Dihydroxyacetone stereospecificity

Due to mechanistic requirements, most of these enzymes are quite specific for the nucleophilic component, which most often is dihydroxyacetone phosphate (DHAP, 3-hydroxy-2-ox-opropyl phosphate) or pyruvate (2-oxopropanoate), while they allow a reasonable variation of the electrophile, which usually is an aldehyde. Activation of the donor substrate by stereospecific deprotonation is either achieved via imine/enamine formation (type 1 aldolases) or via transition metal ion induced enolization (type 2 aldolases mostly Zn2 )2. The approach of the aldol acceptor occurs stereospecifically following an overall retention mechanism, while facial differentiation of the aldehyde is responsible for the relative stereoselectivity. 

Fig. 24 (a, b) Chemo-enzymatic process for synthesis of tetrahydroxylated pyrrolizidines 1-epi-alexine, australine and 3-epi-australine utilising dihydroxyacetone phosphate (DHAP), stereospecific aldol reaction catalysed by fructose-1.6-diphosphate aldolase (FDPA) and acid phosphatase (Pase) [149]... 

As is evident, the reaction proceeds with very high stereoselectivity, since the enzyme can perform stereospecific / -protonation (protonation from only one side) of the enamine leading to dihydroxyacetone phosphate. 

The enzymatic aldol reaction represents a useful method for the synthesis of various sugars and sugar-like structures. More than 20 different aldolases have been isolated (see Table 13.1 for examples) and several of these have been cloned and overexpressed. They catalyze the stereospecific aldol condensation of an aldehyde with a ketone donor. Two types of aldolases are known. Type I aldolases, found primarily in animals and higher plants, do not require any cofactor. The x-ray structure of rabbit muscle aldolase (RAMA) indicates that Lys-229 is responsible for Schiff-base formation with dihydroxyacetone phosphate (DHAP) (Scheme 13.7a). Type II aldolases, found primarily in micro-organisms, use Zn as a cofactor, which acts as a Lewis acid enhancing the electrophilicity of the ketone (Scheme 13.7b). In both cases, the aldolases accept a variety of natural (Table 13.1) and non-natural acceptor substrates (Scheme 13.8). 

Triose phosphate isomerase enzyme catalyses interconversion of the 3-carbon triose phosphate dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (D-GAP). The reaction is just the transfer of the pro-R hydrogen from carbon 1 of DHAP stereospecifically to carbon 2 to form the D-isomer of GAP (Figure 2). Although the equilibrium constant on the enzyme is not known, Keq for the overall reaction is 300 to 1 in favour of DHAP. The large magnitude of this number arises from the combination of an apparent Keq of 22 vith a hydration equilibrium of 29 for the hydrated and unhydrated forms of D-GAP (Trentham ei al., 1969) only the unhydrated forms of the triose phosphates are substrates for or even bind to the isomerase (Vebb eial., 1977). 

Its substrate specificity is very low, catalyzing the turnover of a wide range of compounds from small molecules such as propane-1,2-diol to polysaccharides. The best studied substrate is dihydroxyacetone which is oxidized with triple the speed of D-galactose [154]. Although galactose oxidase has a very low substrate specificity, its stereospecificity is absolute D-glucose and L-galactose are not oxidized [30]. The enzyme s specificity for electron-acceptors is low as well. In... 

We have developed preparative enzymatic syntheses of several unusual hexoketoses using fructose-1,6-diphosphate aldolase (FDP-aldolase, E.C.4.1.2.13) as catalyst and dihydroxyacetone phosphate (DHAP) and an aldehyde as substrates (15). The enzyme appears to be very specific for DHAP but will accept a variety of aldehydes as acceptors. The ketose-1-phosphates prepared are converted to the phosphate free ketoses after removal of the phosphate group by acid- or phosphatase-catalyzed hydrolysis. The ketoses can be isomerized stereospecifically to aldoses catalyzed by glucose isomerase (E.C.5.3.1.5.) from Flavobacteriuum arborescens. The equilibrium mixtures of aldoses and ketoses are then separated by chromatography on Dowex 50 (Ba ) or Dowex 1 (HSO "). Figure 1 illustrates the preparation of a mixture of 6-deoxy-6-fluoro-D-fructose... 

Aldolase catalyzes both a stereospecific exchange of a hydrogen of dihydroxyacetone phosphate and an exchange reaction between carbons 4, 5, and 6 of the fructose and the three carbons of glyceraldehyde. Since glyceraldehyde can also be derived from dihydroxyacetone, the carbons of fructose will randomize (see Fig. 1-8). 

PoYSER and West [503] and Harris, Luscombe and Poyser [274] determined the relative activities of different carbohydrates injected locally in inhibiting the increase in vascular permeability caused by intradermally injected ovomucoid, dextran, dextrin, yeast mannan and z5miosan. With the exception of dihydroxyacetone, galactose, lactose, rhamnose and ribose many hexoses, pentoses, tricses and disaccharides were active. In a further study, Poyser and West [504] elucidated the structural and stereospecific features of topically administered sugars for potent inhibitory activity against intradermal responses to dextran. The monosaccharides most active as inhibitors contained the... 

The (3S,4R) configured probes were prepared by enzymatic routes based on the stereospecific formation of a C-C bond catalyzed by either TK with Li-HPA as donor or by fructose-6-phosphate aldolase (FSA) Ref. [41]. The advantage of FSA for the synthesis of these probes is that acceptor substrates were commercially available, whereas a-hydroxylated TK acceptor substrates had to be prepared first by chemical routes [40, 41]. In particularly, the recently engineered FSA(A129S) variant that was optimized for dihydroxyacetone (DHA) as the donor substrate was found to be a powerful biocatalyst, leading to D-ketose analogs 16a and 16b with 67% and 77% yields, respectively. TK reactions furnished the same products but with lower yields only (37% and 47% respectively) (Scheme 15.17). 

Chain-extended Compounds. - Unusual hexoses and higher carbon sugars have been produced by use of fructose 1,6-diphosphate aldolase which catalysed firstly, in combination with triosephosphate isomerase, the release of dihydroxyacetone phosphate (DHAP) from fructose 1,6-diphosphate and secondly the stereospecific condensation of DHAP with a variety of simple aldehydes. Two examples are given in Scheme 7. ... 

Finally, the use of isotopes in carbon-acid substrates is an invaluable tool for the determination of the stereochemistry of the enzymatic proton transfer. In contrast to organic reactions, stereospecific proton transfers are the rule, rather than the exception, in enzymatic reactions, owing to the inherently asymmetric nature of the protein surface. An example is the pair of isotopic exchange reactions between dihydroxyacetone phosphate and tritiated water catalysed by the enzymes aldolase and triose phosphate isomerase [17]. In the two cases a different a-hydrogen of the ketone is exchanged with water, leading to the two discrete monotritiated derivatives 1 (labelled by the isomerase) and 2 (labelled by the aldolase) ... 

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