Page 366 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

342 16 Aldolases as Catalyst for the Synthesis of Carbohydrates and Analogs

In these reactions, RhuA was the most versatile aldolase, accepting both lin-
ear and branched C-α-substituted N-Cbz-aminoaldehydes, while FucA tolerated
only C-α linear alkane substitutions. Aware of the importance of the reaction
medium, the reactions were assayed in both highly concentrated gel emul-
′
sions [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 sterically more demanding branched alkyl
substituents [16].
The stereochemical outcome of FucA catalysis was controlled by the aldolase,
furnishing always anti(3R,4R) configured aldol adducts (Scheme 16.2). RhuA was
highly stereoselective only for the (S)-N-Cbz-aminoaldehydes acceptors, rendering
the corresponding syn(3R,4S) adducts. For the (R)-configured substrates, different
syn(3R,4S): anti(3R,4R) diastereomeric aldol mixtures were obtained. Interestingly,
it was observed that the amount of anti(3R,4R) adduct increased with the length
of the C-α alkyl chain of the acceptor, being the major diastereomer when (R)-
N-Cbz-2-butylglycinal ((R)-1c) was the acceptor. RAMA did not tolerate any of
the N-Cbz-aminoaldehydes 1a–h in any of the reaction systems assayed. This
behavior was somehow predictable, considering the low conversion observed
in a previous work during RAMA-catalyzed aldolization of (S)- and (R)-N-Cbz-
alaninal [14b] and the fact that the basic amino acids K107 and R109, located
at the RAMA active site to fix its natural aldehyde acceptor d-glyceraldehyde-3-
phosphate, may hamper an effective interaction with hydrophobic C-α substituted
aldehydes.
The recent discovery and synthetic developments of d-fructose-6-phosphate
aldolase (FSA) from E. coli, which gave identical syn(3S,4R)-configured aldol
adducts to those obtainable with RAMA, eclipsed further investigations directed
toward improving the substrate tolerance of the DHAP-dependent aldolase. The
main reason for this decision was the fact that FSA accepts unphosphorylated
DHA donor and its analogs with unprecedented high activity. This was considered
a great advantage from the synthetic point of view, especially when the phosphate
group must be introduced in the substrate and removed from the final product.
This issue will be further discussed below.
As mentioned before, we found that FucA tolerated only C-α linear substi-
tuted (i.e., (S)-, (R)-1a–c) N-Cbz-aminoaldehydes, whereas branched substitutions
(i.e., (S)-, (R)-1d–g) were solely substrates for RhuA. To overcome this limita-
tion, a set of FucA mutants were envisaged to remove bulky amino acid side
chains in the active site to facilitate the accommodation of sterically demand-
ing acceptor aldehydes, including the conformationally restricted prolinal and
hydroxyprolinal derivatives. The corresponding mutants F131A, F206A, and F113A
and the double mutations F131A/F206A and Y113A/F131A were thus obtained.
In addition, Del (207–215), Del (211–215), and the combination F131A/Del
(207–215) were also constructed to eliminate totally or partially the FucA C-
terminal tail [18] which might block the acceptor binding and/or prevent its suitable
positioning (Figure 16.1) [19].