RhuA Stereoselectivity

P-anomeric compounds led cleanly to the expected 6,7-trans adduct 22, a-galac-tosides were practically completely converted to the corresponding 6,7-cis product 24. This finding is particularly surprising, because the locus of structural change is relatively distant from the reaction center. As no conclusive pattern for substrate control of stereoselectivity was evident, a more systematic approach was desirable. 

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Scheme 2.2.5.10 RhuA stereoselectivity screening using structurally defined aldehydes based on carbohydrate scaffolds.

Toward a screening program for RhuA stereoselectivity, structurally more simplified dioxane derivatives 25-27 comprising enantiomeric and diastereomeric 3-hydroxyaldehyde geometries in a conformationally defined environment could be prepared easily from carbohydrate precursors (Scheme 2.2.5.10). First results from product analysis provide further evidence for occasionally biased fixation of... 

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]. 

Although generic 3-hydroxyaldehydes are usually converted by the aldolases ste-reospecifically [1, 4], an interesting, virtually complete reversal of stereoselectivity had been observed upon RhuA-catalyzed reaction of the confermationally restricted galactodialdose derivatives 21 and 23 (Scheme 2.2.5.9) [30]. While several... 

The scheme of thermodynamic equilibration, particularly for 3-hydroxyaI-dehydes 75, as described above for FruA catalysis (Scheme 16) can likewise be applied to reactions with RhuA, the enantiomeric enzyme [189,220]. As is to be expected from the complementarity of the stereoselectivities of these enzymes, the corresponding enantiomeric compounds ent-78 are enriched upon equilibration. Owing to the lower diastereoselectivity of the RhuA versus the FruA enzyme, however, the corresponding FucA configurated isomer 85 accumulates more rapidly at the expense of the thermodynamically less favored diastereomer ent-19 (cf. Scheme 18, Sect. 5.2). 

Like a number of other aldolases the FucA enzyme is now also offered commercially. Overall practical features make the FucA quite similar to the RhuA enzyme, as is illustrated by its high stability in the presence of Zn2 + ions, by its broad substrate tolerance for variously substituted aldehydes at useful reaction rates (Table 5), and by a high asymmetric induction for (3R,4R)-cis stereoselectivity by si-face addition to the aldehyde carbonyl [195,355]. Al-... 

R=OH). With less polar chloride substituents, however, the RhuA dia-stereoselectivity is reduced and a considerable fraction of the FucA configurated product (40%) is also formed. Interestingly, alkaline cyclization in the latter occurs with an inverse preference to furnish 177, which contains the enantiomeric bicyclic [3.2.1] structure shared by the FruA product 175 as well as by (S)-( — )-frontalin 178, the aggregation pheromone of the southern pine beetle Dendroctonus frontalis. 

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]. 

FruA) from different sources, L-rhamnulose-l-phosphate aldolase (RhuA) and l-fuculose-l-phosphate aldolase (FucA) from E. coli have been extensively investigated and applied in the synthesis of carbohydrates and their derivatives such as deoxysug-ars, iminocyclitols and aminocyclitols [44]. An exception was D-tagatose-l,6-bisphos-phate aldolase (TagA), whose lack of stereoselection constitutes a major limitation of this aldolase, which frustrates the attempts to obtain the four possible stereoisomers from a single aldehyde, one of the hallmarks of enzymatic aldol additions [117,118]. 

Please note that in the original article [136] the incorrect (lS)-cQnfiguration was assigned to the polyhydroxy-lated pyrroUzidines. Based on this assignment, the aldol reaction was thought to proceed with low diastere-oselectivity. The correct stereochemical assignment was unequivocally established in a later study [112]. Therefore, RhuA was fully stereoselective in the aldol addition of DHAP to N-Cbz-prolinal derivatives. 

The synthesis of iminocyclitols was also accomplished using aldol additions of the unphosphorylated analogs of DHAP, dihydroxyacetone (DHA). As mentioned before, RhuA ° and RhuA wild type/borate buffers proved their utility with examples reported in the literature [113, 128, 129]. High conversions, for example 90-99%, were accomplished with RhuA/borate buffer, which are comparable to those achieved under different optimized conditions using DHAP donors. Importantly, the full equivalence of the stereochemical outcome with the additions of DHAP indicated the unbiased orientation of DHA in the active site of RhuA catalyst. Remarkably, the additions of DHA-borate to (R)-N-Cbz-aminoaldehydes furnished exclusively the anti (3R,4R) configured adducts, whereas the (S)-N-Cbz-aminoaldehydes always yielded the syn (3R,4S) adducts. This high stereoselectivity toward the R enantiomers of N-Cbz-aminoaldehydes at 25 °C contrasted with the different syn/anti mixtures of aldol adduct obtained using DHAP [24]. 

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