Angewandte
Chemie
Compared to WT enzymes, mutants displayed not only
altered selectivities but also changes in activity for several
of the investigated compounds. Using CDO M232A as
biocatalyst, conversions of 2, 5 and (R)-7 were enhanced
significantly. Remarkably, CDO WT already catalyzed the
oxidation of 2 with 44% product formation, even though it
has been reported in literature that non-conjugated linear
alkenes are generally found to be poor substrates for cis-
dihydroxylation by ROs.[23] With CDO M232A, the conver-
sion was increased additionally by about 2-fold, resulting in
93% product formation and an excellent stereoselectivity
(> 98% ee). Furthermore, the formation of 7b in the CDO
M232A-catalyzed allylic oxidation of (R)-7 was improved
from 46% to > 99% while maintaining a de value > 98% for
(1R, 5S)-7b. Enhanced substrate oxidation up to 7-fold was
also achieved for the linear polyalkene 5 by exchanging
methionine to alanine in CDO M232A and BDO M220 A
(Table 1 and Table S2).
Compared to minor changes in selectivity in the formation
of diol 2a, a significant alteration in stereospecificity was
detected for CDO M232A and BDO M220A during oxy-
functionalization of 4. The arene-substituted cyclic alkene was
converted by WT and mutant ROs yielding diol 4b and the
monohydroxylated product 4a. Interestingly, for both prod-
ucts CDO favored opposite enantiomers compared to NDO
and BDO albeit only low selectivities were obtained for (R)-
4a and (1S,2R)-4b (Table 2). The single amino acid substitu-
tion in the active site of CDO, however, reversed selectivity,
yielding (S)-4a with an improved enantiomeric excess of
87%. Likewise, the increase in ee to 54% for diol 4b was
accompanied by a change in absolute configuration from
(1S,2R)-4b to (1R,2S)-4b, turning CDO and CDO M232A
into enantiocomplementary catalysts for the oxidation of 4.
An altered stereospecificity was also observed for the BDO
variant M220A. Yet, improvement of the enantiomeric excess
from 45% to 82% was only present for (S)-4a while the
stereoselectivity for (1R, 2S)-4b decreased to 33%
(Table S6).
stereoselectivities (Figure 1 and Figure S6). With substrate
4, CDO M232H showed a 3-fold enhancement in ee to 28%
(R)-4a, whereas a switch in stereoselectivity from (R)-4a to
(S)-4a was observed for the other CDO variants compared to
the WT. As demonstrated for 1a, the stereoselectivity for (S)-
4a generally increased with smaller amino acid residues
(Figure S7). Furthermore, large side chains (F, L, I) gave (1S,
2R)-4b with up to 58% ee whereas variants CDO M232V, C,
S, and G yielded (1R,2S)-4b (Figure S8).
To proof the applicability of ROs for organic synthesis,
semi-preparative scale biotransformations (70 mg) of 6 and
(R)-7 were performed with mutant CDO M232A. Without
further optimization of the reaction set-up, 6a and 7b were
isolated in 33% and 38% yield, respectively (35% and 87%
conversion), demonstrating that a single point-mutation was
sufficient to transform WT CDO into an efficient catalyst.
In summary, we demonstrated that ROs can be evolved
for the oxidation of a broad range of olefins. Introduction of
a single point-mutation proved sufficient to generate BDO
and CDO variants displaying remarkable changes in regio-
and stereoselectivity for different aliphatic compounds. The
effect of various amino acid residues at this position on
selectivity highlights the potential for the engineering of ROs.
Of all generated variants, especially CDO M232A gave
excellent stereoselectivities (ꢀ 95%) and conversion rates
> 90% for linear alkenes, which have been reported to be
challenging substrates for RO-catalyzed oxyfunctionaliza-
tions. Furthermore, we demonstrated the preparative oxida-
tion of 6 and (R)-7 yielding 6a and 7b in mg scale. We
therefore suggest that ROs provide a biocatalytic approach to
both the asymmetric cis-dihydroxylation and the regio- and
stereospecific allylic monohydroxylation of various alkenes,
also targeting products difficult to access by Sharpless AD or
Riley oxidation.
Acknowledgements
Next to the alteration in selectivity and conversion, the
amino acid exchange of methionine to alanine resulted in
a CDO variant able to convert terpene 6. A biocatalytic
access to 6a is of particular interest as osmium(VIII) oxide-
catalyzed dihydroxylation of 6 requires elevated temper-
atures ꢀ 708C.[24,25]
We thank Prof. Dr. Rebecca Parales for providing us with the
E. coli JM109 (DE3) (pDTG141) culture, Prof. Dr. David
Leak for providing the plasmid pJRM501, and Prof. Dr.
Hideaki Nojiri for plasmid pIP107D. We gratefully acknowl-
edge financial support by the IMI Joint Undertaking between
the European Union and pharmaceutical industry association
EFPIA under grant agreement no. 115360-2.
In CDO WT productive binding of the substrate might be
sterically hindered due to a clash with the bulky methionine
side chain at position 232 as biotransformations using CDO
M232A gave 33% of 6a with an excellent stereoselectivity of
> 95% de for the (1S,2S,3R,5S)-(++)-isomer.
Keywords: asymmetric dihydroxylation · biocatalysis · cis-diols ·
protein engineering · rieske non-heme iron oxygenases
How to cite: Angew. Chem. Int. Ed. 2015, 54, 12952–12956
Angew. Chem. 2015, 127, 13144–13148
Considering the strong impact on selectivity, the effect of
other amino acid residues at position 232 was examined using
the NDT codon degeneracy.[26] From the NDT mutant library,
11 CDO variants (CDO M232F, L, I, V, Y, H, N, D, C, S and
G) were tested for conversions of 1 and 4 as changes in regio-
and stereoselectivity were most pronounced with these
substrates. For compound 1, the amino acid side chain size
at position 232 was shown to directly influence both the regio-
and stereoselectivity. In general, smaller amino acid residues
favored the formation of 1a accompanied by increased
[1] H. C. Kolb, M. S. Van Nieuwenhze, K. B. Sharpless, Chem. Rev.
[3] R. E. Parales, S. M. Resnick, Biocatal. Pharm. Biotechnol. Ind.
2006, 299 – 331.
[4] A. Karlsson, J. V. Parales, R. E. Parales, D. T. Gibson, H.
Angew. Chem. Int. Ed. 2015, 54, 12952 –12956
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim