DHAP nucleophile

In principle, such processes should also be applicable to bifunctional aldehydes for a two-directional chain elongation in which two equivalents of DHAP nucleophiles would be added sequentially to both the acceptor carbonyls in a fashion that can be classified as a tandem reaction [66,67], without the need for isolation of any intermediates. Depending on the specificity of the enzyme used and on the number and position of hydroxyl functions in the starting material, the isomeric constitution, as well as the absolute and relative stereochemistry should be deliberately addressable. Thus, in a preparatively simple manner, such tandem aldolizations [68] should permit to rapidly construct larger carbohydrate molecules that would rival the carbohydrate core of tunica-mine and related nucleoside antibiotics in structural complexity. 

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. 

Functionally related to FruA is the novel class I fructose 6-phosphate aldolase (FSA) from E. coli, which catalyzes the reversible cleavage of fructose 6-phosphate (30) to give dihydroxyacetone (31) and d-(18) [90]. It is the only known enzyme that does not require the expensive phosphorylated nucleophile DHAP for synthetic purpose. 

Being restricted to DHAP as the nucleophile, aldol additions will only generate ketoses and derivatives from which aldose isomers may be obtained by biocatalytic ketol isomerization (cf. Sect. 7.1) [306]. For a more direct entry to aldoses the inversion strategy may be followed (Scheme 19) [290] which utilizes monoprotected dialdehydes. After aldolization and stereoselective chemical or enzymatic ketone reduction, the remaining masked aldehyde function is deprotected to provide the free aldose. Further examples of the directed, stereodivergent synthesis of sugars and related compounds such as aza- or thiosugars are collected in Sect. 7. 

There is only a single literature example of a bi-directional skeleton elongation performed with an enzyme different from the DHAP dependent aldolases. The 2-deoxy-D-ribose 5-phosphate aldolase (RibA EC 4.1.2.4), a bacterial class I enzyme which requires acetaldehyde as the nucleophilic substrate [42], has been applied to the tandem aldolization of a thioether dialdehyde [95]. The substrate 14 was synthesized from optically homogenous (R)-glycidaldehyde and, because of its Q Synimetry, diastereoselective twofold addition of acetaldehyde led to a symmetrical 5,5 -sulfide-linked dipentofuranose 15. 

Under carefully chosen reaction conditions by using a combination of RibA and another aldolase of different specificity, the intermediate 52 from the first RibA reaction can formally be intercepted by the second aldolase through the addition of a different nucleophile. In the presence of DHAP and FruA indeed predominant formation of the dideoxyketose 55 (17% yield) could be induced [116]. Due to the reversible nature of a thermodynamically unfavorable product, and because of the competition of both enzymes for the same substrate and the long reaction times required, a number of different side products and several stereoisomers have to be accounted for. 

A wide range of natural and unnatural monosaccharides has been generated by exploiting the catalytic capacity of aldolases which perform reactions equivalent to nonenzymatic aldol additions [54]. More than 20 aldolases have been identified so far and can be divided into three main groups, accepting either dihydroxyace-tone phosphate (DHAP), acetaldehyde, or pyruvic acid, and phosphoenolpyruvate as nucleophilic methylene component. A common feature is their high stereocontrol in the formation of the new C-C bond. As presented in Scheme 10 all four possible vicinal diols are accessible by selection of the appropriate DHAP-aldo-lase [2, 55], all of which show a distinct preference for the two stereocenters and a broad substrate tolerance for the aldehyde component. 

The requirement for the electrophilic component (DHAP) is much more stringent than for the nucleophilic component so far investigations have demonstrated that only 1,3-dihydroxy-2-butanone 3-phosphate and l,4-dihydroxy-3-butanone 1-phosphonate are substrates (Table 2). ... 

Fig. 4. Ether phospholipid synthesis from dihydroxyacetone-phosphate. (A) Dihydroxyacetone-P acyl transferase (DHAPAT). The first step of ether phospholipid synthesis is catalyzed by peroxisomal DHAPAT. This enzyme is a required component of complex ether lipid biosynthesis and its role cannot be assumed by a cytosolic enzyme that also forms acyldihydroxyacetone-P. (B) Ether bond formation by alkyl-DHAP synthase. The reaction that forms the 0-alkyl bond is catalyzed by alkyl-DHAP synthase and is thought to proceed via a ping-pong mechanism. Upon binding of acyl-DHAP to the enzyme alkyl-DHAP synthase, the pro-f hydrogen at carbon atom 1 is exchanged by enolization of the ketone, followed by release of the acyl moiety to form an activated enzyme-DHAP complex. The carbon atom at the 1-position of DHAP in the enzyme complex is thought to carry a positive charge that may be stabilized by an essential sulfhydryl group of the enzyme thus, the incoming alkox-ide ion reacts with carbon atom 1 to form the ether bond of alkyl-DHAP. It has been proposed that a nucleophilic cofactor at the active site covalently binds the DHAP portion of the substrate.

