Rhamnulose-1-phosphate 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. 

Synthesis of L-Fructose Using Rhamnulose-1-phosphate Aldolase 205... 

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]. 

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]. 

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. 

The four enzymes of the family of dihydroxyacetone phosphate (DHAP)-dependent aldolases fructose-1,6-bisphosphate aldolase (FruA, EC 4.1.2.13), fuculose-1-phosphate aldolase (FucA, EC 4.1.2.17), rhamnulose-1-phosphate aldolase (RhuA, EC 4.1.2.19) and tagatose-1,6-bisphosphate aldolase (TagA, EC 4.1.2.40), catalyze in vivo the reversible asymmetric addition of DHAP to d-glyceraldehyde-3-phosphate (G3P) or L-lactaldehyde, leading to four complementary diastereomers. DHAP-dependent aldolases create two new stereogenic centers, with excellent enantio and diastereoselectivity in many cases. These enzymes are quite specific for the donor substrate DHAP, but accept a wide range of aldehydes as acceptor substrates. There are only two fructose-6-phosphate aldolase isoenzymes reported to be able to use dihydroxyacetone (DHA) as donor substrate (Schiirmann and Sprenger 2001). 

These systems were tested in the enzymatic aldolization of a variety of A/-Cbz-aminoaldehydes catalyzed by D-fructose-l,6-bisphosphate aldolase from rabbit muscle (RAMA) and L-rhamnulose-1-phosphate aldolase and L-fuculose-1-phosphate aldolase from E. coli (Espelt et al. 2003 a,b, 2005). The largest differences between conventional DMF/water cosolvent systems and gel emulsions were observed with RAMA catalyst (Fig. 6.5.11). 

Vidal L, Ferrer P, Alvaro G et al. (2005a) Influence of induction and operation mode on recombinant rhamnulose-1-phosphate aldolase production by Escherichia coli using the T5 promoter. 1 Biotechnol 118 75-87... 

A combination of glycerol phosphate oxidase (GPO), catalase, and L-rhamnulose-1-phosphate aldolase (RhuA) allowed for an efficient conversion of aminoglycerol into 1-deoxy-l-phosphoramido-L-fructose (Scheme 24). 

Scheme 2.191 Aldol reactions catalyzed by fuculose- and rhamnulose-1-phosphate aldolase...

The reaction of dihydroxyacetone phosphate (DHAP) with racemic or (R)-3-azido-2-hydroxypropanol under the influence of rhamnulose-1-phosphate aldolase or fuculose-1-phosphate aldolase afforded azidoketose intermediates which were converted by way of palladium-mediated reductive amination into a number of novel compounds including 1,6-dideoxy-D-galactojirimycin (24), 1,6-dideoxy-L-altojirimycin (25), 1-deoxy-D-talojirimycin (26), 1-deoxy-L-mannojirimycin (27) and 1-deoxy-L-rhamnojirimycin (28). ... 

For class II aldolases. X-ray structures of the zinc-dependent fuculose 1-phosphate aldolase (FucA, vide infra) [47, 48] and the rhamnulose 1-phosphate aldolase (RhuA, vide infra) [49] from E. coli have recently been solved and confirm close similarity in their overall fold. Both enzymes are homotetramers in which subunits are arranged in C4-symmetry. The active site is assembled in deep clefts at the interface between adjacent subunits and the catalytic zinc ion is tightly coordinated by three His residues. 

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