RhuA reactions

Scheme 2.2.5-9 Stereochemical bias in RhuA reactions with...

This procedure has also been used to prepare a series of hydrolytically stable dipyranoid disaccharide mimetics from homologous dihydroxy-a,oo-dialdehydes37 (Scheme 5.16). While the FruA and FucA reactions are selective for the (W)-aldchydc, the RhuA reactions are selective for the (S)-aldehydes, all giving thermodynamically more stable product with retention of the natural stereochemistry for the two newly formed carbon centers. 

Deprotonation of 1 at C3 yields an ene-diolate intermediate to which phospho-glycolohydroxamate (PGH) 9 bears a close structural resemblance (Scheme 2.2.5.3). In collaboration with J. V. Schloss, 9 was found to be a potent inhibitor not only of FucA, but indeed also of all currently accessible Class 11 aldolases with Ki in the nanomolar range [12]. Obviously, the hydroxamate mimics very effectively an advanced catalytic intermediate or transition state that is shared by these enzymes, and seems to be bound by all Zn -dependent aldolases in a very similar fashion. Interestingly, when the active-site Zn is replaced by Co ions, catalytic activity is restored and actually becomes higher than with native zinc cofactor. However, the RhuA Co complex catalyzes an oxygenase reaction that consumes... 

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-... 

An interesting problem in stereoisomerism is found in the aldol reactions of the achiral aldehydes which are obtained by ozonolysis of the homoallylic alcohols 174. After stereospecific conversion by the FruA [230], the products can be readily induced to form an intramolecular glycoside 175 by acidic (R=OH) or alkaline treatment (R=C1), under which conditions the two equatorial ring hydroxyl groups completely direct the stereogenic acetal formation [234]. The corresponding RhuA catalyzed reactions deliver the enantiomeric... 

Figure 19.3 DHAP-aldolase catalyzed aldol addition of DHAP to N-Cbz-aminoaldehydes. Reaction conversion to aldol adduct in gel emulsion (black bars) and dimethylformamide/ water 1 4 system (gray bars) for reactions catalyzed by (a) RAMA, (b) RhuA and (c) FucA.

Docking simulations carried out with the aldol adducts bound into the active center of RAMA and RhuA suggested that in all cases the bulky N-protecting group cannot get into the catalytic site of the protein. That would explain the small effect of the N-protecting groups with different sizes and shapes on the stereochemical outcome of the reactions, since those groups would remain far from the reactive atoms. 

Scheme 7. Formation of a series of hydrolytically stable dipyranoid disaccharide mimetics from homologous dihydroxy- ,ta-dienes by tandem enzymatic aldolizations. Abbreviations for reaction conditions FruA /RhuA /FucA = 1.) O3, then (CHjljS 2.) aldolase, DHAP 3.) phosphatase. This notation is used throughout subsequent diagrams (except when indicated otherwise)...

Although many aldolases have been characterized for research purposes, these enzymes have not been developed commercially to any significant extent. This is likely due to the availability of the various biocatalysts and the need for dihydroxyacetone phosphate (DHAP) (44), the expensive donor substrate required in nearly all aldolase reactions. A number of chemical and enzymatic routes have been described for DHAP synthesis, which could alleviate these concerns [12], In terms of the enzyme supply issue, this may change with the introduction of products from Boehringer Mannheim and their Chirazyme Aldol reaction kit. They have three kits, each containing a different aldolase fructose-1,6-diphosphate FruA) (EC 4.1.2.13), L-rhamnulose-1-phosphate RhuA (EC 4.1.2.19), and L-fuculose-1-phosphate (FucA) (EC 4.1.2.17). As more screening... 

In these reactions, RhuA was the most versatile aldolase, accepting both linear and branched C-a-substituted N-Cbz-aminoaldehydes, while FucA tolerated only C-a hnear alkane substitutions. Aware of the importance of the reaction medium, the reactions were assayed in both highly concentrated gel emulsions [17] and 1 4 N,N -dimethylformamide (DMF)/water mixtures. In this case, the 1 4 DMF/water mixtures was the reaction medium of choice providing the best conversions especially for stericaUy more demanding branched alkyl substituents [16]. 

Scheme 16.4 RhuA wild type-catalyzed aldol addition reactions of DHAP to N-Cbz piperidine carbaldehyde derivatives for the synthesis of indolizidine and quinolizidine iminocycli-tols. (a) RhuA wild-type catalyst (b) dephosphorylation by acid phosphatase and (c) Pd/C.

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]. 

In addition to protein engineering, the substrate mimicking approach was also appHed for RhuA catalyst It was uncovered that RhuA can perform the aldol addition of DHA to aldehyde at remarkably high rates when the reactions were carried out in the presence of borate [29]. Indeed, when sodium borate was added, the rates of aldol formation improved between 35- and 100-fold [25]. Besides the intrinsic tolerance of RhuA for DHA, the measured retroaldol rates for some aldol adducts in the presence of borate were low or negligible as compared with the synthetic ones, making the process virtually irreversible [29, 30]. Therefore, it was further suggested that the aldol adduct may be trapped by the formation of borate complexes which would be less active substrates for the aldolase [29, 30]. 

Scheme 16.5 RhuA wild type- and RhuA mutant-catalyzed aldol additions reactions of DHA to (S)- and (R)-N-Cbz-alaninal (14a,b), N-Cbz- and N-formyl-glycinal (14c,d). (a) RhuA wild type and RhuA ° mutant and (b) Pd/C.

Conversely, FucA will produce diastereomers at low de, because of the more balanced stability relationships for pyranoid products sharing a as-diol substitution pattern. With racemic 2-hydroxylated aldehydes, thermodynamic control in FruA- or RhuA-catalyzed reactions favors (2R) configured enantiomers but also with lower discrimination (de up to 55%), because of the higher fiexibility of the corresponding ketofuranose rings [25, 206]. 

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. 

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