Aldolase acetaldehyde

Mechanistically similar to the pyruvate lyases, 2-deoxy-D-ribose 5-phosphate aldolase (EC 4.1.2.4) catalyzes the addition of acetaldehyde to D-glyceraldehyde 3-phosphate. 

Threonine catabohsm merges with that of glycine after threonine aldolase cleaves threonine to glycine and acetaldehyde. 

These enzymes catalyse the non-hydrolytic cleavage of bonds in a substrate to remove specific functional groups. Examples include decarboxylases, which remove carboxylic acid groups as carbon dioxide, dehydrases, which remove water, and aldolases. The decarboxylation of pyruvic acid (10.60) to form acetaldehyde (10.61) takes place in the presence of pyruvic decarboxylase (Scheme 10.13), which requires the presence of thiamine pyrophosphate and magnesium ions for activity. 

Aldolases catalyze asymmetric aldol reactions via either Schiff base formation (type I aldolase) or activation by Zn2+ (type II aldolase) (Figure 1.16). The most common natural donors of aldoalses are dihydroxyacetone phosphate (DHAP), pyruvate/phosphoenolpyruvate (PEP), acetaldehyde and glycine (Figure 1.17) [71], When acetaldehyde is used as the donor, 2-deoxyribose-5-phosphate aldolases (DERAs) are able to catalyze a sequential aldol reaction to form 2,4-didexoyhexoses [72,73]. Aldolases have been used to synthesize a variety of carbohydrates and derivatives, such as azasugars, cyclitols and densely functionalized chiral linear or cyclic molecules [74,75]. 

With zymohexase, fructose 1,6-diphosphate, and acetaldehyde, a 5-de-oxypentulose 1-phosphate resulted,66 and, with a pea-aldolase preparation, the product was identified as 5-deoxy-D-ilireo-pentulose (LXI). Using... 

In contrast to transketolase and the DHAP-dependent aldolases, deoxyribose aldolase (DERA) catalyzes the aldol reaction with the simple aldehyde, acetaldehyde. In vivo it catalyzes the formation of 2-deoxyribose-5-phosphate, the building block of DNA, from acetaldehyde and D-glyceraldehyde-3-phosphate, but in vitro it can catalyze the aldol reaction of acetaldehyde with other non-phosphorylated aldehydes. The example shown in Scheme 6.28 involves a tandem aldol reaction... 

The product of the PNP enzyme, FDRP 9 has been purified and characterised. The evidence suggests that FDRP 9 is then isomerised to 5-fluoro-5-deoxyribulose-1-phosphate 10, acted upon by an isomerase (Scheme 7). Such ribulose phosphates are well-known products of aldolases and a reverse aldol reaction will clearly generate fluoroacetaldehyde 11. Fluoroacetaldehyde 11 is then converted after oxidation to FAc 1. We have also shown that there is a pyridoxal phosphate (PLP)-dependent enzyme which converts fluoroacetaldehyde 11 and L-threonine 12 to 4-FT 2 and acetaldehyde in a transaldol reaction as shown in Scheme 8. Thus, all of the biosynthetic steps from fluoride ion to FAc 1 and 4-FT 2 can be rationalised as illustrated in Scheme 7. 

Another promising route was reported in patent and open hterature by both DSM and Diversa [13, 14]. This route employs a 2-deoxy-D-ribose 5-phosphate aldolase (DERA) that catalyzes a tandem aldol addition in which two equivalents of acetaldehyde (AA) are added in sequence to chloroacetaldehyde (CIAA) to produce a lactol derivative that is similar to the 3,5-dihydoxy side chain of synthetic statins (Figure 6.2e). Diversa screened environmental libraries for novel wild-type DERAs and identified an enzyme that was both tolerant to increased substrate concentrations and more active than DERA from E. coli in the target reaction [13]. 

