Biocatalytic single-step alkene cleavage from aryl alkenes: An enzymatic equivalent to reductive ozonization

Article abstract of DOI:10.1002/anie.200601574

(Chemical Equation Presented) O₂ can do: Innocuous molecular oxygen O₂ is the only reagent needed to perform highly chemoselective biocatalytic single-step alkene-cleavage reactions (see scheme). The products are analogous to those of (reductive) ozonization and related metal-based methods. In contrast neither special equipment nor an additional reducing agent is required. The biocatalytic reaction can be performed at ambient temperature. Depending on the substrate, aldehydes or ketones are obtained.

Full text of DOI:10.1002/anie.200601574

Angewandte
Chemie
Biocatalytic Alkene Cleavage
As a consequence, novel or improved methods for alkene
cleavage are being sought.[6b–e,i,8] Enzymatic alkene cleavage
has been observed 1) as a minor (undesired) side-reaction on
the analytical scale catalyzed by peroxidases,[9] 2) with a
combination of lipoxygenases and hydroperoxide lyases for
specific substrates,[10] and 3) on an analytical scale employing
molecular oxygen as oxidant using certain mono-[11] or
dioxygenases[12] displaying high substrate specificity.[13] To
our knowledge, a method for biocatalytic oxidative alkene
cleavage has not been developed to date, which can be
employed on preparative scale.
DOI: 10.1002/anie.200601574
Biocatalytic Single-Step Alkene Cleavage from
Aryl Alkenes: An Enzymatic Equivalent to
Reductive Ozonization**
Harald Mang, Johannes Gross, Miguel Lara,
Christian Goessler, Hans E. Schoemaker,
Georg M. Guebitz, and Wolfgang Kroutil*
In a screening for microorganisms capable of oxidizing
alkenes in allylic position, we were not able to identify any hit.
Nevertheless, for one fungus, namely Trametes hirsuta
GFCC047[14] we observed the disappearance of the starting
material (E)-1-phenyl-1-butene (1a; Scheme 1) and the
Driven by the need for mild and selective oxidation methods
as well as by increased public awareness of hazards derived
from chemical production, environmentally benign oxidation
methods have gained increasing importance.[1] Alkene cleav-
age[2] to give aldehydes or ketones is a very frequently used
method in synthetic organic chemistry[3] to 1) introduce
oxygen functionalities into molecules, 2) split selectively
large compounds, and 3) remove protecting groups.[3g]
Among the methods currently available for the chemical
oxidative cleavage of alkenes, reductive ozonolysis is consid-
ered as the “cleanest”.[4] In practice, however, it has several
drawbacks, since it depends on special equipment (ozonizer),
low temperature (generally ꢀ788C), and a stoichiometric
amount of an additional reducing reagent (e.g. dimethyl
sulfide, zinc, hydrogen, phosphines) for reductive workup.
Furthermore, special precaution is required, since serious
accidents arising from explosions have been reported.[5] Other
methods involving metal-based oxidants[6] require (at least)
stoichiometric amounts of salts or peroxides and are plagued
by limited chemo-, regio-, and stereoselectivity. Over-oxida-
tion of the aldehyde to the corresponding acid as side reaction
is in most cases unavoidable. The only catalytic method
employing molecular oxygen needs a CoII [7] catalyst, is
restricted to isoeugenol-type substrates, and displays moder-
ate chemoselectivity.
Scheme 1. Biocatalytic alkene cleavage employing molecular oxygen.
For 1b!2b see Scheme 2.
formation of an unexpected product, which was identified
as benzaldehyde. To demonstrate that this is a biocatalyzed
reaction, the transformation was repeated with an enzyme
preparation inactivated by heat. In this case no reaction was
observed, which confirmed our assumption of a biocatalytic
reaction. Since biocatalytic conversions in synthetic applica-
tions are becoming increasingly important,[15] we aimed to
develop a novel biocatalytic procedure for reductive ozoni-
zation.
