Aldolases FDP aldolase

Aldolases accept a wide range of aldehydes in place of their natural substrates and permit the synthesis of carbohydrates such as azasugars, deoxy sugars, deoxythio sugars, fluoro-sugars, and C8 or C9 sugars. In the case of D-fructose-1,6-diphosphate aldolase (FDP aldolase, Type A), more than 75 aldehydes have been identified as substrates [143]. 

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... 

Within group-I aldolases, FDP aldolase from rabbit muscle has been extensively used for the synthesis of biologically active sugar analogs on a preparative scale (Scheme 2.187). For example, nojirimycin and derivatives thereof, which have been shown to be potent anti-AIDS agents with no cytotoxicity, have been obtained by a chemoenzymatic approach using RAMA in the key step. As expected, the recognition of the a-hydroxy stereocenter in the acceptor aldehyde was low [1400, 1401]. 

Enzymes with an initially low activity which gradually increased during embryonic development. To this group of enzymes belong fructose-1,6-diphosphate aldolase (FDP-aldolase) and NAD-dependent malate dehydrogenase (NAD-MDH). The activities of these enzymes increased gradually until the morula-blastocyst transition, whereupon the enzyme activity increased more rapidly (Epstein et al., 1969). 

There are two distinct groups of aldolases. Type I aldolases, found in higher plants and animals, require no metal cofactor and catalyze aldol addition via Schiff base formation between the lysiae S-amino group of the enzyme and a carbonyl group of the substrate. Class II aldolases are found primarily ia microorganisms and utilize a divalent ziac to activate the electrophilic component of the reaction. The most studied aldolases are fmctose-1,6-diphosphate (FDP) enzymes from rabbit muscle, rabbit muscle adolase (RAMA), and a Zn " -containing aldolase from E. coli. In vivo these enzymes catalyze the reversible reaction of D-glyceraldehyde-3-phosphate [591-57-1] (G-3-P) and dihydroxyacetone phosphate [57-04-5] (DHAP). 

Fig. 6. FDP-aldolase-catalyzed addition of electrophiles (94) with DHAP (139—146). Representative R groups ia (94) are given as (a—j) (a) methyl, CH (b)...

A tandem enzymatic aldol-intramolecular Homer-Wadsworth-Emmons reaction has been used in the synthesis of a cyclitol.310 The key steps are illustrated in Scheme 8.33. The phosphonate aldehyde was condensed with dihydroxyacetone phosphate (DHAP) in water with FDP aldolase to give the aldol adduct, which cyclizes with an intramolecular Horner-Wadsworth-Emmons reaction to give the cyclo-pentene product. The one-pot reaction takes place in aqueous solution at slightly acidic (pH 6.1-6.8) conditions. The aqueous Wittig-type reaction has also been investigated in DNA-templated synthesis.311... 

Reagents and conditions i, (a) FDP aldolase, H20/DMF (b) acid phosphatase ii, acetone, p-TsOH iii, (a) THF, reflux (b) Si02, hexane/EtOAc, 9/1 iv, DDQ, CH2CI2-H20-acetone v, TsOH, H20-acetone... 

Fig. 39 Chemoenzymatic synthesis of imino sugars exploiting fructose 1,6-diphosppate (FDP) aldolase.

FDP Aldolase. There have been approximately 15 aldolases isolated, each of which catalyzes a distinct aldol reaction (25-26). The aldolases which have been studied the most as synthetic catalysts are fructose-1,6-... 

Figure 1. Synthesis of usual and unusual sugars using FDP aldolase and glucose isomerase as catalysts.

Figure 2. Mechanism of dihydroxyacetone/arsenate reaction with FDP aldolase. Both dihydroxyacetone and inorganic arsenate are not the inhibitor of the aldolase reactions. The rate constant for the arsenate ester formation is determined enzymatically (a plot of 1/v vs 1/E gives a non-zero intercept which is attributed to the rate at infinite enzyme concentration and that rate corresponds to the rate of nonenzymatic formation of the arsenate ester).

Figure 3. Selectivity of the FDP-aldolase reactions using DHAP vs. dihydroxyacetone/arsenate as a substrate. In the former case, the more stable sugar is obtained due to the reversible nature of the reaction. In the later case, both sugars were obtained in nearly equal amounts, because the reaction was found to be virtually irreversible and the formation of the arsenate ester was rate limiting.

