A trispyrazolylborato iron malonato complex as a functional model for the acetylacetone dioxygenase

Article abstract of DOI:10.1002/anie.200802955

(Chemical Equation Presented) Position available: A pentacoordinate iron(II) complex that binds and activates dioxygen shows dioxygenase activity and cleaves diethyl phenylmalonate, in analogy to acetylacetone dioxygenase (see scheme). The mechanism for the model compound allows interesting hypotheses to be made about the enzyme function.

Full text of DOI:10.1002/anie.200802955

Angewandte
Chemie
DOI: 10.1002/anie.200802955
Biomimetic O Activation
2
A Trispyrazolylborato Iron Malonato Complex as a Functional Model
for the Acetylacetone Dioxygenase**
Inke Siewert and Christian Limberg*
Dedicated to Professor Bernt Krebs on the occasion of his 70th birthday
Acetylacetone dioxygenase (Dke1) is an enzyme that can be
isolated from Acinetobacter johnsonii, and it catalyses the
degradation of acetylacetone, which is toxic to various
mammals and to marine creatures and organisms; to achieve
[1]
this process it consumes one equivalent of dioxygen. Dke1 is
also capable of cleaving a whole series of other b diketones
and b ketoesters that are derived from acetylacetone by
substitution at the 1, 3, or 5 position to yield the correspond-
ing carboxylic acids and a-ketoaldehydes. A 1,3-carbonyl
structural motif is decisive for the activity; thus, for example,
related 1-keto-3-hydroxo compounds are not converted.
Dke1 has been investigated with the aid of single crystal X-
ray diffraction and fluorescence and UV/Vis spectroscopy,
and from the results obtained its active site is assumed to
contain iron(II) coordinated by three histidine residues and
Scheme 1. Proposed mechanism for the cleavage of b-dicarbonyl com-
pounds promoted by acetylacetone dioxygenase.
[
4]
On the other hand, it has been shown that a substitution of
2
+
2+
2+
iron(II) by various other metal ions, such as Zn , Co , Mn ,
2
+
2+
[2b]
Cu , or Ni , is accompanied by a loss in activity.
Model compounds can provide valuable support in
answering mechanistic questions arising for the reactions of
[5]
active sites in metalloenzymes. a-Keto acid-dependent non-
[2]
water.
heme iron enzymes may serve as an example. In these
II
The substrate cleavage mechanism is however still largely
unsettled. After preliminary studies based on isotopic label-
ing experiments, an initial deprotonation of acetylacetone
followed by attack of dioxygen or superoxide at the Ca
postion has been suggested. This mechanism would lead to
an organoperoxide unit, which, after nucleophilic attack of its
terminal oxygen atom at the carbonyl carbon atom, should
decompose via a dioxetane species to give the cleavage
proteins (similarly to the acteylacetone dioxygenase), an Fe
center coordinated by three amino acid residues (two
histidines and one asparagine) binds a cofactor through two
oxygen donors for a subsequent oxidation with O . Model
2
complexes containing the Tp ligand (Tp = hydridotrispyrazol-
1-ylborato) have contributed significantly to understanding
the protein function. After precoordination of a bidentate
II
substrate analogue to a {TpFe } unit, one coordination site
[3]
products. In the course of further investigations, it was
proposed that the role of the metal is restricted to breaking
the spin forbiddance in the reaction of triplet dioxygen with
the singlet substrate (by interaction of the HOMOs involved),
so that the organoperoxo species can be generated in a
remains available, as is the case in the enzyme, and it was
shown that dioxygen is activated at the free site for an attack
[
6]
at the cofactor. Herein we investigate whether a similar
scenario is conceivable for acetylacetone dioxygenase with
[
7]
the aid of {TpFe} complexes.