The first step in this sequence is the binding of a molecule of acetaldehyde ( donor ) to the aldolase to form a Schiff base with the active site lysine followed by addition to CIAA, which acts as the acceptor aldehyde. This reaction delivers the mono-addition product, which then acts as an acceptor again to react with a second molecule of AA, yielding the double addition product which cyclizes spontaneously to the stable lactol 1 (Scheme 6.4). 

Controversy remains in the determination of substrate tolerance for KdgA/KhgA aldolases from different sources. Early assay studies with KhgA prepared [127-129] from rat liver concluded that the catalyst had an unusually wide ranging tolerance for nucleophilic components, including a number of 3-substituted pyruvate derivatives as well as pyruvaldehyde, acetaldehyde, and pyruvic esters [135], Later, other workers using enzymes from rat or bovine liver and from E. coli reported their inability to reproduce these results but noted a rather limiting specificity [136]. 

Functionally and mechanistically reminiscent of the pyruvate lyases, the 2-deoxy-D-ribose 5-phosphate (121) aldolase (RibA EC 4.1.2.4) [363] is involved in the deoxynucleotide metabolism where it catalyzes the addition of acetaldehyde (122) to D-glyceraldehyde 3-phosphate (12) via the transient formation of a lysine Schiff base intermediate (class I). Hence, it is a unique aldolase in that it uses two aldehydic substrates both as the aldol donor and acceptor components. RibA enzymes from several microbial and animal sources have been purified [363-365], and those from Lactobacillus plantarum and E. coli could be induced to crystallization [365-367]. In addition, the E. coli RibA has been cloned [368] and overexpressed. It has a usefully high specific activity [369] of 58 Umg-1 and high affinity for acetaldehyde as the natural aldol donor component (Km = 1.7 mM) [370]. The equilibrium constant for the formation of 121 of 2 x 10M does not strongly favor synthesis. Interestingly, the enzyme s relaxed acceptor specificity allows for substitution of both cosubstrates propional-dehyde 111, acetone 123, or fluoroacetone 124 can replace 122 as the donor [370,371], and a number of aldehydes up to a chain length of 4 non-hydrogen atoms are tolerated as the acceptor moiety (Table 6). 

In various mammalian tissues an enzymatic activity has been reported [451-453] which causes the liberation of glycine 149 and acetaldehyde from L-threonine 150 and has therefore been named threonine aldolase (ThrA EC 4.1.2.5). It is curious that a//o-threonine 151 seems to be a more active substrate for this enzyme than is 150. Cleavage of L-3-phenylserine is also catalyzed by mammalian ThrA enzymes [454-456], which in direction of synthesis nonselec-tively produce both threo (152) and erythro (153) configurated adducts from benzaldehyde and 149 [454], The same enzyme preparations were also able to act on 150. Thus, considerable disagreement still exists in the literature about the true nature of these enzymatic activities. 

An interesting enzyme-catalyzed three-component aldolization reaction has been described by Gijsen and Wong [18]. Here, acetaldeyde, 2-substituted acetaldehydes, and dihydroxyacetone phosphate react in the presence of the aldolases 2-deoxyribose-5-phosphate aldolase (DERA) and fructose 1,6-diphosphate aldolase (RAMA) forming the corresponding 5-deoxyketose derivatives (Scheme 9.9). 

In nature, most aldolases are rooted in the sugar metabolic cycle and accept highly functionalized substrates for the aldol reaction. Nevertheless, the scope of enzymatic aldol reactions is limited, since aldolases strictly distinguish between the acceptor and the donor, yielding almost exclusively one product, and is furthermore restricted to only a few different possible natural donors. According to the donor molecules, aldolases are grouped in dihydroxyacetone phosphate-, phosphoenolpyruvate- or pyruvate-, acetaldehyde-, and glycine-dependent aldolases [41]. 

