Rabbit muscle aldolase RAMA

The class I FruA isolated from rabbit muscle aldolase (RAMA) is the aldolase employed for preparative synthesis in the widest sense, owing to its commercial availability and useful specific activity of 20 U mg . Its operative stability in solution is limiting, but the more robust homologous enzyme from Staphylococcus carnosus has been cloned for overexpression [87], which offers unusual stability for synthetic purposes. Recently, it was shown that less polar substrates may be converted as highly concentrated water-in-oil emulsions [88]. 

Scheme 5.22. Chemoenzymatic synthesis of (+) exo-breviocomin by rabbit muscle aldolase (RAMA). Pase = phosphatase.

Schmid, W, Whitesides, G, A new approach to cyclitols based on rabbit-muscle aldolase (RAMA), J. Am. Chem. Soc., 112, 9670-9671, 1990. 

Effenberger and coworkers reported a de novo approach to 5-thio-D-f/zreo-2-pentulofuranose (5-thio-D-xylulose, 20) (Figure 9.7) from 2-mercaptoacetaldehyde, employing rabbit muscle aldolase (RAMA EC 4.1.2.13) and yeast transketolase (EC 2.2.1.1) as the catalysts [32]. 

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). 

Zhu W, Li ZY (2(X)0) Synthesis of perfluoroalkylated sugars catalyzed by rabbit muscle aldolase (RAMA) J Chem Soc Perkin Trans 17 1105-1108... 

To date, the best-studied aldolase has been fructose-1,6-diphosphate aldolase from rabbit muscle. Rabbit muscle aldolase (RAMA) catalyses the condensation of D-glyceraldehyde-3-phosphate (1) with dihydroxyacetone phosphate (2) to yield fructose-1,6-diphosphate (3) (Scheme 5.2). 

Fructose-1,6-Diphosphate Aldolase. Fructose-1,6-diphosphate (FDP) aldolase from rabbit muscle, also commonly known as rabbit muscle aldolase (RAMA), catalyzes the addition of dihydroxyacetone phosphate (DHAP) to o-glyceralde-hyde-3-phosphate to form fructose-1,6-diphosphate (Scheme 2.185) [514, 1382]. 

In our group, a structural variety of N-Cbz-aminoaldehydes were used as acceptor substrates of DHAP-dependent aldolases, namely D-fructose-l,6-phosphate aldolase from rabbit muscle aldolase (RAMA), L-rhamnulose-l-phosphate aldolase (RhuA), and t-fuculose-l-phosphate aldolase (FucA) from Escheridtia coli, for the preparation of structurally diverse pyrrolidine-type iminocyclitols (Scheme 16.2) [16]. 

Rabbit muscle aldolase (RAMA) furnished ketose l ho hates with 3(S)/4(i ) stereochemistry from dihydioxyacetone phosphate (DHAP) and simple aldehydes. When a-hydroxyaldehydes were used kinetic resolution occurred, provided there was a negative charge (e.g. CCX) ) 4 or 5 atoms away from the aldehyde group. Fructose 1,6-diphosphate aldolase from spinach leaves catalysed similar condensations between DHAP and aldehydes with a substrate specifity somewhat different from that of RAMA. A reinvestigation of the stmc course of the hexulose phosphate synthetase-catalysed condensation between ribulose S-phosphate and simple aldehydes is covered in Chapter 2. 

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). 

RAMA rabbit muscle aldolase (fructose-1,6-bis-phosphate aldolase)... 

Scheme 28. RAMA = Rabbit muscle aldolase TPI = Triose phosphate isomerase...

EH Epoxide hydrolase RAMA Rabbit muscle aldolase... 

DHAP-dependent aldolases have also been used as key step in the synthesis of several complex natural products starting from achiral precursors. Thus, the sex pheromone (+)-exo-brevicomin can be synthesized in a multi-step route starting with the stereospecific aldol addition between DHAP and 5-oxohexanal or its 5-dithiane-protected analog catalyzed by FBPA from rabbit muscle ( RAMA ) as the key step by which the absolute configuration of the target is estabUshed (Scheme 4.16) [40]. 

The original preparation of 6-C-perfluoroalkyl-D-fructose has been reported. The first step of this synthesis is the perfluoroalkylation of acrolein acetal. The key step of the synthesis is an aldol condensation between D-3-fluoroalkylglyceraldehyde and dihydroxyacetone phosphate, with RAMA as biocatalyst (RAMA is an aldolase found in rabbit muscles) (Figure 6.43). ... 

Reaction of ribose 5-phosphate 116 with dihydroxyacetone phosphate, catalyzed by fructose 1,6-diphosphate aldolase from rabbit muscle (RAMA) affords the ketose diphosphate 117. Dihydroxyacetone phosphate was formed in situ from fructose 1,6-diphosphate by action of RAMA and triose phosphate isome-rase (TPI). The diphosphate 117 was dephosphorylated enzymatically using acid phosphatase, and the ketose 118 was reduced directly into the a-C-manno-side 119 by treatment with bistrimethylsilyltrifluoroacetamide, trimethylsilyl-triflate and triethylsilane (Scheme 28) [45]. 

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. 

We faced the problem of the poor solubility of most N-protected amino aldehydes in water, which might account for the low reactivity observed with D-fructose-1,6-diphosphate aldolase from rabbit muscle (RAMA) (14, 15, 19-21). Increasing the percentage of organic co-solvent (e.g. dimethylformamide) in the medium to make the aldehyde soluble may lead to either a dramatic enzyme deactivation [22] or an insolubilization of the donor (e.g. dihydroxyacetone (DHA) and DHAP sodium salt). As a result, no reaction or insufficient product yields are often obtained. 

The aldol reaction is extensively used in Nature as in the laboratory to make C-C bonds and some aldolases have been used in asymmetric synthesis. One of the most popular has been the fructose-6-phosphate aldolase44 from rabbit muscle, familiarly known as RAMA. The enzymatic reaction combines the enol from dihydroxyacetone phosphate (DHAP) 142 with glyceraldehyde-3-phosphate 143 in a diastereo- and enantioselective aldol reaction. PO in these diagrams means phosphate. 

Water-in-oil (W/O) gel emulsions has been applied for the first time in a-chymotrypsin-catalyzed peptide synthesis (Clapds et al. 2001) and in aldolic condensation of DHAP with acceptor aldehydes such as phenylacetaldehyde and ben-zyloxyacetaldehyde, catalyzed by D-fructose-l,6-bisphosphate aldolase from rabbit muscle (RAMA). Gel emulsions of the ternary systems such as water/Ci4E4/oil, where C14E4 is a technical grade poly(oxyethylene) tetradecyl ether surfactant, with an average of four moles of ethylene oxide per surfactant molecule and oil can be... 

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