For the identification of the cleavage product(s), a cyclic
alkene, such as indene (1b; Scheme 2), was envisaged as
substrate, since it was expected to give a single product in
which both biooxidation cleavage products are present. Thus,
transformation of 1b yielded the corresponding dialdehyde
2b as shown by GC–MS and comparison with independently
synthesized reference material. Indene (1b) was also used to
elucidate the origin of the two oxygen atoms introduced. By
performing the experiments with 18O-labeled molecular
oxygen with a cell-free extract, we could show by GC–MS
analysis that both oxygen atoms in the two aldehyde frag-
[*] Dr. H. Mang, J. Gross, M. Lara, C. Goessler, Prof. W. Kroutil
Department of Chemistry, Organic and Bioorganic Chemistry
Research Centre Applied Biocatalysis
University of Graz
Heinrichstrasse 28, 8010 Graz (Austria)
Fax: (+43)316-380-9840
E-mail: wolfgang.kroutil@uni-graz.at
Prof. H. E. Schoemaker
DSM Research, Life Science Products
P.O. Box 18, 6160 MD Geleen (The Netherlands)
Prof. G. M. Guebitz
Institute of Environmental Biotechnology
Research Centre Applied Biocatalysis
University of Technology
Petersgasse 12, 8010 Graz (Austria)
[**] This study was financed by the Austrian Science Fund (FWF Project
P18381). Initial experiments were performed within the Research
Centre Applied Biocatalysis supported by DSM, FFG, SFG, Province
of Styria and City of Graz.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Scheme 2. Biocatalytic alkene cleavage of indene (1b) leading to the
corresponding dialdehyde.
Angew. Chem. Int. Ed. 2006, 45, 5201 –5203
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5201

Communications
ments are labeled. As a blank experiment we performed the
entry 3). Most remarkably, no measurable over-oxidation to
the corresponding carboxylic acid was observed.
Monosubstituted alkenes, such as 4-methoxystyrene (1d)
are also converted. Substituting the para-methoxy moiety of
1d by hydrogen led only to a slight decrease in activity
(styrene (1e), Table 1, entry 5). For allyl benzene (1 f) no
experiment in 18O-labeled water using 16O2. In this experiment
none of the two oxygen atoms was labeled; thus both oxygen
atoms originate from molecular O2 (Scheme 1). Although
these experiments are already a strong indication that this is a
one-step reaction catalyzed by a dioxygenase,[12] and not a
stepwise mechanism as found in the cleavage of b-carotene,[11]
possible intermediates in a stepwise transformation of 1a,
such as the diol 3 or the epoxide 4, were tested. However,
=
conversion was observed, probably because the C C bond is
not conjugated to the aromatic system. It is of note that even a
sterically more demanding ortho-substituted styrene, such as
o-methyl styrene (Table 1, entry 7), was converted. With p-
amino styrene (Table 1, entry 9), the amino group was not
touched, and a highly chemoselective alkene cleavage led
exclusively to p-aminobenzaldehyde with > 99% chemo-
selectivity. Even ketones are accessible, for instance, the
transformation of a-methyl styrene (1g) also occurred with
absolute chemoselectivity (> 99%) yielding acetophenone
(Table 1, entry 12).
To increase the solubility of the substrate in the aqueous
phase ethanol can be used as cosolvent up to 25% v/v. The
optimum activity is at 15% v/v. In other reactions the
substrate concentration was varied, and it can be increased
up to 2.7 molLꢀ1 (400 gLꢀ1) for trans-anethole (1c). The high
substrate concentration possible emphasizes the potential of
the method for preparative transformations.
neither 3 nor 4 were transformed to the corresponding
aldehyde; thus, neither of these two compounds is an
intermediate. Therefore the transformation is a single-step
alkene-cleavage reaction catalyzed by an enzyme employing
molecular oxygen as the only oxidant (Scheme 2). In contrast
to non-enzymatic methods, the reaction can be performed in
an aqueous environment at ambient temperature without
additional reagents except molecular oxygen.
To achieve high conversion, oxygen saturation, or even
better elevated oxygen pressure, is required. To reach such
conditions on a one-milliliter scale in parallel for testing, we
exploited the catalase activity, which was also present in the
enzyme preparation. Thus, a hydrogen peroxide solution was
added to the reaction mixture in a GC glass vial (1.5 mL)
which was quickly closed with a rubber seal. The hydrogen
peroxide was disproportionated by the catalase to yield water
and molecular oxygen within seconds giving a theoretical
overpressure of approximately 2 bar. Employing this assay
with a cell-free extract of Trametes hirsuta GFCC047 the
conversion of 1a was increased from 10% to 44% (Table 1,
entry 1). An even better substrate was trans-anethole (1c),
which was converted into anisaldehyde (2c) at 83% con-
version with excellent chemoselectivity (94%, Table 1,
For performing the alkene cleavage on a preparative scale,
a hydrogenation apparatus (Parr 3910) was adapted to work
under oxygen pressure. A constant pressure of 2 bar of
molecular oxygen was applied on the reaction mixture.
Employing 3 g of lyophilized cells, 1c was cleaved with 81%
conversion within 24 h.
