Oxidative decarboxylation of α-hydroxy acids by a functional model of the nonheme iron oxygenase, CloR

Article abstract of DOI:10.1039/b925389k

Iron(II)-α-hydroxy acid complexes of a tripodal N4 ligand undergo oxidative decarboxylation upon exposure to O₂ and mimic the aliphatic C1-C2 cleavage step catalyzed by CloR. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b925389k

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Oxidative decarboxylation of a-hydroxy acids by a functional model
of the nonheme iron oxygenase, CloRw
Tapan Kanti Paine,*a Sayantan Pariaa and Lawrence Que, Jr.*b
Received (in Berkeley, CA, USA) 2nd December 2009, Accepted 25th January 2010
First published as an Advance Article on the web 12th February 2010
DOI: 10.1039/b925389k
Iron(II)–a-hydroxy acid complexes of a tripodal N4 ligand
undergo oxidative decarboxylation upon exposure to O2 and
mimic the aliphatic C1–C2 cleavage step catalyzed by CloR.
The large majority of nonheme iron enzymes that cleave C–C
bonds are catechol dioxygenases, which take advantage of the
electron-rich nature of the aromatic substrates to initiate O2
activation.1 There are now related enzymes that cleave aliphatic
C–C bonds, but a different O2 activation mechanism is
required because of the difference in the substrates.2 A recently
characterized example is 2-hydroxyethylphosphonate (HEP)
dioxygenase (HEPD) that catalyzes the cleavage of the HEP
C1–C2 bond to form hydroxymethylphosphonate and
formate.2,3 The nonheme iron center is bound to a 2-His-1-
carboxylate facial triad4,5 and the substrate binds in a bidentate
manner to the iron via the 2-OH and phosphonate groups.3
Fig. 1 Molecular structure of [(6-Me3-TPA)FeII(mandelate)]+ (1).
All hydrogen atoms except those attached to O3 and the adjacent C23
atom have been omitted for clarity. Selected bond lengths [A] and
angles [1] for 1: Fe1–O1 2.023(2), Fe1–O3 2.178(2), Fe1–N1 2.179(3),
Fe1–N2 2.259(3), Fe1–N3 2.217(3), Fe1–N4 2.287(3), O1–C22
1.273(4), O2–C22 1.243(4), O3–C23 1.431(4); O1–Fe1–O3 76.40(8),
O1–Fe1–N1 166.88(9), O1–Fe1–N3 111.96(9), O3–Fe1–N3 171.00(9).
Another example is CloR, an enzyme involved in the biosynthesis
of clorobiocin,6 an aminocoumarin antibiotic that targets
bacterial DNA gyrase.7,8 Clorobiocin has a 3DMA-4HB
moiety (Scheme 1) that derives from 3DMA-4HPP via two
consecutive oxidative decarboxylation steps catalyzed by
CloR. Of interest to this work is the novel C1–C2 bond
cleavage of 3DMA-4HMA in the second step. By analogy to
HEPD, the mandelate substrate may bind in a bidentate
fashion to the iron to initiate the O2 activation mechanism.
This notion is tested with model complexes in this study.
There are only two reports on the iron(II)–a-hydroxy-
carboxylate complexes,9,10 and no biomimetic iron(II) complexes
that use O2 for the selective oxidative decarboxylation of
mandelic acid to benzoic acid. Thus, to develop an under-
standing of the reaction catalyzed by CloR, we have initiated
the syntheses of a series of iron(II)–a-hydroxy acid complexes
and associated reactivity studies. We report herein the
structure of [(6-Me3-TPA)FeII(mandelate)]+ (1, 6-Me3-TPA =
tris-[(6-methyl-2-pyridyl)methyl]amine) and its reaction with
dioxygen as well as those of other iron(II)–a-hydroxy acid
complexes of the same tetradentate ligand.
