Page 379 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

16.4 Conclusions 355

a second in situ cross-aldol addition of HA to d-threose furnishing 1-deoxy-d-ido-
hept-2-ulose (60b) in 68% yield [5c]. Complementary to these studies on the donor
selectivity, engineering the active site to improve the acceptor tolerance is also of
paramount importance for biocatalyst optimization. We mentioned in the previous
section that the two mutants of FSA, namely A165G and the double A129S/A165G,
showed an improved acceptor tolerance for N-Cbz-aminoaldehydes. Furthermore,
FSA A129S/A165G mutant gave 5-O-benzyl-d-xylulose in >98% conversion compared
with the modest 35% obtained with FSA wild type (Table 16.1, entry 17). Sprenger
and coworkers [32f] engineered the aldehyde binding site of TalB F178Y to improve its
efficiency toward unphosphorylated substrates. After saturation of three positions
corresponding to the putative phosphate-binding site of the acceptor, that is, R181,
S226, and R228, a mutant TalB F178Y/R181E was found to show enhanced tolerance
to GO and d-or l-glyceraldehyde as acceptor substrates. TalB F178Y/R181E achieved
excellent conversions in the synthesis of d-xylulose, d-fructose, and l-sorbose
(Table 16.1, entries 22, 42, and 45) [32f].
Apart from nonionic aldehydes, FSA wild type and FSA A129S were also
found to be excellent catalysts for the addition of GO, HA, and DHA, to
d-glyceraldehyde-3-phosphate toward the preparation of sugar phosphates
(Table 16.1, entries 33–36). Using a multi-enzymatic cascade reaction to generate
the highly sensitive d-glyceraldehyde-3-phosphate in situ, the syntheses of
1-deoxy-d-fructose-6-phosphate, 1,2-dideoxy-d-arabino-hept-3-ulose 7-phosphate,
d-fructose-6-phosphate, and d-arabinose-5-phosphate were accomplished in good
to excellent yields and high purity [36c,d].
Overall, the exploitation of FSA-like aldolases represents a qualitative progress
in aldolase-catalyzed synthesis, as these enzymes accept a large structural variety
of aldehydes including simple aliphatic, haloacetaldehydes, and hydroxyaldehydes,
including d-threose. Additionally, further mutagenic work on the active site may
provide FSA-like aldolases with broader structural tolerance for both donor and
acceptor substrates, which would be of paramount utility in organic synthesis. A
limitation of the FSA-like aldolases is the nonexistence of a set of stereocomple-
mentary enzymes that have not been found in nature yet. Therefore, efforts to
evolve or structure-guided redesign these enzymes toward innovative stereodiverse
catalysts were collaborative projects within the COST CM0701 action and other EU
programs.

16.4
Conclusions
It has been widely demonstrated that aldolases are important biocatalysts for the
asymmetric carbon–carbon bond formation. This is because they possess the
unique characteristics by which they can build up new polyfunctional molecular
frameworks through the assembly of simple molecules. Particularly important in
this point is to design biocatalytic cascade carboligation reactions by a sequential
or one-pot combination of independent aldol additions catalyzed by different