Aqueous oxidation of alcohols catalyzed by artificial metalloenzymes based on the biotin-avidin technology

Article abstract of DOI:10.1016/j.jorganchem.2005.02.001

Based on the incorporation of biotinylated organometallic catalyst precursors within (strept)avidin, we have developed artificial metalloenzymes for the oxidation of secondary alcohols using tert-butylhydroperoxide as oxidizing agent. In the presence of a

Full text of DOI:10.1016/j.jorganchem.2005.02.001

Journal of Organometallic Chemistry 690 (2005) 4488–4491
www.elsevier.com/locate/jorganchem
Aqueous oxidation of alcohols catalyzed by artificial
metalloenzymes based on the biotin–avidin technology
*
Christophe M. Thomas, Christophe Letondor, Nicolas Humbert, Thomas R. Ward
´ ˆ ˆ
Institut de Chimie, Universite de Neuchatel, Rue Emile-Argand 11, Case Postale 2, CH-2007 Neuchatel, Switzerland
Received 28 September 2004; received in revised form 29 December 2004; accepted 6 January 2005
Available online 24 March 2005
Abstract
Based on the incorporation of biotinylated organometallic catalyst precursors within (strept)avidin, we have developed artificial
metalloenzymes for the oxidation of secondary alcohols using tert-butylhydroperoxide as oxidizing agent. In the presence of avidin
as host protein, the biotinylated aminosulfonamide ruthenium piano stool complex 1 (0.4 mol%) catalyzes the oxidation of sec-phen-
ethyl alcohol at room temperature within 90 h in over 90% yield. Gel electrophoretic analysis of the reaction mixture suggests that
the host protein is not oxidatively degraded during catalysis.
Ó 2005 Elsevier B.V. All rights reserved.
Keywords: Artificial metalloenzymes; Ruthenium; Oxidation; Piano stool complex; Biotin–avidin technology
Homogeneous and enzymatic catalysis are in many
respects complementary [1–4]. By incorporating an
organometallic complex into a host protein, we aim at
creating artificial metalloenzymes with properties remi-
niscent both of enzymes and of homogeneous catalysts.
With this goal in mind, we have recently described the
incorporation of achiral biotinylated rhodium–diphos-
phine complexes into (strept)avidin ((strept)avidin refers
to either avidin or streptavidin, used as a host protein)
[5]. These artificial metalloenzymes were used as
catalysts for the enantioselective hydrogenation of N-
protected dehydroaminoacids (ee up to 96%). The per-
spective of improving the performance of such artificial
metalloenzymes using combined chemical- and genetic
optimization strategies (i.e., chemogenetic) provides
the opportunity to focus on more challenging reactions.
For example, it may be interesting to test the power of
such methodology for the oxidation of alcohols. Along
similar lines, several groups have recently reported the
creation of artificial metalloenzymes for the enantiose-
lective oxidation of sulfides to sulfoxides [6–8].
The oxidation of alcohols to the corresponding alde-
hydes and ketones is one of the most important func-
tional group transformations in organic synthesis.
While alcohol dehydrogenases often perform this task
very efficiently [3,4], mild homogeneous catalysts are
scarce. Traditionally, these reactions have been per-
formed with stoichiometric chromium reagents. How-
ever, todayÕs environmental restrictions render most of
the stoichiometric metal oxidants obsolete. For this pur-
pose, the use of tert-butylhydroperoxide (TBHP) as an
oxidizing agent has been investigated in conjunction with
several homogeneous transition-metal catalysts, includ-
ing chromium, ruthenium, rhodium, and copper [9–13].
The present Communication outlines our efforts to
create active artificial metalloenzymes for the oxidation
of alcohols using biotinylated transition metal com-
plexes in the presence of (strept)avidin as host protein.
In contrast to transition-metal catalyzed hydrogena-
tion reactions (where nearly any rhodium–diphosphine
moiety is an active catalyst), the number of versatile
*
Corresponding author. Tel.: +41 32 718 2516; fax: +41 32 718 2511.
E-mail address: thomas.ward@unine.ch (T.R. Ward).
0022-328X/$ - see front matter Ó 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2005.02.001