The reaction of Fe(ClO4)2Á6H2O, 6-Me3-TPA ligand,
mandelic acid and triethylamine in methanol yields a light
yellow iron(II) complex [(6-Me3-TPA)FeII(mandelate)]+ (1)
with a monoanionic mandelate. The X-ray crystal structure
of the monocationic complex 1 reveals a six-coordinate iron(II)
center with a tetradentate tripodal ligand and a bidentate
mandelate anion (Fig. 1). In the structure Fe1 has a distorted
octahedral environment consisting of the tetradentate ligand
and a bidentate mandelate anion coordinated via O1 and O3.
The O3 oxygen is coordinated as hydroxy form and its
hydrogen atom forms an intermolecular hydrogen bond with
the noncoordinated carboxylate oxygen of a neighboring
molecule. The Fe1–O1 bond distance of 2.023 A is typical of
an Fe(II)–carboxylate interaction, such as those found
in the structures of [(6-Me3-TPA)FeII(benzoate)]+ (2) and
[(6-Me3-TPA)FeII(benzoylformate)]+ (3),11,12 and the C–O
bond lengths of the carboxylate group of the coordinated
Scheme 1 Consecutive reactions catalyzed by bifunctional CloR.
a Department of Inorganic Chemistry, Indian Association for the
Cultivation of Science, 2A&2B Raja S. C. Mullick Road, Jadavpur,
Kolkata-700032, India. E-mail: ictkp@iacs.res.in;
Fax: +91 33-2473-2805; Tel: +91 33-2473-4971
b Department of Chemistry and Center for Metals in Biocatalysis,
University of Minnesota, 207 Pleasant Street SE, Minneapolis,
MN 55455, USA. E-mail: larryque@umn.edu;
Fax: +1 612-624-7029; Tel: +1 612-625-0389
w Electronic supplementary information (ESI) available: Crystallo-
graphic data for 1 in CIF format, experimental and spectroscopic
data. CCDC 701933. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/b925389k
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new features in the downfield region can also be associated
with 2 in the presence of 1 equiv. H2O. Extraction of the
reaction solutions after acid treatment into organic solvent
allows the mandelate-to-benzoate ratio to be determined;
this method shows that mandelate is quantitatively converted
to benzoate over the course of 6 h (Fig. S1, ESIw). Thus 1
converts to 2 upon exposure to O2.
Iron(II) complexes of related a-hydroxy acids also undergo
oxidative decarboxylation upon exposure to O2. Replacement
Fig. 2 ESI-MS (positive ion mode in MeCN) of (a) 1, (b) the solution
after oxidation of 1 with O2 and (c) the solution after oxidation of
1 with 18O2.
of the phenyl group with
a methyl group affords
[(6-Me3-TPA)FeII(lactate)]+ (4) that reacts with O2 to form
[(6-Me3-TPA)FeII(acetate)]+, but the reaction takes almost
twice as long (10 h) as the reaction of 1. The ESI-MS of the
oxidized solution of 4 shows a peak at m/z = 447.2 which is
30 mass units less than the lactate complex indicative of the
formation of the acetate complex (Fig. S2, ESIw). In addition
the 1H NMR peaks of the oxidized solution of 4 do match
with those in independently synthesized acetate complex
(Fig. S3, ESIw). The increase in the reaction time upon
replacement of the mandelate a-CH bond with the stronger
lactate a-CH bond suggests that the cleavage of the a-CH
bond must partially contribute to the rate determining step.
The conversion of mandelate to benzoate is a 4eÀ-oxidation.
To ascertain the incorporation of dioxygen in the oxidative
decarboxylation reaction, the oxygenation reaction of 1 was
carried out in an 18O2 atmosphere in dry acetonitrile. The
resulting benzoate product was analyzed by ESI-MS and this
clearly showed the incorporation (495%) of one 18O atom
into the carboxyl group of the product, as demonstrated by the
molecular ion peak at m/z = 511.2 (Fig. 2c). The other 18O
atom involved in this step is most likely converted to water,
which remains bound to the iron(II) center of the product and
affects the chemical shift observed for the a-CH3 resonances of
the 6-Me3-TPA ligand in 2. Thus the conversion of 1 to 2
models the second oxidation reaction catalyzed by CloR.