All the enzymes used in the work described above are quite stable at room temperature and can be used in a free form. They can also be used in an immobilized form to improve the stability and to facilitate the recovery. Many immobilization techniques are available today (25). The recent procedure developed by Whitesides et al using water-insoluble, cross-linked poly(aerylamide-acryloxysuccinimide) appears to be very useful and applicable to many enzymes (37). We have found that the non-crosslinked polymer can be used directly for immobilization in the absence of the diamine cross-linking reagent. Reaction of an enzyme with the reactive polymer produces an immobilized enzyme which is soluble in aqueous solutions but insoluble in organic solvents. Many enzymes have been immobilized by this way and the stability of each enzyme is enhanced by a factor of greater than 100. Horse liver alcohol dehydrogenase and FDP aldolase, for example, have been successfully immobilized and showed a marked increase in stability. 

Two new stereocenters are established in the DHAP-dependent aldolases-cata-lyzed carbon-carbon bond formation. Consequently four different stereoisomers can be formed (Scheme 5.23). Enantioselective aldolases that catalyze the formation of just one of each of the stereoisomers are available fructose 1,6-diphosphate aldolase (FDP A), rhamnulose 1-phosphate aldolase (Rha 1-PA), L-fucu-lose 1-phosphate aldolase (Fuc 1-PA) and tagatose 1,6-diphosphate aldolase (TDP A). In particular the FDP A, that catalyzes the formation of the D-threo stereochemistry, has been employed in many syntheses. One such FDP A that... 

FDP A was employed in a study of pancratistatin analogs to catalyze the formation of the D-threo stereochemistry (Scheme 5.24). When rhamnulose 1-phosphate aldolase (Rha 1-PA) was used the L-threo stereoisomer was obtained with excellent selectivity. Thus these two enzymes allow the stereoselective synthesis of the two threo-stereoisomers [44]. They were also utilised successfully for the synthesis of different diastereoisomers of sialyl Lewis X mimetics as se-lectin inhibitors. Not only the two threo-selective aldolases RAMA and Rha 1-PA, but also the D-erythro-selective Fuc 1-PA was employed. In this way it was possible to synthesise three of the four diastereoisomers enantioselectively (Scheme 5.25). The L-erythro stereochemistry as the only remaining diastereo-isomer was not prepared [45]. This is because the aldolase that might catalyze its formation, TDP A, is not very stereoselective and therefore often yields mixtures of diastereoisomers. 

Scheme 5.1. The two types of aldolase mechanisms The type I Schiff-base forming aldolase is represented by rabbit muscle fructose disphosphate (FDP) aldolase (RAMA, top), and the type II zinc enolate aldolase is represented by E. coli fructose diphosphate (FDP) aldolase (bottom).

Type II FDP aldolases are more stable than their type I counterparts. For example, the enzyme from E. coli has no thiol group in the active site and has a half-life of approximately 60 days in 0.3 mM Zn+2 at pH 7.0. The type I enzyme from rabbit muscle (RAMA), by contrast, has a half-life for the free enzyme of approximately 2 days in aqueous solution at pH 7.O.20 These half-lives can be lengthened by immobilization or enclosure in dialysis membranes. 

In vivo, six known DHAP-dependent aldolases are known to catalyze the reversible enanotioselective aldol addition of dihydroxyacetone phosphate to an acceptor aldehyde. The group is comprised of fructose 1,6-diphosphate (FDP) aldolase (EC 4.1.2.13), L-fuculose 1-phosphate (Fuc 1-P) aldolase (EC 4.1.2.17), tagatose 1,6-diphosphate (TDP) aldolase (EC 4.1.2.2), ketotetrose phosphate aldolase (EC 4.1.2.2), L-rhamnulose 1-phosphate (Rha 1-P) aldolase (EC 4.1.2.19), and phospho-5-keto-2-deoxygluconate aldolase (EC 4.1.2.29). The in vivo catalyzed reactions of this group are shown in Scheme 5.3. 

The aldehyde substrates may be used as racemic mixtures in many cases, as the aldolase catalyzed reactions can concomitantly accomplish kinetic resolution. For example, when DHAP was combined with d- and L-glyceraldehyde in the presence of FDP aldolase, the reaction proceeded 20 times faster with the D-enantiomer. Fuc 1-P aldolase and Rha 1-P aldolase show kinetic preferences (greater than 19/1) for the L-enantiomer of 2-hydroxy-aldehydes. Alternatively, these reactions may be allowed to equilibrate to the more thermodynamically favored products. This thermodynamic approach is particularly useful when the aldol products can cyclize to the pyranose form. Since the reaction is reversible under thermodynamic conditions, the product with the fewest 1,3-diaxial interactions will predominate. This was demonstrated in the formation of 5-deoxy-5-methyl-fructose-l-phosphate as a minor product (Scheme 5.5).20a 25 The major product, which is thermodynamically more stable, arises from the kinetically less reaction acceptor. 