[
4]
iPr2
concerted step (Scheme 1). Thus, a step which plays a
decisive role in conversions of many oxygenating heme and
Kitajiama et al. prepared [Tp Fe(acac)] (acac = acetyl-
iPr2
acetonato, Tp = hydridotris(3,5-ipropylpyrazol-1-yl)bor-
non-heme iron enzymes, the binding of dioxygen at the
ato) without any biomimetic motivation. On exposure to
air, this compound decomposes within one week to yield a
trinuclear iron(III) complex containing m-oxobis(m-acetato)
and m-hydroxobis(m-acetato) ligand constellations between
III
iron(II) center under formation of a Fe -O C entity, has not
2
been considered.
[
10]
the metal centers. The bridging acetato ligands no doubt
have their origin in the acetylacetonato ligands employed,
and the question arises as to whether these acetato ligands
were generated by a dioxygenating cleavage according to the
enzyme reactivity. We therefore synthesized an analogue in
which the isopropyl groups at the ligand are replaced by
[
*] I. Siewert, Prof. C. Limberg
Humboldt-Universität zu Berlin, Institut für Chemie
Brook-Taylor-Strasse 2, 10829 Berlin (Germany)
Fax: (+49)30-2093-6966
E-mail: christian.limberg@chemie.hu-berlin.de
Homepage: http://www.chemie.hu-berlin.de/aglimberg/
[6a]
methyl groups to achieve a higher reactivity. This complex,
[
**] We are grateful to the Fonds der Chemischen Industrie, the BMBF,
and the Humboldt-Universität zu Berlin for financial support. We
thank P. Neubauer and Dr. B. Ziemer for crystal structure analyses
and the Cluster of Excellence “Unifying Concepts in Catalysis” funded
by the DFG for the helpful discussions.
[
Tp*Fe(acac)],
Tp* = hydridotris(3,5-dimethylpyrazol-1-
yl)borato, was then treated with dry dioxygen. The observed
color change from yellow to brown hinted at the formation of
iron(III) compounds, and subsequent ESI-MS studies
+
+
detected the species [FeTp* ] , [Tp*Fe(acac)] , and
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200802955.
2
+
[(Tp*Fe) O(acac)] , that is, the acetylacetonato ligand had
2
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7953

Communications
remained intact and cleavage products could not be detected
see the Supporting Information). Iron complexes containing
(
acetato ligands could only be detected when water was added
to the reaction mixture, so it can be assumed that the acetato
ligands of the previous studies are at least partly due to the
water present under aerobic conditions (hydrolysis of the
[
11]
acetylacetonato ligand following oxidation of the complex).
However, water is clearly not involved in the cleavage of
acetylacetone by Dke1, since in a hydrolytic degradation
acetone would accompany acetate as a product and not—as
observed—pyruvaldehyde. Thus the system [TpFe(acac)]/O2/
H O is not an adequate model.
2
Accordingly, modeling of the b-diketone cleavage with
dioxygen requires a more reactive substrate ligand. Bearing in
mind that it is desirable to detect the 1,2-dicarbonyl product
which is potentially sensitive towards further oxidation, this
ligand should be substituted at least at the Ca position.
Considering that b ketoesters are also cleaved by Dke1, we
chose diethyl malonate with a phenyl substituent in the Ca
position (HPhmal) as a model substrate. This choice was
further inspired by our recent observation for a derivative
that, in the presence of iron(II) without polydentate co-
Scheme 3. Conceivable mechanism for the oxidative cleavage of diethyl
phenylmalonate.
[
8]
ligands, the malonate unit undergoes unselective oxidation ).
The oxidation products expected for cleavage by analogy to
À
Dke1, namely EtOCO2 and ethyl benzoylformate, can
neither be oxidized any further nor enolized for a subsequent
cleavage reaction (Scheme 2).
Scheme 2. Expected products resulting from dioxygenation of diethyl
phenylmalonate according to the reactivity of Dke1.
Figure 1. Molecular structure of 1. All hydrogen atoms were omitted
for clarity. Selected bond lengths [Š] and angles [8]: Fe1–
O1=2.0876(8), Fe1–O2=1.9868(9), Fe1–N1=2.0968(11), Fe1–
N3=2.0906(11), Fe1–N5=2.1678(10), O2–C20=1.2695(14), O1–
C16=1.2444(15), C16–C19=1.4188(17), C19–C20=1.4026(16); C16-
C19-C20=118.74(11), O2-Fe1-N3=142.34(4), O1-Fe1-N5=177.13(4),
C16-C19-C23-C24=71.99(16).
