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346 16 Aldolases as Catalyst for the Synthesis of Carbohydrates and Analogs

N29
DHA/PGH

S75

N32
S116
T115
Figure 16.2 Crystal structure of the RhuA to mutation. A bound PGH and a DHA
wild type active center (PDB code combin- molecule coordinated to the essential Zn(II)
ing 1OJR and 1GT7) [26, 28] showing the are also shown. (Source: Kroemer, M. et al.
five residues on the phosphate binding site 2003 [26], Fig. 2, p. 3. Reproduced with per-
(N29, N32, S75, T115, and S116) subjected mission of the American Chemical Society.)

Among them, RhuA N29D was the most active mutant for the retroaldol reaction of
the natural substrate l-rhamnulose-1-phosphate, although with a residual activity
of just 5.3% of that of the activity of the wild type. This was expected because the
introduction of an anionic charge with an aspartate residue should decrease the
affinity for the phosphate anion. The five RhuA mutants were tested as catalyst
for the aldol addition of DHA to selected model N-protected-aminoaldehydes
(Scheme 16.5). Using (S)-N-Cbz-alaninal (14a, Scheme 16.5) as acceptor substrate,
RhuA N29D resulted in a ∼2.3–3.2-fold increase in speed in the aldol addition
reaction of DHA as compared with the wild-type and S75D or S116D mutants
[25]. The percentage of aldol adduct formation was always higher with RhuA N29D
than with RhuA wild type, the stereochemical outcome being similar for both
biocatalysts (Scheme 16.5). On the other hand, no aldol adduct was detected with
the N32D mutant, while T115D was completely inactive even toward the natural
substrate l-rhamnulose-1-phosphate [25]. The rest of the N-Cbz-aminoaldehyde
examples (14b–d) confirmed the observations with 14a (Scheme 16.5).
In addition to protein engineering, the substrate mimicking approach was also
applied 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].