becomes the nitrUe carbon of ricinine. " Nicotinic acid and nicotinamide are highly incorporated into ricinine, which suggests that these two compounds are closer to ricinine than is succinic acid. Ricinine and nicotine show labeling patterns that are consistent with a pathway where succinic acid or a related dicarboxylic acid is a precursor to nicotinic acid, which is an intermediate in the formation of nicotine and ricinine. The a-carbon of lysine is incorporated into carbon 6 of ricinine and the e-carbon of a-aminoadipic acid is incorporated into carbons 2 and b. " ... 

The condensation of succinate with glycine was postulated by Shemin and Russell (29) to form a-amino-/3-ketoadipic acid. This compoimd de-carboxylates spontaneously to form 3-AL (SO). 5-AL when, supplied to duck erythrocytes was shown to be incorporated into heme more readily than succinate or glycine. With labeled 8-AL the tagged C atoms were found in the predicted positions in the porphyrins by the methods described above (SI). 

In the fermentation of glucose by Propionibacterium arabinosum, the C of both 1-, and 3,4-labeled glucose was found in every carbon of the products—propionic acid, succinic acid, and CO2. The results are consistent with the explanation that glucose is split to two three-carbon compounds and to CO2 plus a five-carbon compound, and that randomization occurs by means of symmetric three- and four-carbon compounds. 

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