Rhamnulose aldolase

Four DHAP converting aldolases are known, these can synthesize different diastereomers with complementary configurations D-fructose (FruA EC 4.1.2.13) and D-tagatose 1,6-bisphos-phate (TagA, F.C 4.1.2.-), L-fuculose (FucA EC 4.1.2.17) and L-rhamnulose 1-phosphate aldolase (RhuA EC 4.1.2.19)3. The synthetic application of the first (class 1 or 2) and the latter two types (class 2) has been examined. 

A solution of 2.25 g (25 mmol) of D-glyccraldehyde in 300 mL of water is combined with a solution of 20 mmol of dihydroxyacetonc phosphate (DIIAP) in 200 mL of water freshly adjusted to pH 6.8. The mixture is incubated with 100 U of L-rhamnulose 1-phosphate aldolase at r.t. for 24 h with monitoring of conversion by TLC (2-propanol/sat. ammonia/water 6 4 2) and by enzymatic assay for DHAP55. 

In the case of L-rhamnulose-1-phosphate aldolase (RhaD), we found that the problem of phosphorylated substrate requirement (dihydroxyactone phosphate (DHAP)) could be overcome by a simple change in buffer. Thus, when using borate buffer, reversible borate ester formation created a viable substrate out of dihydroxyacetone, which is not otherwise accepted by the wild-type enzyme (Figure 6.6) [23]. The process was used in a one-step synthesis of... 

Franke, D., Machajewski, T., Hsu, C.-C. and Wong, C.-H. (2003) One-pot synthesis of L-fructose using coupled multienzyme systems based on rhamnulose-1-phosphate aldolase. The Journal of Organic Chemistry, 68 (17), 6828-6831. 

One-step Synthesis of L-Fructose Using Rhamnulose-l-phosphate Aldolase in Borate Buffer... 

The synthehc applicability of arsenates is restricted by their toxicity that avoids the green aspect of the enzymatic processes. Wong et al. have shown that the use of inorganic borate buffer allows L-rhamnulose-l-phosphate aldolase (Rha-IPA) to accept DHA as substrate, although the of the reaction is about 50 times lower than with the natural substrate [10]. In spite of this fact, these authors have successfully used this approach for the one-step synthesis of L-fructose and L-rhamnulose, and for the facile two-step synthesis of several L-iminocyclitols. 

D-fructose 1,6-bisphosphate 2 (FruA E.C. 4.1.2.13), D-tagatose 1,6-bisphosphate 4 (TagA E.C. 4.1.2.40), L-fuculose 1-phosphate 5 (FucA, E.C. 4.1.2.17), and L-rhamnulose 1-phosphate 4 (RhuA, E.C. 4.1.2.19). From previous studies, we have DHAP aldolases with all four possible specificities readily available, we have demonstrated their broad substrate tolerance for variously substituted aldehydes, and we have investigated their stereoselectivity profile with non-natural substrates [3-6]. 

Besides FDPA, three other aldolases using DHAP as the donor are known each aldolase generates a new C3-C4 bond with a different stereochemistry u-erythro for fuculose-l-phosphate aldolase, i.-threo for rhamnulose 1-phosphate aldolase, and D-erythro for tagatose 1,6-diphosphate aldolase [7]. These aldolases accept a great variety of electrophilic substrates, which has been widely exploited in synthesis of sugar analogues [8,9]. 

W. D. Fessner, G. Sinerius, A. Schneider, M. Dreyer, G. E. Schulz, J. Badia, and J. Arguilar, Diastereoselective enzymatic aldol additions L-Rhamnulose and L-fuculose 1-phosphate aldolases from E. coli, Angew. Chem, lnt. Ed. Engl. 30 555 (1991). 

While the lyases that transfer a pyruvate unit form only a single stereogenic center, the group of dihydroxyacetone-phosphate-(DHAP, 41)-dependent aldolases create two new asymmetric centers, one each at the termini of the new C-C bond. A particular advantage for synthetic endeavors is the fact that Nature has evolved a full set of four stereochemically-complementary aldolases [189] (Scheme 6) for the retro-aldol cleavage of diastereoisomeric ketose 1-phosphates— D-fructose 1,6-bisphosphate (42 FruA), D-tagatose 1,6-bisphosphate (43 TagA), L-fuculose 1-phosphate (44 FucA), and L-rhamnulose 1-phosphate (45) aldolase (RhuA). In the direction of synthesis this formally allows the... 

Fig. 8. Stability of the rhamnulose 1-phosphate aldolase from Escherichia coli (RhuA) vs. that of the fructose 1,6-bisphosphate aldolase from rabbit muscle (FruA) in phosphate buffer (pH 7.2 25°C ca. 1 Uml ) a) RhuA b) O RhuA, 30% EtOH c) RhuA, 50% DMSO d) FruA...

The L-rhamnulose 1-phosphate aldolase (RhuA EC 4.1.2.19) is found in the microbial degradation of L-rhamnose which, after conversion into the corresponding ketose 1-phosphate 44, is cleaved into 41 and L-lactaldehyde (l-16). The RhuA has been isolated from E. coli [336-339], and characterized as a metallo-protein [194,340,341]. Cloning was reported for the E. coli [342,343] and Salmonella typhimurium [344] genes, and construction of an efficient overexpression system [195,220] has set the stage for crystallization of the homotetrameric E. coli protein for the purposes of an X-ray structure analysis [345]. 

Table 5. Substrate tolerance of L-rhamnulose 1-phosphate and L-fuculose 1-phosphate aldolases [195,347]...

Enzymology,29 techniques of isolation, and descriptions of a number of them. Apparently, only three have been considered for preparative chemistry, that is, aldolase, sialyl aldolase, and Kdo synthetase. However, whole cells of some strains of Escherichia coli have been used as sources of fucu-lose 1-phosphate aldolase (E.C. 4.1.2.17) or rhamnulose 1-phosphate aldolase (E.C. 4.1.2.19).30 Extraction, and concentration to a suitable degree of homogeneity, of noncommercially available aldolases are not difficult. The examination of their synthetic possibilities could be very rewarding for we already observe that the wealth of chemicals prepared with the help of aldolase and sialyl aldolase far exceeds what they make in Nature. Still, not any aldehyde, however hydrophilic, is a substrate for aldolases. 

For the described approach, it is important to note that aldolases of different origin were tested and that, in contrast to L-rhamnulose-1-phosphate aldolase (RhuA), the D-fructose-1,6-biphosphate aldolase from rabbit muscle and L-fucu-lose-1-phosphate aldolase from E. coli were not active for DHAP/(R)-N- and (S)-iV-Cbz-prolinal condensation. Since RhuA accepts both, (S)-N- and (R)-N-Cbz prolinals, the chemo-enzymatic synthesis of both, hyacinthacines A and A2 isomers could be achieved. In conclusion, the origin and the particular enzyme itself... 

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. 

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. 

An efficient asymmetric total synthesis of L-fructose combines the Sharpless asymmetric dihydroxylation with an enzyme-catalyzed aldol reaction. L-Glyceraldehyde, prepared from acrolein, is condensed to DHAP in a buffered water suspension of lysed cells of KI2 Escherichia coli containing an excess of L-rhamnulose-1-phosphate (Rha) aldolase E. coli raised on L-rhamnose as sole carbon source). The L-fructose phosphate obtained is hydrolyzed to L-fructose with acid phosphatase. Similarly, the RAMA-catalyzed condensation of D-glyceraldehyde with DHAP,... 

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