The synthesis of complex [Tp*Fe(Phmal)] (1; Scheme 3)
was thus pursued and finally achieved by treatment of KTp*
and LiPhmal dissolved in acetonitrile with FeCl . After
2
workup, analytically pure 1 was obtained in 51% yield.
Compound 1 was crystallized by slow cooling of a saturated
solution of 1 in hexane. The result of a single crystal X-ray
[12]
diffraction study is given in Figure 1.
The iron(II) ion is located in a distorted trigonal
the function of Dke1 (see Scheme 2). The second product,
[13]
À
bipyramidal coordination sphere (t = 0.58)
that is com-
EtOCO2 , appears to decompose during the reaction to give
posed of the three nitrogen atoms of the Tp* ligand and the
oxygen atoms of the substrate. As expected, a vacant
coordination site is available at the iron(II) center for the
complexation and activation of dioxygen.
ethanolate and carbon dioxide; the latter could be detected by
IR spectroscopy. To obtain further evidence for dioxygenase
reactivity, additional experiments were carried out in which
1
8
compound 1 was treated with O-enriched dioxygen (95%).
After treating of an acetonitrile solution of 1 with dry
dioxygen at room temperature, workup with 0.5 mL of 3m
HCl, followed by filtration employing a silica gel column and
acetonitrile as eluent, led to a solution containing the HPhmal
cleavage products. A GC-MS (CI) analysis revealed only one
Subsequent GC-MS analysis revealed the incorporation of
1
8
one O atom into the a keto ester with an efficiency of
[15]
94%. Incorporation of the second oxygen atom into carbon
dioxide was confirmed by IR spectroscopy; the n absorption
as
À1
18
16
[16]
band shifted by about 16 cm , as expected for OC O.
1
13
1
peak (m/z = 179.1) which, as confirmed by H and C NMR
The reaction was also monitored by H NMR spectrosco-
[14]
spectroscopy,
corresponds to ethyl benzoylformate. As
py. If a solution of 1 in CD CN is exposed to dioxygen, the
3
outlined above, ethyl benzoylformate is one of the two
paramagnetically shifted signals of 1 disappear within 2 h and
products expected for dioxygenase activity of 1 analogous to
signals corresponding to ethyl benzoylformate and two sets of
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Angew. Chem. Int. Ed. 2008, 47, 7953 –7956

Angewandte
Chemie
signals for ethanolate species appear in their place. Further
signals could not be detected, so the fate of the {Tp*Fe}
fragment was initially unclear. However, within a few hours,
be in accordance with the results of kinetic studies performed
with the enzyme.
[
4]
crystals of [Tp* Fe] precipitated from the solution. The
2
formation of this species requires the generation of a Tp*- Experimental Section
free iron compound, which then carries the ethanolate
ligands.
All manipulations were carried out in a glove box, or by Schlenk
techniques involving the use of a dry argon atmosphere. Solvents
[
17]
[18]
were purified, dried, and degassed prior to use. Me PzH, KTp*,
2
Next, the question of whether malonate cleavage requires
the activation of dioxygen at the iron(II) center was
addressed. LiPhmal was reacted with dioxygen. After
workup in an analogous manner to the reaction of 1 with
dioxygen, products from cleavage could not be identified, so
that the C=C cleavage appears to require more than just a
[
19]
and lithium diisopropylamide (LDA) were prepared according to
literature procedures. O-enriched dioxygen (95%) was purchased
18
from Chemotrade. The lithium salt of diethyl phenylmalonate was
prepared by stirring 1 equivalent of LDA with diethyl phenylmalo-
nate in THF.
1
: FeCl (376 mg, 2.97 mmol) was added to a solution of lithium
2
diethyl phenylmalonate (720 mg, 2.97 mmol) and of KTp* (1.00 g) in
acetonitrile (50 mL). The reaction mixture was stirred overnight and a
Lewis acidic metal center. Moreover, 1 was oxidized with
NOPF , and the resulting iron(III) compound was treated
6
small quantity of [Tp* Fe] was then filtered off. The filtrate was
2
with dioxygen. Again, the usual workup did not lead to the
detection of cleavage products by GC/MS analysis, so we
assume that, in contrast to the mechanism proposed for the
enzyme, the reaction of 1 takes place by activation of
dioxygen at the iron(II) center to give an iron(III)–super-
oxido species. The terminal oxygen atom then attacks at the
electrophilic carbonyl carbon atom of the Phmal ligand,
leading to an iron-organoperoxido unit (Scheme 3), as
similarly suggested for model complexes for a-keto acid-
evaporated to dryness, and the resulting white residue was washed
twice with hexane (20 mL). Thereafter 1 was extracted three times
with hexane (60 mL), and all volatiles were removed from the
combined solutions in vacuo. The resulting white powder was washed
twice with hexane (15 mL) to give analytically pure 1 (904 mg, 51%
yield, 1.54 mmol). Crystals of 1 suitable for single crystal X-ray
diffraction studies were obtained by cooling a saturated solution of 1
1
in hexane. H NMR (CD CN, 258C) d = À13.66 (4H, CH ), À2.03
3
2
(
6H, CH ), 5.93 (1H, CH ), 6.03 (9H, Pz-CH ), 6.96 (2H, CH ),
3 Ar 3 Ar
8
.46 (2H, CHAr), 12.25 (9H, Pz-CH ), 56.59 ppm (3H, 4H-Pz). IR:
3
[6]
dependent iron enzymes. Subsequently, either a dioxetane
species is formed, decomposing by cycloreversion to give the
cleavage products, or OÀO bond cleavage (homolytic or
n˜ = 2984 (w), 2928 (w), 2523 (s), 1624 (vs), 1599 (m), 1543 (m), 1450
(s), 1410 (s), 1379 (s), 1333 (s), 1313 (s), 1271 (vw), 1198 (m), 1011 (w),
À1
8
06 (m), 789 cm
(m). Elemental analysis (%) calcd for
À1
C H BFeN O (588.23 gmol ): C 57.17, H 6.34, N 14.29; found:
2
8
37
6
4
heterolytic) occurs. The latter would have to proceed with the
concomitant formation of a high-valent iron oxido species,
C 56.72, H 6.25, N 14.20. UV/Vis (MeCN) l = 260 nm; c = 1.05 ”
m
À2
3
À1
10
cm mol , meff = 5.00 m (m = 4.90 m ).
B so B
which should oxygenate the C atom. Attack of the terminal
a
oxygen atom of the iron superoxido species directly at the
nucleophilic Ca atom seems unlikely. The fact that, after
Received: June 20, 2008
Published online: September 3, 2008
oxidation with NOPF and subsequent reaction with dioxy-
6
gen, cleavage was not observed also excludes a mechanism
involving intramolecular electron transfer and formation of a
substrate-centered radical by analogy to the intradiol-cleav-
Keywords: diketones · iron · models · oxygen · oxygenases
.
[6c–f]
ing catechol dioxygenases.
Scheme 3 clearly shows that all four oxidation equivalents
of dioxygen are compensated exclusively by the ligand, such
that after the cleavage, the iron center has the oxidation state
[
1] G. Straganz, L. Brecker, H.-J. Weber, W. Steiner, D. W. Ribbons,
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Egger, G. Aquino, S. D’Auria, B. Nidezky, J. Mol. Catal. B 2006,
+
II again. Consequently, catalytic conversions should be
possible, and to test this hypothesis a mixture of PhmalLi and
(5 mol%) was treated with dioxygen. Workup and analysis
1
39, 171 – 178.
revealed selective conversion into ethyl benzoylformate,
carbon dioxide, and ethoxide as before; now, however, the
[
3] G. D. Straganz, H. Hofer, W. Steiner, B. Nidetzky, J. Am. Chem.