In conclusion, the described novel biocatalytic alkene-
cleavage method is a simple, benign, and “green” alternative
to chemical methods. It does not require specialized equip-
ment (ozonizer) or chemicals (reducing agents, oxidizing
salts) and does not produce any waste. It requires the most
innocuous oxidant, namely oxygen. The highest possible
achievable atom efficiency[16] for alkene cleavage can only be
reached by using molecular oxygen, which is achieved in the
biocatalytic method presented. We have demonstrated that
the biocatalytic alkene cleavage described extends the
repertoire of biocatalytic reactions available for organic
synthesis.
Table 1: Results of the biocatalytic alkene cleavage of various alkenes.
Entry
Substrate[a]
Prod. [%][b]
Chemosel. [%][c]
1
2
(E)-1-phenyl-1-butene (1a)
indene (1b)
44
71
72
88
Experimental Section
3
4
5
6
7
8
9
10
11
12
trans-anethole (1c)
4-methoxystyrene (1d)
styrene (1e)
allylbenzene (1 f)
o-methylstyrene
p-methylstyrene
p-aminostyrene
p-tBu styrene
m-chlorostyrene
a-methylstyrene (1g)
83
32
25
<0.1
10
26
11
26
20
94
89
94
n.a.
>99
90
>99
92
Preparative-scale biocatalytic alkene cleavage under oxygen pres-
sure: Lyophilized cells of Trametes hirsuta GFCC047 (3 g) were
rehydrated with Bis–Tris buffer (125 mL, 50 mm, pH 6; Bis–Tris = 2,2-
bis(hydroxyethyl)iminotris(hydroxymethyl)methane) for 30 min on a
rotary shaker (258C, 130 rpm). The mixture was placed into the
reaction vessel (450 mL) of the “Hydrogenation Apparatus
Parr3910” and trans-anethole (1c; 0.6 mL, 0.59 g, 3.9 mmol) and
EtOH (1.7 mL) were added. The atmosphere was saturated with pure
O2, and then the oxygen pressure was adjusted to 2 bar. After 24 h of
agitation at 228C under constant oxygen pressure (2 bar), the reaction
mixture was extracted with EtOAc (4 ” 50 mL) and centrifuged after
each extraction step (8000 rpm, 20 min) to achieve phase separation.
The cells were removed by filtration from the aqueous solution. The
aqueous solution was then extracted with EtOAc (50 mL). The
combined organic phase was dried with Na2SO4, filtered, and
88
>99
26
[a] Conditions: cell-free enzyme preparation of Trametes hirsuta
GFCC047, performed in GC vials under oxygen pressure, with 6 gLꢀ1
substrate concentration, 258C, pH 6.0. [b] GC yield, 24 h reaction time,
ratio of formed aldehyde/ketone to initial substrate concentration.
[c] Ratio of aldehyde/ketone formed to all compounds formed; n.a. not
applicable.
concentrated.
A
conversion of 81% p-anisaldehyde (2c) was
5202
www.angewandte.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 5201 –5203

Angewandte
Chemie
determined by GC analysis. Column chromatography (50 g silica gel,
petroleum ether/ethyl acetate 20:1) gave 0.31 g of 2 c (57% yield of
isolated product).
[7] R. S. Drago, B. B. Corden, C. W. Barnes, J. Am. Chem. Soc. 1986,
108, 2453 – 2454.
[8] a) A. G. Shoair, R. Mohamed, Synth. Commun. 2006, 36, 59 – 64;
b) W. P. Griffith, E. Kwong, Synth. Commun. 2003, 33, 2945 –
2951; c) A. K. Sinha, B. P. Joshi, R. Acharya, Chem. Lett. 2003,
32, 780 – 781.
Received: April 21, 2006
Published online: July 20, 2006
[9] Myeloperoxidase and Coprinus cinereus peroxidase with elec-
tron-deficient styrene derivatives: a) A. Tuynman, J. L. Spel-
berg, I. M. Kooter, H. E. Schoemaker, R. Wever, J. Biol. Chem.
2000, 275, 3025 – 3030; Horseradish peroxidase in the presence
of phenol as cosubstrate: b) P. R. Ortiz de Montellano, L. A.
Grab, Biochemistry 1987, 26, 5310 – 5314; Horseradish perox-
idase with indoles: c) K.-Q. Ling, L. M. Sayre, Bioorg. Med.
Chem. 2005, 13, 3543 – 3551; engineered horseradish peroxidase:
d) S.-i. Ozaki, P. R. Ortiz de Montellano, J. Am. Chem. Soc.
1995, 117, 7056 – 7064; chloroperoxidase from Caldariomyces
fumago with tert-butyl hydroperoxide: e) D. J. Bougioukou, I.
