Hydroxy functionalization of non-activated Ci-H and Ci=C Bonds: New perspectives for the synthesis of alcohols through biocatalytic processes

Article abstract of DOI:10.1002/anie.201308556

New perspectives through enzymes: Recent breakthroughs have been achieved in the selective hydroxy functionalization of non-activated Ci-H and Ci=C bonds. Enzymes turned out to be suitable catalysts for the ω-hydroxylation of (substituted) alkanes and regioselective hydroxylation of aromatic hydrocarbons with atmospheric oxygen as the oxidant, and the asymmetric addition of water to non-activated alkenes.

Full text of DOI:10.1002/anie.201308556

Angewandte
Chemie
DOI: 10.1002/anie.201308556
Enzyme Catalysis
À
Hydroxy Functionalization of Non-Activated C H and
C C Bonds: New Perspectives for the Synthesis of
=
Alcohols through Biocatalytic Processes**
Harald Grçger*
Dedicated to Professor Werner Hummel on the
occasion of his 65th birthday
alcohols · biocatalysis · enzyme catalysis · hydra-
tion · hydroxylation
I
n organic chemistry, biocatalysis has become a valuable tool
for academic and industrial synthesis and provides a comple-
ment to “classic chemical” and chemocatalytic processes.[1] In
particular, enzyme-catalyzed processes are applied with
tremendous success in the production of fine chemicals and
active pharmaceutical ingredients.[2,3] A current focus in
biocatalysis research is on the realization of chemical trans-
formations, for which efficient and sustainable solutions do
not exist. The demand for such new methodologies has been
expressed in detail by representatives of the pharmaceutical
industry.[4] One of the challenges is the selective hydroxy
À
=
functionalization of non-activated C H and C C bonds.
Examples of such “dream reactions” include the w-hydrox-
ylation of (substituted) alkanes and the regioselective hy-
droxylation of aromatic hydrocarbons with atmospheric oxy-
gen as the oxidant in both cases as well as the asymmetric
addition of water to non-activated alkenes (Scheme 1).
Although in general enzymes appear to be predestined for
this purpose, hydroxylations in particular are often hampered
by low enzyme activities and stabilities, thus leading to
biotransformations with low efficiency. In the past few months
impressive solutions based on enzyme catalysis have been
presented for the reactions mentioned above. In this High-
light this is exemplified by three pioneering studies,[5–8] which
can all be considered to be breakthroughs also from the
perspective of synthetically practical catalytic processes.
A current challenge in enzyme catalysis is broadening its
application range in the production of specialty and bulk
chemicals exceeding an annual output of 1000 tonnes, since in
this field its use is still very limited.[2] One of the few examples
is the production of acrylamide with a nitrile hydratase.[9]
Currently, w-aminolauric acid and its esters are interesting
substitutes for laurin lactam,[10] which is required for the
Scheme 1. Selected synthetic challenges in the field of hydroxy func-
tionalization.
manufacture of polyamide-12 on a multi-10000 tonne scale
annually. In a joint project with Evonik Industries AG the
research group of Schmid and Bꢀhler has now developed
a new process for the synthesis of w-aminolauric acid methyl
ester based on sustainable feedstocks and the use of a mono-
oxygenase.[5,6] The resulting catalytic selective hydroxylation
of an alkyl chain as a reaction type, which up to now has been
known to be synthetically difficult and in general connected
with low efficiency, proceeds here with remarkably high
process efficiency (Scheme 2). An E. coli whole-cell catalyst
is used bearing a monooxygenase and an AlkL membrane
protein (which is responsible for the transport of the ester
through the hydrophobic membrane and into the cell) in
recombinant form. Upon application of this catalyst dodec-
anoate 1 is first terminally hydroxylated and then transformed
in part into the aldehyde 3. In the process, a product
concentration of 159 mm of alcohol 2 is obtained in the
organic phase and additionally about 50 mm of the aldehyde 3
is formed by further oxidation.[5] Besides the monooxygenase,
the AlkL protein also plays a key role: in its presence the
[*] Prof. Dr. H. Grçger
Faculty of Chemistry, Bielefeld University
Universitꢀtsstrasse 25, 33615 Bielefeld (Germany)
E-mail: harald.groeger@uni-bielefeld.de
À1
whole-cell activity increases by 62-fold up to 87 Ugcdw
(cdw = cell dry weight).
The subsequent conversion of aldehyde 3 with a trans-
aminase and l-alanine as an amine donor then furnishes the
desired w-aminododecanoate, for which a whole-cell process
[**] I thank the German Federal Ministry of Education and Research
(Bundesministerium fꢁr Bildung und Forschung; BMBF) for
generous support within the project “BISON—Biotechnologische
Synthese von Carboxyaminen und Carboxyalkoholen” (grant num-
ber 0316044A).
Angew. Chem. Int. Ed. 2014, 53, 3067 – 3069
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3067