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À1
reaction occurs catalytically with a TOF of 55 h .
[4] G. D. Straganz, B. Nidetzky, J. Am. Chem. Soc. 2005, 127, 12306 –
2314.
[
1
Thus, compound 1 is an excellent model for the active site
of acetylacetone dioxygenase, as it meets three criteria:
5] Concepts and Models in Bioinorganic Chemistry (Eds.: H.-B.
Kraatz, N. Metzler-Nolte), Wiley-VCH, Weinheim, 2006.
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1
) structural similarity: the Tp* ligand mimics the (His)3
[
coordination sphere of the iron(II) center, and diethyl
phenylmalonate is a suitable substrate analogue for b diket-
onates and b ketoesterates; 2) the function is also simulated:
upon contact with dioxygen, ethyl benzoylformate and carbon
dioxide are selectively formed, and the dioxygenase activity
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1
8
was confirmed by O experiments; and 3) as in the case of
2
104, 939 – 986.
the enzyme, the model also acts catalytically.
[
7] Hints to the viability can be derived from previous investigations
in which diethyl malonate derivates coordinated to iron(II)
centers were unselectively oxygenated and cleaved by dioxy-
Consequently, the dioxygen activation mechanism
deduced for the model compound 1 is an interesting new
hypothesis for the enzyme function, especially as it would also
[
8]
gen, and from work by Que et al., in which the reaction of an
Angew. Chem. Int. Ed. 2008, 47, 7953 –7956
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7955

Communications
a keto acid–iron(II) compound with dioxygen led to partial CÀC
hydrogen atoms were added geometrically and refined by using
a riding model. CCDC-692202 contains the supplementary
crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
[
9]
bond cleavage adjacent to the carbonyl group.
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[
[
2
4
6
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073 – 6081.
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[
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1
6
18
11] Early work by Pearson et al. showed that deprotonated acetyl-
acetone and methylacetylacetone are cleaved by hydroxide ions
to give acetic acid and acetone/acetic acid and butanone,
respectively. This reaction is initiated by the formal addition of
water to the double bond of the enol unit, and subsequent CÀC
[15] To exclude an O/ O exchange with water during workup, the
reaction mixture was treated with neat H PO instead of 3m
3
4
HCl. It had been confirmed beforehand that this modification of
the workup procedure has no influence on the result of the
product analysis.
À
bond cleavage leads to CH C(O)CH . R. G. Pearson, E. A.
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2545 – 2549.
2
3
Mayerle, J. Am. Chem. Soc. 1951, 73, 926 – 930.
[
12] X-ray structure analysis of 1: C H BFeN O , M = 588.30,
2
8
37
6
4
r
monoclinic,
P2
1
/c,
a = 9.8480(4),
b = 19.0788(8),
c =
3
1
1
6.2832(6) Š, b = 101.146(3)8, V= 3001.7(2) Š , Z = 4, T=
00(2) K, F000 = 1240, m = 0.545 mm , V= 2.55–29.008, reflec-
À1
tions collected 30965, independent reflections 7909 (Rint
=
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[20] G. M. shelxs-97, Program for Crystal Structure Solution,
University of Gꢀttingen 1997.
0
.0373), GoF = 0.992, R = 0.0297, wR = 0.0709, largest diffrac-
1
2
À3
tion peak/hole 0.348/À0.300 eŠ . The crystal was mounted on a
glass fiber and then transferred into the cold nitrogen gas stream
of the diffractometer (Stoe IPDS2N). MoKa radiation, l =
0
.71073 Š. The structure was solved by direct methods
[21] G. M. shelxl-97, Sheldrick, Program for Crystal Structure
Refinement, University of Gꢀttingen 1997.
[
20]
2
[21]
(
SHELXS-97), refined versus F (shelxl-97) with aniso-
tropic temperature factors for all non-hydrogen atoms. All
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