Thiamine isolation

The reduction of a number of thiazolium salts had been shown to yield diastereomeric mixtures of thiazolidines from which it has been possible, in some cases (including that of thiamine), to isolate one pure species (Schemes 94 and 94a). 

This resonance-stabilized intermediate can be protonated to give hydroxyethyl-TPP. This well-characterized intermediate was once thought to be so unstable that it could not be synthesized or isolated. However, its synthesis and isolation are actually routine. (In fact, a substantial amount of the thiamine pyrophosphate in living things exists as the hydroxyethyl form.)... 

Nevertheless, the isolation of these metabolites was interesting in two respects. First, the structure of the thiazole glycol stimulated the research of functionalized carbohydrate chains as precursors of thiazole. Second, the thiazolecar-boxylic acid 40 can be secreted by derepressed cells in relatively high amounts, 0.24 nmol per mg of dried cells, which is nearly half the amount of synthesized thiamine. The presence of this free thiazolic derivative in the cells contrasts with... 

Enterobacterbacter aerogenes adth Mutants have been isolated,77 and adenine inhibits the synthesis of thiamine.74,77 [l4C]Formate incorporates at C-2 of pyramine.78... 

In the 1930s, Peters and co-workers showed that thiamine deficiency in pigeons resulted in the accumulation of lactate in the brainstem [ 15]. Furthermore, they showed that the addition of small quantities of crystalline thiamine to the isolated brainstem tissue from thiamine-deficient birds in vitro resulted in normalization of lactate levels. These findings led to the formulation of the concept of the biochemical lesion in thiamine deficiency. Subsequent studies showed that the enzyme defect responsible for the biochemical lesion was a-KGDH rather than pyruvate dehydrogenase (PHDC), as had previously been presumed. a-KGDH and PHDC are major thiamine diphosphate (TDP)-dependent enzymes involved in brain glucose oxidation (Fig. 34-4). 

This factor is particularly significant in OFBD since biological samples or isolates are used. In addition to background interference, fluorescence quenching has been demonstrated in a variety of biomolecules such as thiamine (vitamin Bi),(27) nicotinamide/28 nucleosides/nucleotides,(29) and pyruvate/30 To circumvent the obvious limitations associated with the use of UV or visible fluorophores in OFD, the potential... 

This thiamin pyrophosphate-dependent enzyme [EC 2.2.1.1], also known as glycolaldehyde transferase, catalyzes the reversible reaction of sedoheptulose 7-phos-phate with D-glyceraldehyde 3-phosphate to produce D-ribose 5-phosphate and o-xylulose 5-phosphate. The enzyme exhibits a wide specificity for both reactants. It also can catalyze the reaction of hydroxypyruvate with R—CHO to produce carbon dioxide and R—CH(OH)—C(=0)—CH2OH. Transketolase isolated from Alkaligenes faecalis shows high activity with D-erythrose as the acceptor substrate. 

Vitamin Bj or thiamine (Figure 19.21) is 3-(4-amino-2-methylpyrimidin-5-ylmethyl)-5-(2-hydroxyethyl)-4-methylthiazolium. It is isolated, synthesized, and used in food supplements and in food fortifications as a solid thiazolium salt in the form of thiamine hydrochloride or thiamine mononitrate [403]. 

The isolation of 5-formyl-2-methylpyrimidine (62) and 5-amino-2-methylpyrimidine (63) from thiamin requiring mutants of Neurospora has led to a suggestion that these may be late intermediates in the biosynthesis of (45), a contention supported in part by the demonstration of conversion of the amino compound (63) to the hydroxymethylpyrimidine (45) by yeast cell-free extracts. 

Nearly all the water-soluble vitamins are heterocyclic compounds. Among the first to be isolated was thiamine (vitamin Bi) (62), deficiency of which causes degenerative changes in the nervous system, including the multiple peripheral neuritis characteristic of beriberi. Thiamine deficiency can arise from decomposition of the vitamin by bacteria in the gut. In mammalian metabolism the hydroxy group of thiamine is esterified to give cocarboxylase (thiamine pyrophosphate) which catalyzes the decarboxylation of a-keto acids to aldehydes, acyloins or acids, and their transformation into acyl phosphates. 

Progress in isolation of the vitamins was slow, principally because of a lack of interest. According to R. R. Williams, when he started his work on isolation of the antiberiberi factor in 1910 most people were convinced that his efforts were doomed to failure, so ingrained was Pasteur s idea that diseases were caused only by bacteria. In 1926, Jansen isolated a small amount of thiamin, but it was not until 1933 that Williams, working almost without financial support, succeeded in preparing a large amount of the crystalline compound from rice polishings. Characterization and synthesis followed rapidly.c d... 

Why do we need vitamins Early clues came in 1935 when nicotinamide was found in NAD+ by H. von Euler and associates and in NADP+ by Warburg and Christian. Two years later, K. Lohman and P. Schuster isolated pure cocarboxylase, a dialyz-able material required for decarboxylation of pyruvate by an enzyme from yeast. It was shown to be thiamin diphosphate (Fig. 15-3). Most of the water-soluble vitamins are converted into coenzymes or are covalently bound into active sites of enzymes. Some lipid-soluble vitamins have similar functions but others, such as vitamin D and some metabolites of vitamin A, act more like hormones, binding to receptors that control gene expression or other aspects of metabolism. 

Most known thiamin diphosphate-dependent reactions (Table 14-2) can be derived from the five halfreactions, a through e, shown in Fig. 14-3. Each halfreaction is an a cleavage which leads to a thiamin- bound enamine (center, Fig. 14-3) The decarboxylation of an a-oxo acid to an aldehyde is represented by step b followed by a in reverse. The most studied enzyme catalyzing a reaction of this type is yeast pyruvate decarboxylase, an enzyme essential to alcoholic fermentation (Fig. 10-3). There are two 250-kDa isoenzyme forms, one an a4 tetramer and one with an ( P)2 quaternary structure. The isolation of ohydroxyethylthiamin diphosphate from reaction mixtures of this enzyme with pyruvate52 provided important verification of the mechanisms of Eqs. 14-14,14-15. Other decarboxylases produce aldehydes in specialized metabolic pathways indolepyruvate decarboxylase126 in the biosynthesis of the plant hormone indoIe-3-acetate and ben-zoylformate decarboxylase in the mandelate pathway of bacterial metabolism (Chapter 25).1243/127... 

Jansen and Donath isolated vitamin B, (thiamine) from rice polishings. 

Transketolase from common yeast (Saccharomyces cerevisiae) is commercially available, but it is possible to work with a partially purified enzyme, isolated with little expense from spinach leaves.54 Transketolase catalyzes the transfer of a hydroxyacetyl group, reversibly from a ketose phosphate, or irreversibly from hydroxypyruvate to an acceptor aldose, phosphorylated or not.55 It requires thiamine pyrophosphate as a coenzyme, but only in catalytic amounts. In all the cases listed in Table V, the new chiral center, C-3 of the ketose, has the l-glycero configuration. 

Lebova S (1990) Characterization of pyruvate decarboxylases isolated from germinating seeds. In Bisswanger H, Ullrich J (eds) Biochemistry and physiology of thiamin diphosphate enzymes. VCH, Weinheim, p 133... 

Reaction of an aqueous solution of cystine with thiamin, glutamate, and ascorbic acid produces a complex mixture of compounds with an overall flavor resembling that of roasted meat. The reaction was carried out at 120 C for 0.5h at pH 5.0 in a closed system. The aroma compounds were isolated by means of the simultaneous steam distillation/solvent extraction method. The flavor concentrate was pre-separated by liquid chromatography on silica gel and subsequently analysed by GC and GC/MS. Unknown flavor components were... 

When 0.5% thiamin hydrochloride in degerminated yellow corn meal was extruded on a Werner Pfleiderer ZSK-30 co-rotating twin-screw extruder at the final product temperature of 180°C, numerous volatile components were generated. Table II lists the volatile compounds isolated and identified by purge-and-trap GC/MS method. 

The molar masses of the 2-oxoacid ferredoxin oxidoreductases are 200,000-300,000 g/mol and they are composed of four subunits of the kind a2p2. It has been shown that halobacteria have only these systems of 2-oxoacid ferredoxin oxidoreductases. The two enzymes of H. halobium (pyruvate and oxoglutarate) were isolated and characterized by Kerscher and Oesterhelt (1981a). These systems proved to be thiamin diphosphate-containing iron-sulfur proteins. The relative stability of the halobacterial enzymes enabled detailed analysis of the various steps of the catalytic cycles (Kerscher and Oesterhelt, 1981b), demonstrating two distinct steps of one-electron transfer reactions. 

A number of investigators (15, 16, 17), working with pyruvate decarboxylase, actually isolated the pyruvate adduct to thiamin pyrophosphate, and demonstrated that the enzyme will then decar-boxylate that adduct to yield C02 and acetaldehyde. In an elegant biomimetic study, Lienhard (18, 19) and his collaborators synthesized an analog of this adduct, and showed that it undergoes decarboxylation upon heating in water. Further, the rate is enhanced 105-fold by carry-... 

The ALS isolated as described in Table III displayed typical Michaelis-Menten kinetics with respect to pyruvate with a Km of 2.44 mM. Substrate concentrations as high as 50x Km had no effect on the rate of the reaction. Thiamine pyrophosphate, FAD and Mg(2+) were an absolute requirement for catalysis by the purified enzyme. These properties are consistent with observations made by others (30). Optimum activity was obtained at pH 7.1 and 37C, which were also the best conditions for inhibition by TP. There was no significant difference in the 1(50) value of TP whether ALS was taken after step 2 or 5, indicating low potential for non-specific binding of the herbicide to other proteins. 

An X-ray structure of a thiamine dependent transketolase enzyme was determined by Schneider et al. after isolation from Saccharomyces cerevisiae in the 1990s and is shown in Fig. 10 (Sundstrom et al. 1993 Nilsson et al. 1997). The thiamine cofactor is embedded in a narrow channel in the centre of the enzyme. From the complex surrounding of the heart of this enzyme it seems to be obvious that chemical reactions at the catalytically active site in this channel proceed inevitably with high selectivities. 

Thiamine (= Vitamin B ) (pyrimidinylmethyl thiazole) dietary deficiency yields beriberi involving oedema, pain, neuritis, paralysis death detected by Christiaan Eijkman as polyneuritis in hens fed polished rice isolated from polishings by Jansen Donath... 

The synthetic potential of thiamin diphosphate dependent enzymes is now recognized and has been reviewed recently.84-85 TK has been isolated from... 

In practice, donor substituents make it possible actually to isolate a range of carbenes 4.105. With somewhat less stabilisation, the carbene 4.106, although it is only found as a reactive intermediate, is exceptionally easy to form. It is the key intermediate in all the metabolic steps catalysed by thiamine coenzymes, and its reactions are characterised by its nucleophilicity. Similarly, dimethoxycarbene 4.107 reacts as a nucleophile with electrophiles like dimethyl maleate to give the intermediate 4.108, and hence the cyclopropane 4.109, but it does not insert into unactivated alkenes. 

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