Journal of the American Chemical Society
Article
Figure 2. (A) Structure of the active site of the ketopantoate complex
of wild-type KPHMT (chain A, PDB 1M3U).15 Ketopantoate is
shown with yellow C atoms, and the three residues proposed for
mutation are shown with orange C atoms. Models of the prereactive
complexes of (B) KPHMT V214G with (S)-1b and 2a, (C) KPHMT
I202A/V214G with (S)-1d and 2a, and (D) KPHMT I212A with 1f
and 2h. Nucleophiles 1b, 1d, and 1f are shown with yellow C atoms,
electrophiles 2a and 2h with green C atoms, and the mutated residues
with orange C atoms. Nucleophiles are shown in their enolate form
(1b and 1d as E-enolates) coordinated to the metal cofactor (purple
sphere), which is also coordinated by residues D45 and D84 and by
two water molecules. 2a and 2h are displayed with their carbonyl
oxygen atom forming hydrogen bonds with residue E181 and one of
the metal-coordinated waters. 2h is shown approaching the enolate
from its si-face. The surfaces of the active site cavities near the
nucleophile substrates are shown in cyan. Interactions are shown with
dashed lines: H-bond in yellow, salt bridges in magenta.
Figure 3. Screening of KPHMT-catalyzed aldol addition of selected
3,3-disubstituted 2-oxoacids 1 to aldehydes 2. Conversions to aldol
adducts (%) at 24 h of reaction measured by HPLC after
derivatization of the substrates and products via oxime formation
(see SI). Other nucleophile/electrophile combinations were not
performed due to the low activity in the screening with methanal as
detected.
hydroxyethanal (2c) (50 and 42%, respectively, with I212A)
were used as electrophiles, whereas they were not converted
using 2b and 2e−h. Nucleophiles 1m−n and electrophiles 2i−
k were not converted by any KPHMT catalysts. The results are
consistent with the initial reaction rates (v0) of the
KPHMT(Co2+) catalyst loading was 0.068 mol %. This
catalytic efficiency in addition to their high expression levels
make these enzymes highly valuable for preparative-scale
synthesis.
Preparative Synthesis. The positive reactions were run at
a preparative scale (2.5 mmol of the limiting 3,3-disubstituted
2-oxoacid (1) substrate), providing, after the adequate
treatment (see below), the corresponding 2-oxolactones
(4a−g) (Scheme 3A), 3-hydroxy acid derivatives (5a−j)
(Scheme 3B), bicyclic lactones (6) (Scheme 4A), and ulosonic
acid type products (7a−q) (Scheme 4B), all of them bearing
gem-dialkyl, gem-cycloalkyl, and spirocyclic quaternary centers,
with final product amounts ranging from 50 to 390 mg. The 2-
oxolactones4a−g were produced during workup (acidic
aqueous−organic solvent extraction). Thus, when quantitative
conversions were achieved, this methodology was enough to
afford pure material (Scheme 3A). Otherwise, an additional
extraction with aqueous buffer at neutral pH removed the
remaining starting 2-oxoacid 1, which was the main impurity
present. The 2-oxo group can be reduced,16 affording α-
hydroxy-γ-butyrolactones (e.g., (R)- or (S)-pantolactone and
analogues), chiral auxiliaries, and important precursors for the
synthesis of naturally occurring and synthetic biologically
active compounds.17 Elaborated chemical transformations on
the carboxylate group such as oxidative decarboxylation
followed by esterification lead to different 3-hydroxyesters
(5a−i and amide 5j, Scheme 3B) with application as building
blocks in the synthesis of pharmaceutical compounds18 and
polymers, coating oligomers, and dendrimers, e.g., 2,2-
bis(hydroxymethyl) propionic acid derivative 5g.19
Using 2-hydroxyaldehydes, 2c and 2e−h, as electrophiles,
the spontaneous formation of cyclic hemiketals effectively
shifted the reaction equilibrium to the aldol products 6a,b and
7a−q (Scheme 4A,B). The resulting adduct products were
easily purified by anion exchange chromatography, eluting with
formic acid. These ulosonic acid type products, bearing a
quaternary center at C3, have not been hitherto reported, and
their preparation using conventional chemical procedures may
be anticipated to be elusive because of the number of
functional groups and the intrinsic reactivity of the 2-oxoacid
moiety.20 Furthermore, KPHMT would facilitate access to a
great variety of ulosonic acids (e.g., sialic acid family), which
constitute important biologically active compounds involved in
cellular recognition and communication.21
The addition of 1j to 2c (Scheme 4A) gave a mixture of the
two diastereomeric lactones 6a and 6b (91:9, respectively),
identified by NMR after anion exchange purification. The
acidic elution conditions (i.e., HCOOH, 1 M) and the
C
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX