Page 376 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

352 16 Aldolases as Catalyst for the Synthesis of Carbohydrates and Analogs

diverse carbohydrate, deoxysugars, and sugar phosphate analogs, as well as poly-
hydroxylated molecules with exquisite stereoselectivity (Table 16.1) [5c, 32d–f,h,
36]. Not all the possible combinations are yet documented in the literature but the
examples presented should give a clear idea of the enormous potential of these two
aldolase types.
Recent reports have focused on the different donor substrate preferences of FSA
wild type and TalB F178Y and their mutants in connection with their X-ray structure.
As mentioned before, it has been observed that the acceptor substrate tolerance is
dependent on the donor substrate. Therefore, a good donor quality may, in some
instances, favor good conversion of weak acceptor substrates. For FSA wild type,
the donor substrate quality followed the order HA > HB, GO > DHA [5c], whereas
for TalB F178Y the donor quality follows the order DHA > HA > GO, HB being a
strong inactivating substrate (Table 16.1, entries 5–7, 16, 17, 24–27, and 37–39)
[32f,h]. This is a consequence of the residues surrounding the binding site of the
variable moiety of the donor substrates: a highly hydrophobic environment for
FSA (L107, A129), whereas polar residues are found in the equivalent positions of
TalB F178Y (N154, S176) [6i, 32h]. This facilitates the preference of FSA toward the
more hydrophobic substrates HA and HB and diminishes the efficacy for DHA,
whereas TalB F178Y has a strong preference for DHA. As mentioned before, FSA A129S
exhibited improved tolerance toward DHA with a donor preference that followed
the order DHA > HA > GO, which was similar to that observed for TalB F178Y [32e].
Interestingly, the aldol addition of DHA to GO catalyzed by FSA A129S proceeded
in 80% aldehyde conversion to d-xylulose (i.e., 42% isolated yield, Table 16.1 entry
22), which is comparable to that found with TalB F178Y catalysis [32e,h]. On the other
hand, while using FSA wild type under the same conditions, d-threose was the
major product, which arose from the self-aldol addition reaction of GO, whereas the
cross-aldol addition, d-xylulose, was not detected [5c]. FSA wild type accomplishes
the cross-aldol additions of HA to GO furnishing 1-deoxy-d-xylulose [32d], which
is in agreement with its donor preference [5c].
In the complementary direction, the poor tolerance of TalB F178Y toward HA
could be improved by the mutation S176A (i.e., the double mutant TalB F178Y/A176S ),
the equivalent A129 in FSA, which generates a more hydrophobic environment
and facilitates the accommodation of the ethyl moiety of HA [32h]. Indeed, in a
competition aldol addition reaction of equal concentrations of both DHA and HA to
3-hydroxypropionaldehyde, TalB F178Y/S176A showed practically identical conversion
of the HA aldol adduct to the DHA-derived one [32h]. However, no aldol adduct
was detected using HB as donor substrate and, although the variant appeared to be
more stable, enzyme inactivation still occurred at concentrations >50 mM [32h].
As pointed out before, the donor quality influences the acceptor tolerance. For
instance, FSA A129S mutant was found to furnish 5-O-benzyl-d-xylulose in 60%
conversion while FSA wild type gave only a 35% under optimized conditions
(Table 16.1, entry 17, other examples in entries 3, 4, 22, 38, 42, and 45). d-Threose
(Table 16.1, entry 47) was an excellent acceptor when HA was the donor, whereas no
product was detected either with DHA and GO. In this case, a nice cascade reaction
was accomplished consisting of, first, the homoaldol addition of GO, followed by