C.M. Thomas et al. / Journal of Organometallic Chemistry 690 (2005) 4488–4491
4489
OH
in DMF (31 ll), streptavidin (0.08 lmol of the tetra-
meric protein) in a mixture of water (500 ll) and acetone
(150 ll), acetophenone was obtained with 81% conver-
sion after 90 h at room temperature (Table 1, entry 1).
Extraction of the reaction mixture with pentane al-
lows to isolate the product devoid of any traces of the
hybrid catalyst which remains exclusively in the aqueous
phase. Following this treatment, the aqueous orange-red
solution was subjected to non-denaturing gel-electro-
phoresis. Inspection of the gel suggests that, despite
the presence of a strong oxidizing agent, the host protein
remains tetrameric (Fig. 1). There is no evidence for pro-
tein degradation by oxidative cleavage or denaturation
(to afford monomeric streptavidin). Performing the reac-
tion in the absence of host protein under the same con-
ditions, the reaction is nearly quantitative, but affords a
black residue, suggesting the formation of RuO2, which
is supposed to result from the degradation of an arene-
ruthenium complex by tert-butylhydroperoxide [17].
As control experiments, the catalytic reaction was
carried out in the absence of either the ruthenium cata-
lyst or TBHP. The presence of both compounds was
found to be essential. No oxidized product was detected
in the absence of TBHP (thus excluding an Oppenauer-
type oxidation with acetone acting as a hydrogen accep-
tor [15]), and very low conversions were obtained in the
absence of the ruthenium complex (<1% based on sec-
phenethyl alcohol). Carrying out the reaction in the
presence of air revealed no difference, while substitution
of TBHP with H2O2 afforded no trace of oxidized
product.
O
artificial metalloenzyme
TBHP
(strept)avidin
ηn-CnRn
M
N
L
N
biotin
spacer
artificial metalloenzyme
Scheme 1. Artificial metalloenzymes for the oxidation of sec-phenethyl
alcohol based on biotinylated d6 piano stool complexes.
organometallic catalysts for the mild oxidation of sec-
ondary alcohols in water is limited [14–16]. With no
homogeneous system to build upon, we decided to focus
on d6 piano stool complexes as catalyst precursors as
illustrated in Scheme 1. Biotinylation of the metal was
introduced by means of a chelating ligand, thus ensuring
stability of the coenzyme as well as its quantitative
incorporation within (strept)avidin [5].
Initial oxidation experiments using sec-phenethyl
alcohol as substrate were performed with biotinylated
amino-sulfonamide Ru(II) catalyst 1 (Scheme 2). A
detailed experimental procedure is provided in the Sup-
porting Information. Using streptavidin as a host pro-
tein, we found that, in the presence of 1, the oxidation
proceeded efficiently using tert-butylhydroperoxide
(TBHP) as terminal oxidant. When sec-phenethyl alco-
hol (250.0 equiv., 62.5 lmol) was treated with aqueous
TBHP (300.0 equiv., 75.0 lmol) in the presence of the
ruthenium coenzyme 1 (1.0 equiv., 0.25 lmol) dissolved
While sec-phenethyl alcohol was oxidized to afford
exclusively acetophenone, oxidation of benzyl alcohol
(entry 2) afforded a mixture of benzaldehyde and
+
O
Ru
HN
NH
O
Ru
Cl
N
O
S
H
H
Cl
N
H
O
O
HN
NH
H2N
N
N
S
NH
H
H
O
EtO
O
H
N
S
O
1
2
O
O
S
HN
NH
HN
NH
Ir
Rh
Cl
H
H
H
H
O
O
S
Cl
H2N
H
H
N
S
O
N
N
H2N
N
S
O
O
O
3
4
Scheme 2. Biotinylated organometallic coenzymes tested for the oxidation of alcohols.