To date, 2-deoxy-D-ribose 5-phosphate aldolase (DERA) is the only acetaldehyde-dependent aldolase being applied in organic synthesis. Thus the stereoselectivity of DERA is significant, all known enzymes from different organisms showing the same preferences, limiting the field of application to syntheses in which specifically the DERA-catalyzed enantiomer is needed. 

The similarity between mechanisms of reactions between proline- and 2-deoxy-ribose-5-phosphate aldolase-catalyzed direct asymmetric aldol reactions with acetaldehyde suggests that a chiral amine would be able to catalyze stereoselective reactions via C-H activation of unmodified aldehydes, which could add to different electrophiles such as imines [36, 37]. In fact, proline is able to mediate the direct catalytic asymmetric Mannich reaction with unmodified aldehydes as nucleophiles [38]. The first proline-catalyzed direct asymmetric Mannich-type reaction between aldehydes and N-PMP protected a-ethyl glyoxylate proceeds with excellent chemo-, diastereo-, and enantioselectivity (Eq. 9). 

Kuzuhara et al. synthesized an optically resolved pyridoxal analog having an ansa chain" between the 2 - and 5 -positions (45) [46]. The aldolase-type reaction of 45 and glycine with either acetaldehyde or propionaldehyde afforded the corresponding P-hy-droxy-a-amino acid with 27-77% ee. The erythro isomers were 1.2-1.8 times dominant over threo ones. The (S) -enantiomer of the pyridoxal derivative furnished the (S)-amino acid in excess. Accordingly, the reaction occurred on the same face as was occupied by the ansa chain. We have confirmed these results [47]. 

The chiral 2,4-dideoxyhexose derivative required for the HMG CoA reductase inhibitors has also been prepared using 2-deoxyribose-5-phosphate aldolase (DERA).The reactions start with a stereospecific addition of acetaldehyde (44) (Fig. 18.14) to a substituted acetaldehyde to form a 3-hydroxyl-substituted butyraldehyde 45, which reacts subsequently with another acetaldehyde to form a 2,4-dideoxyhexose derivative 46. DERA has been expressed in Escherichia coli (Gijsen and Wong, 1995). 

Scheme 5.20 Aldolases are classified according to the donor molecule they activate DHAP, pyruvate, glycine and acetaldehyde.

Acetaldehyde-dependent aldolases are the only aldolases that can catalyze the aldol formation between two aldehydes, i.e. with an aldehyde both as donor and acceptor [2-4, 40, 43]. More importantly, since they utilize, with high selectivity,... 

Scheme 5.2. The four main groups of aldolase reactions classified by their donor substrate (1) Dihydroxyacetone phosphate (DHAP)- dependent aldolases, (2) phosphoenol pyruvate (PEP)-and pyruvate-dependent aldolases, (3) 2-deoxyribose-5-phosphate aldolase (DERA), a member of the acetaldehyde-dependent aldolases, and (4) glycine-dependent aldolases (GDA).

The enzyme DERA, 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), is unique among the aldolases in that the donor is an aldehyde. In vivo it catalyzes the reversible aldol reaction of acetaldehyde and D-glyceraldehyde 3-phosphate, forming 2-deoxyribose 5-phosphate, with an equilibrium lying in the synthetic direction (Scheme 5.41). DERA, the only well-characterized member of this type I aldolase, has been isolated from both animal tissue and microorganisms.67... 

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... 

The dephosphorylation of 5-chloro and 5-bromo-D-xylulose-l-phosphate was carried out by the addition of acid phosphatase. After purification, 5-chloro-D-xylu-lose and 5-bromo-D-xylulose were recovered as pure compounds in 47 and 12% yields, respectively, from DHAP. In this study, we have shown that DHAP generated from glycidol 7 can be used in situ as a donor substrate of FruA in the presence of 2-halo-acetaldehydes 20 as acceptor substrates for the synthesis of 5-halo-D-xylulose 19. Given that DHAP aldolases display a broad specificity towards acceptor substrates, this strategy can be applied generally to the synthesis of various analogs of monosaccharides. 

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