Acylation, nucleophiles by 1-acyl-DHAP chloride, 242f Alcohols... 

Synthesis of DHAP. Both RAMA and Fuc-l-P aldolase require DHAP as the nucleophilic component. Although DHAP is commercially available, it is too expensive for synthetic use, and must be synthesized for preparative-scale enzymatic reactions. DHAP has been prepared via three major routes enzymatically from FDP using a combination of RAMA and triose isomerase (TIM, E.C. 5.3.1.1) (25), chemically, by phosphorylation of dihydroxyacetone dimer (26), and enzymatically by phosphorylation of dihydroxy acetone catalyzed by glycerokinase (Scheme 8) (25). 

Since many of the aldolases use DHAP as the nucleophilic component of the reaction, attention has been given to devising efficient methods for its preparation starting from dihydroxyacetone. The synthesis of DHAP can be achieved in high overall yield according to the procedure shown... 

One of the drawbacks of DHAP aldolases is their strict specificity toward the donor substrate DHAP. DHAP is chemically unstable, particularly under alkaline conditions, and decomposes into inorganic phosphate and methyl glyoxal, both of which may inhibit the aldolase [4cj. Although the preparation [22] and synthetic applications of DHAP have reached a high degree of sophistication and efficiency [4h, 6e,i, 23], the preferred choice is by far the inexpensive unphosphorylated DHA nucleophile, which reduces costs and improves the atom economy of the process, especially when the phosphate group of the product must be removed in a separate reaction. In this connection, we focused our efforts on RhuA and FSA from E. coli [24]. 

For class I type enzymes, the (/ia)8-barrel structure of the class I fructose 1,6-bisphosphate aldolase (FruA, vide infra) from rabbit muscle was the first to be uncovered by X-ray crystal-structure analysis [33] this was followed by those from several other species [34-37]. A complex of the aldolase with non-covalently bound substrate DHAP (dihydroxyacetone phosphate) in the active site indicates a trajectory for the substrate traveling towards the nucleophilic Lys229 N [38, 39]. There, the proximity of side-chains Lysl46 and Glul87 is consistent with their participation as proton donors and acceptors in Schiff base formation (A, B) this was further supported by site-directed mutagenesis studies [40]. 

The DHAP aldolases are quite specific for the phosphorylated nucleophile 41, tvhich must therefore be prepared independently or generated in situ (cf. Section 5.4.4). Initial aldol products tvill thus contain a phosphate ester moiety, vhich facilitates product isolation, for example by barium salt precipitation or by use of ion-exchange techniques. The corresponding phosphate free compounds can be easily obtained by mild enzymatic hydrolysis using an inexpensive alkaline phosphatase at pH 8-9 [148], vhereas base-labile compounds might require vorking at pH 5-6 using a more expensive acid phosphatase [149]. 

Aldol addition of DHAP to aldehydes is catalyzed by DHAP-dependent aldolases. Two stereogenic centers are formed and therefore four possible stereoisomers can be obtained. Although nature has evolved a set of four distinct stereocomplementary types (Scheme 10.3), so far, only three of the known DHAP-dependent aldolases, namely the D-fructose-l,6-bisphosphate aldolase (FruA), L-rhamnulose-1-phosphate aldolase (RhuA), and L-fuculose-1-phosphate aldolase (FucA), have found broad synthetic applicability due to their high stereoselectivity and broad acceptor tolerance [5,77]. DHAP-dependent aldolases are highly selective for the nucleophilic substrate DHAP, tolerating only few isosteric modifications [84-88]. 

The aminocyclitol synthesis mediated by DHAP-dependent aldolases consists of a double aldol reaction, the first one enzymatically controlled by aldolases and the second one a spontaneous intramolecular nitro-aldol reaction (i.e., the Henry reaction) (Scheme 10.18). The latter makes use of the electrophilic carbonyl unit introduced by the first aldol addition on the nucleophilic character of carbon bonded to a nitro group installed from the acceptor (e.g., 62). This twofold C—C bond-forming reaction cascade was shown to deliver, after nitro group reduction, aminocyclitol analogs of valiolamine (63), of interest as inhibitors of intestinal glycosidases [141]. 

While pyruvate aldolases form only a single stereogenic center, the aldolases specific for dihydroxyacetone phosphate (DHAP, 22) as a nucleophile create two new asymmetric centers at the termini of the new C—C bond. Particularly useful for synthetic applications is the fact that nature has evolved a full set of four stereochemically unique aldolases [27] for the retroaldol cleavage of ketose 1-phosphates 23-26 (Fig. 12). In the direction of synthesis this formally allows the deliberate preparation of any one of the possible four diastereomeric aldol adducts in a building block fashion [15,22,27] by simply choosing the complementary enzyme and starting materials for full control over constitution and absolute configuration of the desired product. 

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