The preparation of DHAP for synthetic applications has been accomplished both enzymatically and chemically (Scheme 5.7).27 DHAP can be generated enzymatically in situ from fructose 1,6-diphosphate, using FDP aldolase acting in its catabolic mode, and triosephosphate isomerase (TPI). Glyceraldehyde 3-phosphate (G3P) and DHAP are produced in this reaction, with the G3P rapidly undergoing isomerization to DHAP. 

Thiosugars can be rapidly synthesized when sulfur-substituted aldehydes are condensed with DHAP under the influence of FDP aldolase.42 The synthesis of these heterocycles is completed by phosphate cleavage, acetylation and reduction of the resultant ketone (Scheme 5.20). Cyclitols, another interesting class of biologically active compounds, have been prepared through the reaction of phosphonate- and nitro-substituted aldehydes with DHAP under the influence of FDP aldolase (Scheme 5.21) 43,436... 

Stereoselective synthesis of the C11-C16 fragment of the polyene macro-lide antibiotic, pentamycin, has also been accomplished under the aldolase protocol.45 A formyl and benzyl protected aldehyde, available from D-glucose by chemical methods, reacts with DHAP under the influence of FDP aldolase. After phosphatase hydrolysis the essential C11-C16 skeleton of pentamycin is generated. Removal of an additional hydroxyl group at position 1 and isolation of the C11-C16 fragment as a thioacetal, is accomplished in several steps (Scheme 5.23). 

A formal total synthesis of (+)-aspicilin, an 18-membered ring lactone isolated from the lichen Aspicilia gibbosa, is accomplished by the FDP aldolase protocol.46 The three carbon chain extension of benzyl protected 4-hydroxy-butanal is achieved with DHAP under the influence of FDP aldolase. The acid... 

In DERA reactions, where acetaldehyde is the donor, products are also themselves aldehydes. In certain cases a second aldol reaction will proceed until a product has been formed that can cyclize to a stable hemiacetal.71 For example, when a-substituted aldehydes were used, containing functionality that could not cyclize to a hemiacetal after the first aldol reaction, these products reacted with a second molecule of acetaldehyde to form 2,4-dideoxyhexoses, which then cyclized to a hemiacetal, preventing further reaction. Oxidation of these materials to the corresponding lactone, provided a rapid entry to the mevinic acids and compactins (Scheme 5.43). Similar sequential aldol reactions have been studied, where two enzyme systems have been employed72 (Scheme 5.44). The synthesis of 5-deoxy ketoses with three substitutents in the axial position was accomplished by the application of DERA and RAMA in one-pot (Scheme 5.44). The long reaction time required for the formation of these thermodynamically less stable products, results in some breakdown of the normally observed stereoselectivity of the DERA and FDP aldolases. In a two-pot procedure, DERA and NeuAc aldolase have... 

FDP by aldolase is split reversibly To phosphoglyceraldehyde, also DHAP. 

Diphosphofructoaldolase is a soluble glycolytic enzyme especially abundant in skeletal muscle, occurring also in the myocardium and to a lesser extent in liver and erythrocytes, so that hemolysis of blood specimens elevates the serum aldolase activity and must therefore be avoided. The molecular weight of muscle aldolase is 147,000-180,000 (DIO), Its function is specifically the reversible splitting of D-fructose-1,6-diphosphate (FDP) into equimolecular amounts of the trioses D-glyceralde-hyde-3-phosphate (G-3-P) and dihydroxyacetone phosphate (DAP). 

For both methods, serum of very high aldolase activity may be suitably diluted with normal saline just prior to assay, a method proved valid by testing serial dilutions. Even high activities are retained in semm with little change for at least a fortnight at — 17°C. Semm aldolase activity may be conventionally expressed by both methods as pi of FDP split per hour by 1 ml of semm at 37°C (B21), and in 50 healthy adults the normal range has been found (R13) to be 2.3-8.8 units per ml (mean 5.7 units). For conversion to International Units, I pl/hour/ml = 0.745 pmole/minute/liter (C2). 

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