4490
C.M. Thomas et al. / Journal of Organometallic Chemistry 690 (2005) 4488–4491
Table 1
Oxidation of alcohols catalyzed by different artificial metalloenzymesa
To optimize the catalytic activity of such metalloen-
zymes, two complementary approaches can be pursued:
chemical modification of the first coordination sphere of
the biotinylated catalyst or genetic modification of the
protein.
Our first attempts focused on the use of a biotinyl-
ated bipyridine Ru(II) catalyst 2 (Table 1, Entry 5).
With this coenzyme, a slight decrease in catalytic
activity was observed (69% conversion, compared to
80% conversion for the sulfonamide Ru(II) complex
1).
We also examined the oxidation of sec-phenethyl
alcohol in the presence of [Rh(g5-C5Me5)(Biot-Ligand)]
3 and [Ir(g5-C5Me5)(Biot-Ligand)] 4. The results dis-
played in Table 1 suggest that the iridium catalyst 4
was more active that its rhodium analog 3. However,
both systems are much less active than the corrrespond-
ing ruthenium-based catalysts 1 and 2: acetophenone
was obtained in 3% yield and 12% yield after 140 h with
catalyst precursors 3 and 4, respectively (Table 1,
Entries 6–7).
Entry Coenzyme Substrate
Host protein Conversion
(%)b
1
1
1
1
1
2
3
4
1
1
1
Phenethyl alcohol Streptavidin
Benzyl alcohol
Cyclohexanol
81
68
80
43
69
3
2
Streptavidin
Streptavidin
3c
4d
5
Phenethyl alcohol Streptavidin
Phenethyl alcohol Streptavidin
Phenethyl alcohol Streptavidin
Phenethyl alcohol Streptavidin
Phenethyl alcohol Ser112Gly
Phenethyl alcohol Pro64Gly
Phenethyl alcohol Avidin
6c
7c
8
12
75
70
92
9
10
a
Unless otherwise stated, the reaction was carried out under nitro-
gen at room temperature for 90 h with alcohol (62.5 lmol), aqueous
TBHP (75.0 lmol), coenzyme (0.25 lmol) dissolved in DMF (31 ll),
protein (0.08 lmol) dissolved in a mixture of water (500 ll) and ace-
tone (100 ll).
b
Determined by GC integration.
The reaction was performed for 140 h.
c
d
The coenzyme was dissolved in DMSO (31 ll).
Having identified the most promising organometallic
fragment (Table 1, Entry 1), we subjected the host pro-
tein to site-directed mutagenesis. Two streptavidin
mutants were tested with the most promising organome-
tallic catalyst precursor 1. Neither Ser112Gly nor
Pro64Gly streptavidin mutants displayed enhanced
activity (Table 1, Entries 8–9). Using avidin as the host
protein, we found that, in the presence of 1, the oxida-
tion of sec-phenethyl alcohol proceeds nearly to comple-
tion (Table 1, Entry 10).
In summary, we have developed active artificial met-
alloenzymes for the oxidation of alcohols in water based
on the non-covalent incorporation of biotinylated d 6
piano stool complexes in (strept)avidin. Having estab-
lished that the protein does not suffer from oxidative
damage despite the harsh reaction conditions, our next
goal is to develop selective artificial metallo-alcohol
dehydrogenases for the kinetic resolution of secondary
alcohols.
Fig. 1. Non-denaturing gel-electrophoresis demonstrating the integrity
of the host protein following a catalytic run. (Lane 1: denatured pure
streptavidin, revealing monomeric protein; Lane 2: non-denatured
pure streptavidin, revealing tetrameric and oligotetrameric protein;
Lane 3: aqueous reaction mixture resulting from a catalytic run,
identical to Lane 2; Lanes 4 and 5: supernatant and precipitate,
respectively, resulting from centrifugation at 16000g of a catalytic run).
benzoic acid in a ratio of 4:1 (68% total conversion
(Table 1, Entry 2)).
With cyclic alcohols such as cyclohexanol (Table 1,
Entry 3), the corresponding ketone was obtained with-
out the formation of ring-opened or lactone products
(80% conversion).
Acknowledgements
Using DMSO instead of DMF to solubilize the cata-
lyst precursor, led to a decrease in conversion (Table 1,
Entry 4). Dimethyl sulfoxide can act as a reductant in
certain aerobic oxidation reactions catalyzed by late
transition metals and dimethyl sulfone is produced dur-
ing the reaction [18]. In contrast, DMSO is also a stoi-
chiometric oxidant in a variety of chemical [19,20] and
biological [21] oxidation reactions, yielding dimethyl
sulfide as a byproduct. However, in our case there is
no evidence for presence of either dimethyl sulfone or
dimethyl sulphide (GC analysis). Presumably DMSO
lowers the oxidation rate by coordinating to the Ru(II)
center.
This work was supported by the Swiss National
Science Foundation and CERC3. We thank Belovo
Egg Science and Technology for a generous gift of egg
white avidin.
Appendix A. Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/
j.jorganchem.2005.02.001.

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