Angewandte Chemie International Edition
10.1002/anie.201914877
COMMUNICATION
3
, 4 or 5, the formed lactone rapidly hydrolyzes into the stable
AOX* was highly selective towards the S-enantiomer of 1-phenyl-
ethanol (no conversion for the R-enantiomer).
The steady-state kinetic parameters for a selection of the
discovered AOX* substrates were determined (Table 2). The
observed values for 2 and its corresponding lactol (int-2) support
the proposed catalytic mechanism of the double oxidation going
hydroxy acid. We also monitored the conversion of 4. This
revealed that the first detectable product intermediate exists in its
hemiacetal form. According to these data, we conclude that the
AOX*-catalyzed double oxidation of aliphatic diols proceeds via
formation of the corresponding hemiacetal which is subsequently
oxidized into the lactone, which is prone to hydrolysis into the
corresponding 5-hydroxy acids (Scheme 1).
through the lactol intermediate. The rate limiting step (lower kcat
/
M
K ) seems to be the second oxidation step, the oxidation of the
Substrate 6 was also tested, because the corresponding
product lactone (ε-caprolactone) is of value as polymer building
block.[ Somewhat unforeseen, AOX* was found to oxidize both
hydroxyl groups to aldehyde groups resulting in the production of
adipaldehyde. In the employed buffer, adipaldehyde sponta-
neously reacted via aldol condensation (non-enzymatic reaction,
see Supporting information) while also some further oxidation into
lactol. This was confirmed by substrate 1 and also by shorter
conversion experiments for substrates 2-5. Shorter incubations
revealed the accumulation of the respective lactols.
20]
Table 2. Apparent Steady-State Kinetic Parameters for AOX* [a]
.
a]
[b]
[a]
Entry
Substrate
K [
M
K
I
k
cat
k
cat / K
34.1
24.3
M
6-oxohexanoic acid was observed. Similarly to substrate 6, 1,8-
1
2
198
n.d.
n.d.
6.74
1.28
octanediol (7) and triethylene glycol (8) underwent oxidation on
both hydroxyl groups yielding the corresponding dialdehydes,
which then got further oxidized into oxocarboxylic acids. For these
two substrates the aldol condensation product was not obtained.
This is probably due to the unfavorable formation of a seven-
membered ring product. For the latter substrates (6-8), AOX* is
able to perform a triple oxidation by oxidizing the gem-diol form of
the formed dialdehydes. NMR analysis also confirmed that these
aliphatic aldehydes are significantly hydrated (10%-50%) in the
employed buffer. For the selective oxidation on only one hydroxyl
group, as observed for 3-5, formation of a very stable hemiacetal
intermediate seems to be crucial. If the hemiacetal is not formed
upon the first oxidation, the enzyme will oxidize the other hydroxyl
group resulting in the dialdehyde. Subsequently, one aldehyde
group is oxidized to carboxylic acid via the gem-diol. Once that
the carboxylic acid is obtained, the enzyme does not accept this
compound for the further oxidation towards a diacid. Along these
lines, substrate 9 was found to yield hexanoic acid, supporting our
hypothesis that AOX* can further oxidize the initially formed
aldehyde via its gem-diol. Different from the other substrates, 9
and the intermediate product formed from 9 (Int-9) exhibited
substrate inhibition (Table 2 and Supporting information). This
may be due to alternative binding pockets for these relatively
apolar substrates when compared with the tested diols.
52.6
int-2
95.5
69.6
12.3
119
n.d.
n.d.
n.d.
n.d.
n.d.
56
1.13
3.48
0.73
3.56
4.07
0.56
0.15
11.8
50.0
59.3
29.9
316
3
4
5
6
12.9
3.0
9
187
Int-9
0.20
5.5
750
[
a] Values obtained using the HRP-coupled assay in 50 mM potassium
phosphate, pH 7.5. K values are presented in units of mM, kcat values are
presented in units of s−1, and kcat/K values are presented in units of M−1 s−1
b] When no substrate inhibition was observed, this is indicated as n.d. (not
detected).
M
M
.
[
Substrates 4 and 5 were also tested as substrates with
several other flavoprotein alcohol oxidases: alditol oxidase
(
HotAldO) from Acidothermus cellulolyticus 11B, chitooligo-
Aminoalcohol (10) was used to explore the substrate
promiscuity of AOX*. Interestingly, complete conversion was
saccharide oxidase (ChitO) from Fusarium graminearum, 5-
hydroxymethylfurfural oxidase (HMFO) wild type and variant
1
observed for 10. From H NMR spectra it can be concluded that
8BxHMFO from Methylovorus sp. strain MP688, methanol
the reaction occurred as selective single oxidation on the hydroxyl
group. A singlet at 7.88 ppm clearly indicated that the product
exists as an imine (in situ formed spontaneously from
oxidase from Hansenula sp. (EC 1.1.3.13), glucose oxidase form
Aspergillus niger (EC 1.1.3.4), and choline oxidase wild type and
an engineered choline oxidase variant (AcCO6) from Arthrobacter
1
aminoaldehyde). The H NMR spectra appeared complex and the
[
16,17,22–24]
chlorophenolicus.
Except for (AcCO6), all these
product could not be extracted with ethyl acetate to confirm the
product by GC-MS analysis. Nevertheless Braekman and
coworker describe that Δ-piperideine under reaction conditions
oxidases did not convert 4 and 5. The six fold mutant of choline
oxidase, AcCO6, proved to be able to perform the double
oxidation of 4 and 5, albeit with low yields: 5% product (38% lactol
intermediate) with 4, and 28% of product (68% lactol intermediate)
with 5, using the same reaction conditions as with AOX.*
Furthermore, AcCO6 was found to suffer from severe substrate
inhibition.
In conclusion, this work demonstrates the potential of AOX*
as biocatalyst to produce, in one pot, hydroxy acids from 1,5-diols
through a selective double oxidation. By this, diethylene glycol
and thiodiglycol can be converted into the corresponding hydroxy
(
pH 7.5) analogous to ours preferably dimerizes to form tetra-
1
[21]
hydroanabasine, which correlates with our H NMR spectra.
It is known that alcohol oxidases can oxidize secondary
alcohols, though with poor efficiency. We selected just a few to
test AOX* (substrates 11, 12, and 13). Cyclohexanol (11) was
oxidized to cyclohexanone with a low yield. Initially, we tested
racemic 1-phenylethanol and its conversion was rather low,
similar to cyclohexanol. After that, we tested separately each
enantiomer of this alcohol (substrates 12 and 13). Remarkably,
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