.
Angewandte
Highlights
Scheme 2. Selective w-hydroxylation of dodecanoate 1 using a whole-cell catalyst.
was also developed by Schmid and Bꢀhler et al.[6] The
preparative utility and industrial potential of the process
concept to produce biotechnological w-aminolauric acid from
palm kernel oil for polyamide-12 synthesis are underlined by
a recent press release from Evonik Industries AG,[11] accord-
ing to which a pilot plant for this purpose started operation in
Slovenska Lupka (Slovakia) in early 2013.
example halogenated arenes. The resulting substituted phe-
nols are valuable intermediates for the synthesis of natural
products and active pharmaceutical ingredients. A remaining
challenge for this process, which proceeds with molecular
oxygen in water at room temperature, consists in the increase
of the product concentration, which is so far ꢀ0.67 gLÀ1.
In connection with the selective enzymatic hydroxylation
of arenes the recent contribution of Shoji and Watanabe et al.
should also be mentioned at this stage: here with the P450
BM3 wild-type enzyme a high ortho selectivity was achieved
when in addition perfluorinated carboxylic acids were used as
so-called “decoy molecules”.[12]
The catalytic enantioselective hydration of non-activated
alkenes, for example styrene, represents an attractive syn-
thetic approach for obtaining enantiomerically pure alcohols.
In contrast to many other asymmetric reactions, however, to
date no suitable chiral chemocatalyst is known for this type of
reaction.[13] Recently, the enantioselective transformation of
styrene to 1-phenylethanol was achieved by means of
a chemoenzymatic one-pot synthesis.[14] However, this “for-
mal addition of water” proceeds in two steps by means of
a Wacker oxidation and subsequent enzymatic ketone reduc-
In analogy to aliphatic molecules the hydroxy function-
alization of aromatic hydrocarbons also presents a challenge
for the synthetic chemist. Up to now selective hydroxylations
of substituted arenes have been considered to be difficult and
are often accomplished only under harsh reaction conditions
and with side-product formation and low selectivity. Here,
again, enzyme catalysis provides an interesting means to solve
this problem. Recently the group of Schwaneberg succeeded
in developing a highly regioselective ortho-hydroxylation of
substituted arenes 4 by protein engineering (Scheme 3).[7]
The P450-monooxygenase-catalyzed substitution of ani-
sole (4a) proceeds with a high turnover frequency of
38.6 sÀ1 [7] This corresponds to 19.5 UmgÀ1, which represents
.
the highest enzyme activity observed so far for aromatic
hydroxylations with P450 monooxygenases. In addition, the
product ortho-methoxyphenol (6a) is formed with an ex-
cellent ortho/para regioselectivity of > 95:5 and the meta-
substituted form does not occur. High activities and ortho/
para selectivities are also obtained with other substrates, for
=
tion. Although direct biocatalytic hydrations of C C bonds
are known and applied on an industrial scale for the
production of fumaric acid,[15] in this case the substrates are
activated alkenes, for example enoates. Accordingly, the
direct asymmetric catalytic addition of water to styrene and
other non-activated alkenes remained a challenge, for which
now a solution has been found by Faber et al. by means of
enzyme catalysis (Scheme 4).[8] Interestingly hydroxybenzoic
acid decarboxylases turned out to be suitable for this purpose
and enabled the asymmetric addition of water to p-hydrox-
ystyrene (6a) and substituted derivatives with conversions of
up to 82% and enantioselectivities of up to 71% ee. For
example, p-hydroxystyrene was transformed into the product
(S)-7a with 82% conversion and 43% ee. In contrast, the
carboxylation, which could be expected from this enzyme
class, took place at only a low level (< 5%) even in the
presence of a high concentration of carbonate buffer of 3m.
Remarkable is the high proportion of enzymes suitable for
=
this hydration of non-activated C C bonds with six out of
Scheme 3. Selective ortho-hydroxylation of arenes using P450 mono-
oxygenases.
seven studied decarboxylases. In view of the broad applica-
3068 www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 3067 – 3069

