Pyruvate adduct formation

The product, a-acetolactate, is formally derived from the acyl carbanion generated by the decarboxylation of pyruvate with a second molecule of pyruvate. The formation of lactylthiamin diphosphate on the enzyme generates initially the unprotonated adduct, which contains the enamine equivalent of a carbanion. This can add to the carbonyl group of a second molecule of pyruvate to form acetolactate (Scheme 35). 

The equilibrium mixture contains 94% keto and 6% diol (2S4) and only traces of enol. Only the keto form (XVIII) is a substrate 234-236). The rate at which the diol (XX) dehydrates to the ketone (XVIII) correlates well with the slow appearance of active substrate when a solution of the diol (XX) is brought to pH 6.9 235). Pyruvate enolizes too slowly for the enol form to be the true substrate 236). Pyruvate transiently freed of enolpyruvate is a better substrate than the equilibrium mixture of forms XVIII, XIX, and XX 237) since the formation of the enzyme NAD-pyruvate adduct is prevented. 

However, in MCRs based on pyruvic acids no formation of a,(3-unsaturated carbonyl compounds was observed. Instead of Knoeveganel condensation, the first step of the process was the formation of the corresponding azolmethine 34, which when treated with enole form of pyruvic acid gave adduct 35. The final stage was cyclization into compounds 29 or 32 [51, 53] (Scheme 14). 

Knoevenagel adduct 239 of oxohomophthalimide 240 with malononitrile 27a in reactions with CH-acids behaves ambiguously (82CPB1215). Reactions of 239 with acetylacetone, ethyl esters of acetoacetic and ben-zoylacetic acids, as well as methyl pyruvate led to the formation of the desired spiropyrans 241. However, benzoylacetone, dibenzoylmethane, cyanacetamide, and oxindole always gave the same 242. Authors explain this feature in terms of a retro-cleavage of adducts of Michael product 239... 

Experimental support for the mechanism of Eq. 15-26 has been obtained using D-chloroalanine as a substrate for D-amino acid oxidase.252-254 Chloro-pyruvate is the expected product, but under anaerobic conditions pyruvate was formed. Kinetic data obtained with a-2H and a-3H substrates suggested a common intermediate for formation of both pyruvate and chloro-pyruvate. This intermediate could be an anion formed by loss of H+ either from alanine or from a C-4a adduct. The anion could eliminate chloride ion as indicated by the dashed arrows in the following structure. This would lead to formation of pyruvate without reduction of the flavin. Alternatively, the electrons from the carbanion could flow into the flavin (green arrows), reducing it as in Eq. 15-26. A similar mechanism has been suggested for other flavoenzymes 249/255 Objections to the carbanion mechanism are the expected... 

In addition, the ability of dibenzoylethylene to act as a hydride ion acceptor became apparent in the formation (3-adduct 171, dibenzoylethane 172 and triazolopyrimidines 173 and 174, while their dihydro analogues were not observed as reaction products. Another example, involving pyruvic acid derivatives and 3-amino-1,2,4-triazole, will be shown below. 

By considering the above-mentioned solution studies and the refined three-dimensional structure of the S. cerevisiae flavocytochrome 62 active site, Lederer and Mathews proposed a scheme for the reverse reaction (the reduction of pyruvate) (39). They did not discuss how the transfer of electrons took place except to say that the structure did not rule out the possibility of a covalent intermediate (39). Ghisla and Massey (116) considered the anionic flavin N5 to be too close to the pyruvate carbonyl (3.7 A) without the formation of a covalent adduct taking place. Covalent intermediates between substrate and flavin have been observed for lactate oxidase (117, 118) and o-amino acid... 

FORMATION OF CARBON-CARBON BONDS - The anion derived from the tosylate of CF3CH2OH readily adds to ketones or nonenolizable aldehydes. Sequential treatment of the adduct with acid and base gives the substituted pyruvic acid. 

Formation of Portisins in model solution The formation of a Portisin was monitored at 35 °C in 20% aqueous ethanol (pH=2.0) in a 2 mL screw cap vial containing 0.2 mg of malvidin-3-coumaroylglucoside pyruvic acid adduct previously isolated (11) and 0.33 mg of (+)-catechin. The total volume of die solution was set to 50% of the vial capacity. After 15 days of reaction, the solution was analyzed by HPLC using the conditions described above. 

Anthocyanin-pyruvic acid adducts are known to be more abundant in Port wines than in red table wines, as seen from previous analysis in our laboratories (data not shown) and as referred by other authors (77). This feature may be related to the higher levels of pyruvic acid expected in fortified wines as a result of a shortened fermentation. In fact, when wine spirit is added in order to stop fermentation, the pyruvic acid concentration is expected to be higher than when the fermentation is allowed to go to dryness. Effectively, the pyruvic acid excreted by the yeast at the beginning of the fermentation is further used in the yeast metabolism (35). Therefore, could favor the formation of anthocyanin-pyruvic acid adducts. 

In these reactions, the C2-atom of ThDP must be deprotonated to allo v this atom to attack the carbonyl carbon of the different substrates. In all ThDP-dependent enzymes this nucleophilic attack of the deprotonated C2-atom of the coenzyme on the substrates results in the formation of a covalent adduct at the C2-atom of the thiazolium ring of the cofactor (Ila and Ilb in Scheme 16.1). This reaction requires protonation of the carbonyl oxygen of the substrate and sterical orientation of the substituents. In the next step during catalysis either CO2, as in the case of decarboxylating enzymes, or an aldo sugar, as in the case of transketo-lase, is eliminated, accompanied by the formation of an a-carbanion/enamine intermediate (Ilia and Illb in Scheme 16.1). Dependent on the enzyme this intermediate reacts either by elimination of an aldehyde, such as in pyruvate decarboxylase, or with a second substrate, such as in transketolase and acetohydroxyacid synthase. In these reaction steps proton transfer reactions are involved. Furthermore, the a-carbanion/enamine intermediate (Ilia in Scheme 16.1) can be oxidized in enzymes containing a second cofactor, such as in the a-ketoacid dehydrogenases and pyruvate oxidases. In principal, this oxidation reaction corresponds to a hydride transfer reaction. 

The mechanism of decarboxylation of pyruvic acid is shown in Scheme Xll. The first step is formation of the anion, which then adds to C-2 of pymvate, forming a covalent adduct. This compound has been prepared chemically and its... 

Sequential treatment of 3-acetylthiazolidine-2-thione (1055) with stannous trifluoro-methanesulfonate, diamine 1057, and methyl pyruvate results in the formation of adduct 1058 with S5Vo ee. The 1-naphthyl group on the pyrrolidine system is essential to ensure high asymmetric induction. Other groups such as cyclohexyl, phenyl, 2,6-xylyl, or 2-naphthyl... 

The kinetics of the Michael addition reaction with acetonitrile on reduced protein studied by Cavins and Friedman (19) served as an excellent model for later studies with N-acetyldehydroalanine methyl ester (20) where the formation of a variety of dehydroalanine adducts of amino acids were reported. It is necessary to use the dehydroalanine methyl ester in these studies because the reaction kinetics are much faster than with the free N - acetyl dehydroalanine (21). The free amino compounds decomposes to ammonia and pyruvic acid when synthesis is attempted. 

Copper complexes of the bisoxazoline ligands have been shown to be excellent asymmetric catalysts not only for the formation of carbocyclic systems, but also for the hetero-Diels-Alder reaction. Chelation of the two carbonyl groups of a 1,2-dicarbonyl compound to the metal atom of the catalyst sets up the substrate for cycloaddition with a diene. Thus, the activated diene 20 reacts with methyl pyruvate in the presence of only 0.05 mol% of the catalyst 66 to give the adduct 138 with very high enantiomeric excess (3.99). 

At the same time, both pyruvate and NAD are in oxidized states. Similar formation of an adduct can easily be accomplished by a nonenzymatic base-catalyzed reaction without, of course, any stereospecificity. In the case of the mimetic reaction, further bond formation between the amide nitrogen and the pyruvate carbonyl carbon occurs to give a cyclized product. The cyclization also... 

Gunsalus and Sagers (1958), in studying this sequence for C. acidiurici, propose that dissimilation of glycine proceeds either by condensation with FH4 and oxidation of the adduct, or else by way of serine to pyruvate and thence oxidative decarboxylation to acetate and COs. Formate and CO2 are in equilibrium, thus providing another cyclic mechanism for oxidation of Cl units. A similiar reaction sequence is stated to hold for the dissimilation of ycine by the anaerobe Diplococcua glyeinophilus. Glyoxylic acid is inert in these systems. 

---