Wild-type FucA

Figure 16.1 X-ray structure of the active site (Y113 ) denotes a residue from a neigh-of wild-type FucA in complexation with the boring FucA subunit. The model shows the transition state analog phosphoglycolohy- target residues FI 31, F206, and F113 which droxamate (PCH) bound to the active center were independently substituted by alanine.

The stereochemical outcome of the new FucA was indistinguishable compared to that obtained for the wild type. However, while the (J )-N-Cbz-aminoaldehydes yielded the anti(3i ,4i )-configured aldol adduct in high diastere-oselectivity (>2 98 syn iR,4-S)/anti(iR,4R) ratio), the (S) enantiomers depended on the aldehyde. In the extreme situation, R)-N-Chz prolinal derivatives ((J )-7a,b) gave exclusively the anti 3R,4-R) adduct whereas the S counterparts ((S)-7a,c,d) rendered the syn(3i ,4S) one. Protein molecular models were built to gain insight into the acceptor binding mode that led to this distinct stereochemical outcome [19]. 

R)-lOa) and rac-N-Cbz-2-(piperidin-4-yl)acetaldehyde ((rac)-lOb) as aldehyde acceptors (Scheme 16.4), which can be accessed from the commercially available alcohol precursors. fucA wild type as weU as FucA ", FucA , and mutants provided very low yields, and thus were not satisfactory from a preparative point of view. RhuA gave the best results using DHAP as donor (Scheme 16.4). The aldol addition of DHAP to (S)-N-Cbz-piperidin-2-carbaldehyde (S)-10a furnished the sy (3 R,4S)-configured aldol adduct, which is consistent with the results obtained with (S)-N-Cbz-proUnal derivatives [19]. On the other hand, its enantiomer (R)-10a furnished the (5R)-lla adduct as 2 3 syn(3R,4S)/anti(3R,4R) mixture. This was not observed in the case of the (R)-N-Cbz-proHnal, which exclusively provided the syn... 

FruA from rabbit muscle (RAMA) RhuA and FucA from coli. FucA FI31A substitution of the phenylalanine131 by alanine in FucA wild-type. Yield of unphosphorylated material after treatment with acid phosphatase. 

Besides protein engineering, it was uncovered that RhuA wild type can improve the aldol addition of DHA to aldehyde between 35- and 100-fold when the reactions were carried out in the presence of borate [113,128]. Moreover, retroaldol rates for some aldol adducts in the presence of borate were low or negligible as compared with the aldol ones, making the process irreversible [128, 129]. However, the tolerance to unphosphorylated DHA by of a Class II DHAP-dependent aldolase appears to be an exclusive property of RhuA, since the stereocomplementary FucA had no detectable activity with DHA either with or without borate added [113]. 

Example of multistep chemo-enzymatic synthesis of pipecolic acid derivatives (60) and homoiminocyclitols (61) by two consecutive enzymatic aldol addition reactions (a) i-serine hydroxy-methyltransferase from Streptococcus thermophilus (lSHMTj ) (b) Cbz-OSu CHjCN/aqueous HCI (c) Cbz-OSu MeOH/SO CIj CaCI, NaBH, EtOH/THF CHjCN/aqueous HCI (d) o-threonine aldolase from Achromobacterxylosoxidans (oThrA j (e) FucA F131A (f) RhuA wild-type (g) acid phosphatase from potato type II and (h) H Pd/C. 

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