Angewandte
Chemie
[1] Enzyme Catalysis in Organic Synthesis, Vol. 1 – 3, 3rd ed. (Eds.:
K. Drauz, H. Grçger, O. May), Wiley-VCH, Weinheim, 2012.
[2] A. Liese, K. Seelbach, C. Wandrey, Industrial Biotransforma-
tions, 2nd ed., Wiley-VCH, Weinheim, 2006.
[3] U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz,
J. C. Moore, K. Robins, Nature 2012, 485, 185 – 194.
[4] D. J. C. Constable, P. J. Dunn, J. D. Hayler, G. R. Humphrey, J. L.
Leazer, Jr., R. J. Linderman, K. Lorenz, J. Manley, B. A. Pearl-
man, A. Wells, A. Zaks, T. Y. Zhang, Green Chem. 2007, 9, 411 –
420.
[5] M. K. Julsing, M. Schrewe, S. Cornelissen, I. Hermann, A.
Schmid, B. Bꢀhler, Appl. Environ. Microbiol. 2012, 78, 5724 –
5733.
[6] M. Schrewe, N. Ladkau, B. Bꢀhler, A. Schmid, Adv. Synth. Catal.
2013, 355, 1693 – 1697.
[7] A. Dennig, N. Lꢀlsdorf, H. Liu, U. Schwaneberg, Angew. Chem.
2013, 125, 8617 – 8620; Angew. Chem. Int. Ed. 2013, 52, 8459 –
8462.
Scheme 4. Asymmetric addition of water to substituted styrenes using
decarboxylases.
[8] C. Wuensch, J. Gross, G. Steinkellner, K. Gruber, S. M. Glueck,
K. Faber, Angew. Chem. 2013, 125, 2349 – 2353; Angew. Chem.
Int. Ed. 2013, 52, 2293 – 2297.
[9] Y. Asano, P. Kaul in Comprehensive Chirality, Vol. 7 (Eds.: E. M.
Carreira, H. Yamamoto), Elsevier, Amsterdam, 2012, p. 122 –
142.
[10] T. Schiffer, G. Oenbrink in Ullmannꢀs Encyclopedia of Industrial
Chemistry, 6th ed., Electronic Release, Wiley-VCH, Weinheim,
2000.
[11] Evonik Industries AG, press release “An alternative raw
material for polyamide 12: Evonik is operating a pilot plant
for bio-based w-amino lauric acid”, July 30, 2013.
[12] O. Shoji, T. Kunimatsu, N. Kawakami, Y. Watanabe, Angew.
Chem. 2013, 125, 6738 – 6742; Angew. Chem. Int. Ed. 2013, 52,
6606 – 6610.
tion of this method, future research tasks include increasing
substrate scope (currently limited to para-hydroxy-substitut-
ed styrenes) and improving the enantioselectivity.
In conclusion, recent pioneering research has led to the
development of synthetically practical catalytic processes for
several hydroxy functionalizations of (substituted) alkanes
and alkenes. Thereby the w-hydroxylation of fatty acid esters
and the regioselective ortho-hydroxylation of aromates are
accomplished with atmospheric oxygen. A biocatalytic pro-
cess was also established for the asymmetric addition of water
to non-activated alkenes. These contributions offer promising
perspectives for the increased application of such enzyme-
catalyzed reactions in organic synthesis.
[13] L. Hintermann, Top. Organomet. Chem. 2010, 31, 123 – 155.
[14] I. Schnapperelle, W. Hummel, H. Grçger, Chem. Eur. J. 2012, 18,
1073 – 1076.
Received: October 1, 2013
Published online: March 3, 2014
[15] J.-F. Jin, U. Hanefeld, Chem. Commun. 2011, 47, 2502 – 2510.
Angew. Chem. Int. Ed. 2014, 53, 3067 – 3069
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 3069