Stability thiamin

By protodetritiation of the thiazolium salt (152) and of 2 tritiothiamine (153) Kemp and O Brien (432) measured a kinetic isotope effect, of 2.7 for (152). They evaluated the rate of protonation of the corresponding yiides and found that the enzyme-mediated reaction of thiamine with pyruvate is at least 10 times faster than the maximum rate possible with 152. The scale of this rate ratio establishes the presence within the enzyme of a higher concentration of thiamine ylide than can be realized in water. Thus a major role of the enzyme might be to change the relative thermodynamic stabilities of thiamine and its ylide (432). 

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.)... 

The pentagon stabilization has been found in a biochemical phenomenon [80], The hydrogen on the thiazolium ring 9 (Scheme 7) is easily ionized to afford the corresponding carbene 10, a key catalyst in enzymatic reactions for which thiamine (vitamin B-1,11) pyrophosphate is the cofactor. The pentagon stability is expected to contribute to this unusual deprotonation. A lone pair generated on the carbon atom in 10 can similarly delocalize through the vicinal C-N and C-S a bonds in a cyclic manner. 

Bell et al. (2002) investigated the relationship between water mobility as measured by oxygen-17 NMR (transverse relaxation rate obtained from linewidth at half-height) and chemical stability in glassy and rubbery polyvinylpyrrolidone (PVP) systems. Reported results suggest that water mobility in PVP model systems was not related to Tg. The study did not find a link between water mobility and reaction kinetics data (half-lives) for degradation of aspartame, loss of thiamin and glycine, and stability of invertase. 

Kluger and Brandi (1986b) also studied the decarboxylation and base-catalysed elimination reactions of lactylthiamin, the adduct of pyruvate and thiamin (Scheme 2). These reactions are nonenzymic models for reactions of the intermediates formed during the reaction catalysed by the enzyme pyruvate decarboxylase. The secondary j3-deuterium KIE for the decarboxylation was found to be 1.09 at pH 3.8 in 0.5 mol dm-3 sodium acetate at 25°C. In the less polar medium, 38% ethanolic aqueous sodium acetate, chosen to mimic the nonpolar reactive site in the enzyme, the reaction is significantly faster but the KIE was, within experimental error, identical to the KIE found in water. This clearly demonstrates that the stabilization of the transition state by hyperconjugation is unaffected by the change in solvent. 

The finding that thiamine, and even simple thiazolium ring derivatives, can perform many reactions in the absence of the host apoenzyme has allowed detailed analyses of its chemistry [33, 34]. In 1958 Breslow first proposed a mechanism for thiamine catalysis to this day, this mechanism remains as the generally accepted model [35]. NMR deuterium exchange experiments were enlisted to show that the thiazolium C2-proton of thiamine was exchangeable, suggesting that a carbanion zwitterion could be formed at that center. This nucleophilic carbanion was proposed to interact with sites in the substrates. The thiazolium thus acts as an electron sink to stabilize a carbonyl carbanion generated by deprotonation of an aldehydic carbon or decarboxylation of an a-keto acid. The nucleophilic carbonyl equivalent could then react with other electro-... 

A novel and more general method to enable biocatalyzed conversion and synthesis of hydrophobic compounds involves the use of gel-stabilized aqueous-organic two-phase systems [8], Features, advantages, disadvantages, and perspectives of this method in asymmetric synthesis will be discussed in this chapter, illustrated for the stereoselective benzoin condensation and the reduction of ketones catalyzed by thiamine pyrophosphate (TPP)-dependent lyases and NAD(P)H-dependent alcohol dehydrogenases, respectively. 

The stability of some vitamins is influenced by aw. In general, the stability of retinol (vitamin A), thiamin (vitamin Bj) and riboflavin (vitamin B2) decreases with increasing aw. At low av (below 0.40), metal ions do not have a catalytic effect on the destruction of ascorbic acid. The rate of loss of ascorbic acid increases exponentially as aw increases. The photodegradation of riboflavin (Chapter 6) is also accelerated by increasing aw. 

In the case of two flavoenzyme oxidase systems (glucose oxidase (18) and thiamine oxidase s where both oxidation-reduction potential and semiquinone quantitation values are available, semiquinone formation is viewed to be kinetically rather than thermodynamically stabilized. The respective one-electron redox couples (PFl/PFl- and PFI7PFIH2) are similar in value (from essential equality to a 50 mV differential) which would predict only very low levels of semiquinone (32% when both couples are identical) at equilibrium. However, near quantitative yields (90%) of semiquinone are observed either by photochemical reduction or by titration with dithionite which demonstrates a kinetic barrier for the reduction of the semiquinone to the hydroquinone form. The addition of a low potential one-electron oxidoreductant such as methyl viologen generally acts to circumvent this kinetic barrier and facilitate the rapid reduction of the semiquinone to the hydroquinone form. 

Transketolase requires the cofactor thiamine pyrophosphate (TPP), which stabilizes a two-carbon car-banion in this reaction (Fig. 14—26a), just as it does in the pyruvate decarboxylase reaction (Fig. 14-13). Transaldolase uses a Lys side chain to form a Schiff base with the carbonyl group of its substrate, a ketose,... 

For stability reasons it would be better to substitute thiamine hydrochloride by thiamine mononitrate in formulation No. 2. 

Nucleophilic catalysis is a specific example of covalent catalysis the substrate is transiently modified by formation of a covalent bond with the catalyst to give a reactive intermediate. There are also many examples of electrophilic catalysis by covalent modification. It will be seen later that in the reactions of pyridoxal phosphate, Schiff base formation, and thiamine pyrophosphate, electrons are stabilized by delocalization. 

Thiamine pyrophosphate (structure 2.51) is another coenzyme that covalently bonds to a substrate and stabilizes a negative charge. 

T4 lysozyme 33,497 helix stability of 528, 529 hydrophobic core stability of 533, 544 Tanford j8 value 544, 555, 578, 582-Temperature jump 137, 138, 541 protein folding 593 Terminal transferase 408,410 Ternary complex 120 Tertiary structure 22 Theorell-Chance mechanism 120 Thermodynamic cycles 125-131 acid denaturation 516,517 alchemical steps 129 double mutant cycles 129-131, 594 mutant cycles 129 specificity 381, 383 Thermolysin 22, 30,483-486 Thiamine pyrophosphate 62, 83 - 84 Thionesters 478 Thiol proteases 473,482 TNfn3 domain O-value analysis 594 folding kinetics 552 Torsion angle 16-18 Tbs-L-phenylalanine chloromethyl ketone (TPCK) 278, 475 Transaldolase 79 Tyransducin-o 315-317 Transit time 123-125 Transition state 47-49 definition 55... 

The nitrogen atom can also stabilize by delocalizing a negative charge on the adduct of thiamine with many compounds, as, for example, in hydroxy-ethylthiamine pyrophosphate, a form in which much of the coenzyme is found in v vivo (equation 2.53). 

The acidity of the ring proton of the thiamine ring is a consequence of the adjacent positive nitrogen and the known ability of sulfur to stabilize an adjacent carbanion. Nucleophilic attack of the anionic carbon of 10 on C2 of 2-oxopro-panoic acid is followed by decarboxylation ... 

The only vitamins likely to be found in unfortified soft drinks are vitamin C (either added as an antioxidant or deriving from fruit materials) and vitamin A precursor (beta-carotene, added as a colour). However, soft drinks provide a good medium for vitamin fortification, the limitations being solubility (for fat-soluble vitamins), flavour impairment (for example the meaty notes of thiamine) and stability. 

Intermediates of this type have the necessary chemical reactivity for cleaving the bonds indicated in figure 10.1b and c. The decarboxylated product of the pyruvate adduct shown in equation (2) is resonance-stabilized by the thiazolium ring (fig. 10.2a). This intermediate may be protonated to a-hydroxyethyl thiamine pyrophosphate (fig. I0.2d) alternatively, it may react with other electrophiles, such as the carbonyl groups of acetaldehyde or pyruvate, to form the species in figure 10.2b and c or it may be oxidized to acetyl-thiamine pyrophosphate (fig. 10.2e). The fate of the intermediate depends on the reaction specificity of the enzyme with which the coenzyme is associated. 

Mechanism of thiamine pyrophosphate action. Intermediate (a) is represented as a resonance-stabilized species. It arises from the decarboxylation of the pyruvate-thiamine pyrophosphate addition compound shown at the left of (a) and in equation (2). It can react as a carbanion with acetaldehyde, pyruvate, or H+ to form (b), (c), or (d), depending on the specificity of the enzyme. It can also be oxidized to acetyl-thiamine pyrophosphate (TPP) (e) by other enzymes, such as pyruvate oxidase. The intermediates (b) through (e) are further transformed to the products shown by the actions of specific enzymes. 

Decarboxylation of an a-keto acid like pyruvate is a difficult reaction for the same reason as are the ketol condensations (see fig. 12.33) Both kinds of reactions require the participation of an intermediate in which the carbonyl carbon carries a negative charge. In all such reactions that occur in metabolism, the intermediate is stabilized by prior condensation of the carbonyl group with thiamine pyrophosphate. In figure 13.5 thiamine pyrophosphate and its hydroxyethyl derivative are written in the doubly ionized ylid form rather than the neutral form because this is the form that actually participates in the reaction even though it is present in much smaller amounts. 

In the first step of the conversion catalyzed by pyruvate decarboxylase, a carbon atom from thiamine pyrophosphate adds to the carbonyl carbon of pyruvate. Decarboxylation produces the key reactive intermediate, hydroxyethyl thiamine pyrophosphate (HETPP). As shown in figure 13.5, the ionized ylid form of HETPP is resonance-stabilized by the existence of a form without charge separation. The next enzyme, dihydrolipoyltransacetylase, catalyzes the transfer of the two-carbon moiety to lipoic acid. A nucleophilic attack by HETPP on the sulfur atom attached to carbon 8 of oxidized lipoic acid displaces the electrons of the disulfide bond to the sulfur atom attached to carbon 6. The sulfur then picks up a proton from the environment as shown in figure 13.5. This simple displacement reaction is also an oxidation-reduction reaction, in which the attacking carbon atom is oxidized from the aldehyde level in HETPP to the carboxyl level in the lipoic acid derivative. The oxidized (disulfide) form of lipoic acid is converted to the reduced (mer-capto) form. The fact that the two-carbon moiety has become an acyl group is shown more clearly after dissocia-... 

Based on the conventional analysis of the mechanism of decarboxylation of thiamin-derived intermediates, there is no role for a catalyst in the carbon-carbon bond-breaking step of this reaction. The thiazolium nitrogen is at its maximum electron deficiency with no available coordination sites. Ultimately, there is no place for a proton or other cation to position itself in order to promote the reaction by stabilizing a transition state that resembles the product of the reaction. Since there is no role for an acid, base, or metal to accelerate the decarboxylation of these intermediates by stabilizing the transition state for C-C bond-breaking, the means by which this could be achieved became a source of interest and speculation. 

Figure 15 Stability of thiamine hydrochloride in a cellulose-magnesium stearate tablet containing various amounts of moisture following storage at 55°C.

PDC increases the rate of decarboxylation of pyruvate by thiamine alone by a factor of 3 x 1012 at pH 6.2 and 30 °C [52], The capacity of ThDP to catalyse the decarboxylation of a-keto acids depends mainly on two properties of the thiazolium ring of ThDP (a) its capacity to ionize to form a nucleophilic anion and thus bind to the a-carbonyl group of pyruvate, and (b) its ability to stabilize the negative charge upon cleavage of carbon dioxide. 

Analytical Properties Useful for the separation of relatively polar compounds such as phenols, carboxylic acids, organic anions, nucleosides, alkylarylketones, chlorophenols, barbiturates, thiamine derivatives good stability under high and low pH reasonable mechanical integrity at high carrier pressure compatible with buffered liquid phases Reference 36-44... 

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. 

I have been pursuing enzyme mimics, artificial enzymes that perform biomimetic chemistry, since starting my independent career in 1956. In the first work [52-59] my co-workers and I studied models for the function of thiamine pyrophosphate 1 as a coenzyme in enzymes such as carboxylase. We discovered the mechanism by which it acts, by forming an anion 2 that we also described as a stabilized carbene, one of its resonance forms. We examined the related anions from imidazolium cations and oxazolium cations, which produce anions 3 and 4 that can also be described as nucleophilic carbenes. We were able to explain the structure-activity relationships in this series, and the reasons why the thiazolium ring is best suited to act as a biological... 

The proton at C(2) in oxazole is acidic, that at C(5) less so. Instantaneous deuteration at C(2) was observed when oxazole was dissolved in DMSO- 6 containing sodium methoxide a slower exchange occurred at C(5). The effect is enhanced by the presence of electron-attracting substituents at C(4) and even more in oxazolium ions, which owe their reactivity to the stability of ylides such as (145). Unlike thiamine and other thiazolirm... 

Most early clinical descriptions of apparent thiamine-responsive PDC deficiency were not characterized biochemically to ascertain true thiamine dependence. In subsequent reports, immunochemical analyses have demonstrated varied patterns of a- and P-subunit expression, and in vitro studies of cultured cells have sometimes found altered El enzyme kinetics (high Km, low Vmax) for TPP. When molecular genetic analyses have been undertaken, different mutations have been identified within the conserved TPP-binding motif that are considered to lead to diminished binding affinity for TPP or to decreased stability of the oc2P2 tetramer. 

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