Smonou, Tetrahedron Lett. 2002, 43, 339 – 342; f) D. J. Bougiou-
kou, I. Smonou, Tetrahedron Lett. 2002, 43, 4511 – 4514; g) M.
Takemoto, Y. Iwakiri, Y. Suzuki, K. Tanaka, Tetrahedron Lett.
2004, 45, 8061 – 8064.
[10] G. Bourel, J.-M. Nicaud, B. Nthangeni, P. Santiago-Gomez, J.-M.
Belin, F. Husson, Enzyme Microb. Technol. 2004, 35, 293 – 299.
[11] Employing b-carotene monooxygenase for epoxidation in a
cascade with a hydrolase and further enzymes: M. G. Leuen-
berger, C. Engeloch-Jarret, W.-D. Woggon, Angew. Chem. 2001,
113, 2684 – 2687; Angew. Chem. Int. Ed. 2001, 40, 2614 – 2617.
[12] For reviews see: a) T. D. H. Bugg, Tetrahedron 2003, 59, 7075 –
7101; b) M. Sono, M. P. Roach, E. D. Coulter, J. H. Dawson,
Chem. Rev. 1996, 96, 2841 – 2888.
[13] Quercetin 2,3-dioxygenase from Bacillus subtilis: a) M. R.
Schaab, B. M. Barney, W. A. Francisco, Biochemistry 2006, 45,
1009 – 1016; Quercetin 2,3-dioxygenase from Aspergillus niger:
b) H.-K. Hund, J. Breuer, F. Lingens, J. Hüttermann, R. Kappl, S.
Fetzner, Eur. J. Biochem. 1999, 263, 871 – 878; indoleamine 2,3-
dioxygenase and tryptophan 2,3-dioxygenase from mammals:
c) D. H. Munn, M. Zhou, J. T. Attwood, I. Bondarev, S. J.
Conway, B. Marshall, C. Brown, A. L. Mellor, Science 1998,
281, 1191 – 1193, and references therein; a dioxygenase from
Acinetobacter johnsonii is restricted to 1,3-diones: d) G. D.
Straganz, H. Hofer, W. Steiner, B. Nidetzky, J. Am. Chem. Soc.
2004, 126, 12202 – 12203; e) G. Straganz, A. Glieder, L. Brecker,
D. W. Ribbons, W. Steiner, Biochem. J. 2003, 369, 573 – 581;
Lignostilbene-a,b-dioxygenase isozymes cleave various substi-
tuted stilbene derivatives: f) S. Kamoda, T. Terada, Y. Saburi,
Biosci. Biotechnol. Biochem. 1997, 62, 2575 – 2576; g) S.
Kamoda, M. Samejima, Agric. Biol. Chem. 1991, 55, 1411 –
1412; h) S. Kamoda, Y. Saburi, Biosci. Biotechnol. Biochem.
1993, 57, 931 – 934; i) S. Kamoda, T. Terada, Y. Saburi, Biosci.
Biotechnol. Biochem. 2003, 67, 1394 – 1396; j) S. Kamoda, T.
Terada, Y. Saburi, Biosci. Biotechnol. Biochem. 2005, 69, 635 –
637; Heme-dependent oxygenase cleaves double bonds of
rubber (poly(cis-1,4-isoprene)): k) R. Braaz, P. Fischer, D.
Jendrossek, Appl. Environ. Microbiol. 2004, 70, 7388 – 7395;
review on b-diketone cleavage: l) G. Grogan, Biochem. J. 2005,
388, 721 – 730.
Keywords: alkenes · biocatalysis · oxidation · oxygen · ozonolysis
.
[1] a) Modern Oxidation Methods (Ed.: J.-E. Bäckvall), Wiley,
Weinheim, 2004; b) G.-J. ten Brink, I. W. C. E. Arends, R. A.
Sheldon, Chem. Rev. 2004, 104, 4105 – 4123; c) R. Noyori, M.
Aoki, K. Sato, Chem. Commun. 2003, 1977 – 1986; d) M. Poliak-
off, J. M. Fitzpatrick, T. R. Farren, P. T. Anastas, Science 2002,
297, 807 – 810; e) K. Sato, M. Hyodo, M. Aoki, X.-Q. Zheng, R.
Noyori, Tetrahedron 2001, 57, 2469 – 2476.
