Aldehyde-lyases

In several recent applications of enzyme catalysis, the snbstrates on which the enzymes act are not the kind of snbstrates that are natnral to the enzyme. However, enzyme catalysed synthesis of hexoses in the laboratory depends solely on enzymes acting on natural or near natnral snbstrates. The relevant enzymes are the aldolases (EC 4.1.2 aldehyde-lyases) since they catalyse an aldol type of C-C bond forming aldol addition reaction. The aldolases most commonly join two C-3 units, called donor and acceptor, and two new stereocentra are formed with great stereoselectivity. 

For this type of C-C bond formation both stereoisomers of the hydroxyphenyl-propanone can be obtained using either the BFD mentioned above or the benz-aldehyde lyase (BAL). Both of these enzymes are dependent on thiamine diphosphate (ThDP) as cofactor [22]. For the enantioselective reduction of the intermediate also, both stereoisomers can be obtained by using two different ADH enzymes. Thus all four possible stereoisomers of the diol can be obtained in high optical purity (see Scheme. 3.1.1) [23]. 

Enzymes of Aldehyde Production. Lipoxygenases are discussed elsewhere in this volume. The aldehyde-lyase has only recently been obtained even in crude extracts 145), However, use of 13- and 9-hydroper-oxyoctadecanoic acids to form Cg and C9 aldehydes and corresponding w-oxoacids is evidence that the pathway is correct (145). As with alliinase and myrosinase these reactions occur only when leaves or fruit are cut or crushed (138,143,144,145). If they are heated before blending or blended under N2 (138), little carbonyl compound is formed (142). H2O2 inhibits (138) this reaction as expected for lipoxygenase (153). lrarw-2-Hexenal is formed nonenzymatically from m-S-hexenal (138). 

Aldehyde-lyases (a few utilize coenzymes, including pyridoxal phosphate)... 

Aldolases are a specific group of lyases (aldehyde-lyases EC 4.1.2) catalyzing the reversible stereoselective addition of a donor compound (nucleophile) to an acceptor compound (electrophile). 

According to the information available, then, three enzymes take part in the aromatization of neutral Cig-stcroids. These are a 19-hydroxylase, a 19-oxida.so and an aldehyde lyase (desmolase), and all are to be found in the microsomal fraction of human placenta. 

A subclass of lyases, involved in amino acid metabolism, utilizes pyridoxal 5-phosphate (PLP, 3-hydroxy-2-methyl-5-[(phosphonooxy)methyl]-4-pyridinecarbaldehyde) as a cofactor for imine/ enamine-type activation. These enzymes are not only an alternative to standard fermentation technology, but also offer a potential entry to nonnatural amino acids. Serine hydroxymethyl-tansferase (SHMT EC 2.1.2.1.) combines glycine as the donor with (tetrahydrofolate activated) formaldehyde to L-serine in an economic yield40, but will also accept a range of other aldehydes to provide /i-hydroxy-a-amino acids with a high degree of both absolute and relative stereochemical control in favor of the L-erythro isomers41. 

A number of lyases are known which, unlike the aldolases, require thiamine pyrophosphate as a cofactor in the transfer of acyl anion equivalents, but mechanistically act via enolate-type additions. The commercially available transketolase (EC 2.2.1.1) stems from the pentose phosphate pathway where it catalyzes the transfer of a hydroxyacetyl fragment from a ketose phosphate to an aldehyde phosphate. For synthetic purposes, the donor component can be replaced by hydroxypyruvate, which forms the reactive intermediate by an irreversible, spontaneous decarboxylation. 

Typically, lyases are quite specific for the nucleophilic donor component owing to mechanistic requirements. Usually, approach of the aldol acceptor to the enzyme-bound nucleophile occurs stereospedfically following an overall retention mechanism, while the facial differentiation of the aldehyde carbonyl is responsible for the relative stereoselectivity. In this manner, the stereochemistry of the C—C bond formation is completely controlled by the enzymes, in general irrespective of the constitution or configuration of the substrate, which renders the enzymes highly predictable. On the other hand, most of the lyases allow a reasonably broad variation of the electrophilic acceptor component that is usually an aldehyde. This feature... 

N-Acetylneuraminic acid aldolase (or sialic acid aldolase, NeuA EC 4.1.3.3) catalyzes the reversible addition of pyruvate (2) to N-acetyl-D-mannosamine (ManNAc (1)) in the degradation of the parent sialic acid (3) (Figure 10.4). The NeuA lyases found in both bacteria and animals are type I enzymes that form a Schiff base/enamine intermediate with pyruvate and promote a si-face attack to the aldehyde carbonyl group with formation of a (4S) configured stereocenter. The enzyme is commercially available and it has a broad pH optimum around 7.5 and useful stability in solution at ambient temperature [36]. 

A biochemically related benzaldehyde lyase (BAL) (EC 4.1.2.38) catalyzes the same carboligation reactions, but with opposite (J )-selectivity (mf-110) [178]. All these enzymes seem to display a rather useful substrate tolerance for variously substituted aldehyde precursors. 

The hydroxynitrile lyase (HNL)-catalyzed addition of HCN to aldehydes is the most important synthesis of non-racemic cyanohydrins. Since now not only (f )-PaHNL from almonds is available in unlimited amounts, but the recombinant (S)-HNLs from cassava (MeHNL) and rubber tree (HbHNL) are also available in giga units, the large-scale productions of non-racemic cyanohydrins have become possible. The synthetic potential of chiral cyanohydrins for the stereoselective preparation of biologically active compounds has been developed during the last 15 years. 

R)-Benzoins and (/ )-2-hydroxypropiophcnonc derivatives are formed on a preparative scale by benzaldehyde lyase (BAL)-catalyzed C-C bond formation from aromatic aldehydes and acetaldehyde in aqueous buffer/DMSO solution with remarkable ease in high chemical yield and high optical purity (Eq. 8.112).303 Less-stable mixed benzoins were also generated via reductive coupling of benzoyl cyanide and carbonyl compounds by aqueous titanium(III) ions.304... 

Hydroxynitrile lyases (HNLs or oxynitrilases) catalyze C—C bond-forming reactions between an aldehyde or ketone and cyanide to form enantiopure cyanohydrins (Figure 1.15), which are versatile building blocks for the chiral synthesis of amino acids, hydroxy ketones, hydroxy acids, amines and so on [68], Screening of natural sources has led to the discovery of both... 

In plant tissues, various enzymes convert the hydroperoxides produced by LOX to other products, some of which are important as flavor compounds. These enzymes include hydroperoxide lyase, which catalyzes the formation of aldehydes and oxo acids hydroperoxide-dependent peroxygenase and epoxygenase, which catalyze the formation of epoxy and hydroxy fatty acids, and hydroperoxide isomerase, which catalyzes the formation of epoxyhydroxy fatty acids and trihydroxy fatty acids. LOX produces flavor volatiles similar to those produced during autoxidation, although the relative proportions of the products may vary widely, depending on the specificity of the enzyme and the reaction conditions. 

Roberge, C., Eleitz, E., Pollard, D. and Devine, P., Asymmetric synthesis of cyanohydrin derived from pyridine aldehyde with cross-linked aggregates of hydroxynitrile lyases. Tetrahedron Lett., 2007, 48, 1473-1477. 

The production of optically active cyanohydrins, with nitrile and alcohol functional groups that can each be readily derivatized, is an increasingly significant organic synthesis method. Hydroxynitrile lyase (HNL) enzymes have been shown to be very effective biocatalysts for the formation of these compounds from a variety of aldehyde and aliphatic ketone starting materials.Recent work has also expanded the application of HNLs to the asymmetric production of cyanohydrins from aromatic ketones. In particular, commercially available preparations of these enzymes have been utilized for high ee (5)-cyanohydrin synthesis from phenylacetones with a variety of different aromatic substitutions (Figure 8.1). 

Chapter 8 describes the application of hydroxyl nitrile lyases to the synthesis of new chiral cyanohydrins and a-hydroxy acids and includes new approaches to the transformation of difficult aldehyde and ketone substrates using substrate engineering and immobilization techniques. 

A less common reactive species is the Fe peroxo anion expected from two-electron reduction of O2 at a hemoprotein iron atom (Fig. 14, structure A). Protonation of this intermediate would yield the Fe —OOH precursor (Fig. 14, structure B) of the ferryl species. However, it is now clear that the Fe peroxo anion can directly react as a nucleophile with highly electrophilic substrates such as aldehydes. Addition of the peroxo anion to the aldehyde, followed by homolytic scission of the dioxygen bond, is now accepted as the mechanism for the carbon-carbon bond cleavage reactions catalyzed by several cytochrome P450 enzymes, including aromatase, lanosterol 14-demethylase, and sterol 17-lyase (133). A similar nucleophilic addition of the Fe peroxo anion to a carbon-nitrogen double bond has been invoked in the mechanism of the nitric oxide synthases (133). 

Miiller and co-workers have developed an enantioselective enzymatic crossbenzoin reaction (Table 2) [43, 44], This is the first example of an enantioselective cross-benzoin reaction and takes advantage of the donor-acceptor concept. This transformation is catalyzed by thiamin diphosphate (ThDP) 23 in the presence of benzaldehyde lyase (BAL) or benzoylformate decarboxylase (BFD). Under these enzymatic reaction conditions the donor aldehyde 24 is the one that forms the acyl anion equivalent and subsequently attacks the acceptor aldehyde 25 to provide a variety of a-hydroxyketones 26 in good yield and excellent enantiomeric excesses without contamination of the other cross-benzoin products 27. The authors chose 2-chlorobenzaldehyde 25 as the acceptor because of its inability to form a homodimer under enzymatic reaction conditions. 

Ammonia lyases catalyze the enantioselective addition of ammonia to an activated double bond. A one-pot, three-step protocol was developed for the enantioselective synthesis of L-arylalanines 50 using phenylalanine ammonia lyase (PAL) in the key step (Scheme 2.20). After formation of the unsaturated esters 48 in situ via a Wittig reaction from the corresponding aldehydes, addition of porcine Ever esterase and basification of the reaction mixture resulted in hydrolysis to the carboxylic acids 49. Once this reaction had gone to completion, introduction of PAL and further addition of ammonia generated the amino acids 50 in good yield and excellent optical purity [22]. 

Aldolases are part of a large group of enzymes called lyases and are present in all organisms. They usually catalyze the reversible stereo-specific aldol addition of a donor ketone to an acceptor aldehyde. Mechanistically, two classes of aldolases can be recognized [4] (i) type I aldolases form a Schiff-base intermediate between the donor substrate and a highly conserved lysine residue in the active site of the enzyme, and (ii) type II aldolases are dependent of a metal cation as cofactor, mainly Zn, which acts as a Lewis acid in the activation of the donor substrate (Scheme 4.1). 

Chiral hydroxynitriles are useful synthetic intermediates. They can be prepared using errzymatic synthesis in reactions between aldehydes or ketones and hydrogen cyanide (Griengl et al., 1997) (Figme 9.10). There are different kinds of ertzymes (hydroxynitrile lyases) catalysing the formation of the (R)- and the (S)-enantiomers, respectively. It is a problem that the reactants can react spontaneously as well. 

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