[2] a) F. E. Kühn, R. W. Fischer, W. A. Hermann, T. Weskamp in
Transition Metals for Organic Synthesis, Vol. 2 (Eds.: M. Beller,
C. Bolm), Wiley-VCH, Weinheim, 2004, pp. 427 – 436; b) D. G.
Lee, T. Chen in Comprehensive Organic Synthesis, Vol. 7 (Eds.:
B. M. Trost, I. Fleming), Pergamon, Oxford, 1991, pp. 541 – 591;
c) M. Hudlicky, Oxidation in Organic Chemistry, ACS Mono-
graph 186, American Chemical Society, Washington, DC, 1990.
[3] A selection of recent examples yielding aldehydes: employing
ozone: a) I. Paterson, G. J. Florence, A. C. Heimann, A. C.
Mackay, Angew. Chem. 2005, 117, 1154 – 1157; Angew. Chem.
Int. Ed. 2005, 44, 1130 – 1133; b) A. Wrobleski, K. Sahasrabudhe,
J. Aube, J. Am. Chem. Soc. 2004, 126, 5475 – 5481; c) X. Peng, D.
Bondar, L. A. Paquette, Tetrahedron 2004, 60, 9589 – 9598;
employing OsO4/NaIO4: d) T. E. Nielsen, M. Meldal, Org. Lett.
2005, 7, 2695 – 2698; e) S. Takahashi, K. Souma, R. Hashimoto,
H. Koshino, T. Nakata, J. Org. Chem. 2004, 69, 4509 – 4515;
employing RuCl3/NaIO4: f) L. G. Arini, P. Szeto, D. L. Hughes,
R. A. Stockman, Tetrahedron Lett. 2004, 45, 8371 – 8374;
employing RuCl3/NaIO4 for deprotection: g) B. Alcaide, P.
Almendros, J. M. Alonso, Tetrahedron Lett. 2003, 44, 8693 –
8695.
[4] R. A. Berglund in Encyclopedia of Reagents for Organic Syn-
thesis, Vol. 6 (Ed.: L. A. Paquette), Wiley, New York, 1995,
pp. 3837 – 3843.
[5] a) K. Koike, G. Inoue, T. Fukuda, J. Chem. Eng. Jpn. 1999, 32,
295 – 299; b) R. A. Ogle, J. L. Schumacher, Process Saf. Prog.
1998, 17, 127 – 133.
[6] One of the most widely used reagents is OsO4/NaIO4 (Lemieux–
Johnson reagent): a) R. Pappo, D. S. Allen, Jr., R. U. Lemieux,
W. S. Johnson, J. Org. Chem. 1956, 21, 478 – 479; b) W. Yu, Y.
Mei, Y. Kang, Z. Hua, Z. Jin, Org. Lett. 2004, 6, 3217 – 3219;
further reagents are: OsO4/oxone: c) B. R. Travis, R. S. Narayan,
B. Borhan, J. Am. Chem. Soc. 2002, 124, 3824 – 3825; cat. RuCl3/
(NaIO4 or Oxone): d) D. Yang, C. Zhang, J. Org. Chem. 2001, 66,
4814 – 4818; ruthenium nanoparticles/NaIO4: e) C.-M. Ho, W.-Y.
Yu, C.-M. Che, Angew. Chem. 2004, 116, 3365 – 3369; Angew.
Chem. Int. Ed. 2004, 43, 3303 – 3307; other ruthenium-derived
catalysts: f) V. Kogan, M. M. Quintal, R. Neumann, Org. Lett.
2005, 7, 5039 – 5042; chromium(VI): g) J. March, Advanced
Organic Chemistry, Wiley, New York, 1992, p. 1181; Permanga-
nate: h) D. Arndt, Manganese Compounds as Oxidizing Agents
in Organic Chemistry, Open Court, LaSalle, IL, 1981, pp. 241 –
246; i) S. Lai, D. G. Lee, Synthesis 2001, 1645 – 1648; for
autooxidation see: j) R. A. Sheldon, J. K. Kochi, Metal-Cata-
lyzed Oxidation of Organic Compounds, Academic Press, New
York, 1981; k) Ti-zeolite with hydrogen peroxide: W. Adam, A.
Corma, A. Martinez, M. Renz, Chem. Ber. 1996, 129, 1453 –
1455; peroxotungstate complexes with H2O2: l) A. Haimov, H.
Cohen, R. Neumann, J. Am. Chem. Soc. 2004, 126, 11762 –
11763.
[14] In-house culture collection.
[15] H. E. Schoemaker, D. Mink, M. G. Wubbolts, Science 2003, 299,
1694 – 1697.
[16] R. A. Sheldon, Pure Appl. Chem. 2000, 72, 1233 – 1246.
Angew. Chem. Int. Ed. 2006, 45, 5201 –5203
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5203