Some insight into the mechanism of the oxidative decarboxyl-
ation of 1 can be gleaned from a comparison of the
susceptibility of three [FeII(6-Me3-TPA)(X)] complexes to
dioxygen, where X is mandelate (1), benzoate (2), and benzoyl-
formate (3). Interestingly, 2 is air stable,12 suggesting that the
O2 affinity of the iron(II) center is quite low. On the other hand,
3 reacted with O2 over a period of 4 days resulting in the
oxidative decarboxylation of the coordinated benzoylformate
to give 2 in almost quantitative yield.11 Complex 3 differs from 2
in having an a-keto group that binds to the iron(II) center. It
seems unlikely that the introduction of this additional ligand
would be sufficient to increase significantly the O2 affinity of the
iron(II) center in 3 relative to 2. However what the a-keto group
provides is an electrophilic carbonyl group to trap the nascent
nucleophilic superoxide that forms upon O2 binding to the
iron(II) center, which drives the reaction forward and irreversibly
towards oxidative decarboxylation. Remarkably, 1 reacted
even faster with dioxygen, producing the same oxidatively
decarboxylated product 2 over a period of only 6 h, a factor of
20-fold faster than 3. The coordination of an alcohol as the
sixth ligand in this complex, which is a better Lewis base than
the corresponding carbonyl oxygens in 2 and 3, may introduce
sufficient electron density into the iron(II) center to increase its
affinity for O2. The nascent superoxide would then have to
mandelate clearly indicate charge delocalization of the
carboxylate. The longer Fe1–O3 distance of 2.178 A is
associated with the 2-OH ligand and consistent with a neutral
hydroxyl oxygen comparable to the Fe–O distance found in
[(TPA)FeII(MeOH)2]2+ 13
A shorter Fe–O bond distance
.
(1.970 A) for a negatively charged enolate oxygen is observed
for an iron(II) complex with the same tripodal ligand.14
The ESI-MS spectrum of 1 in acetonitrile shows a dominant
signal at m/z = 539.2 with the expected isotope distribution
pattern indicating a monocationic complex with composition
[(6-Me3-TPA)FeII(mandelate)]+ in solution (Fig. 2a).
Complex 1 reacts with pure dioxygen in acetonitrile at ambient
temperature over a period of 6 h. Monitoring the reaction by
ESI-MS shows the time-dependent disappearance of the
m/z = 539.2 peak concomitant with the appearance of a peak
at m/z = 509.2 (Fig. 2b), with an isotope distribution pattern
indicative of a mononuclear monocationic complex with an m/z
value that matches that of [(6-Me3-TPA)FeII(benzoate)]+ (2).
The transformation of 1 to 2 can also be monitored by
1H NMR. The a-CH3 resonances of the 6-Me3-TPA ligand in
1 appear as a broad paramagnetically shifted peak at À45 ppm
due to the presence of four unpaired electrons on the high-spin
iron(II) center (Fig. 3a). Upon exposure to O2, the broad peak
slowly diminishes in intensity and is replaced by a sharper
resonance at À43 ppm (Fig. 3b–d). We associate this latter
peak with the a-CH3 resonances of independently prepared 212
in CD3CN but only after the addition of 1 equiv. H2O. The
Fig. 3 1H NMR spectral changes of complex 1 in CD3CN (a) during
the reaction with O2 at 25 1C after 2 h (b), 4 h (c) and 6 h (d) at 25 1C.
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(a) coupling between the a-Cꢁ radical and HOOꢁ or (b)
electronic reorganization to generate an FeII(OOH)(a-ketoacid)
complex followed by nucleophilic attack of HOOÀ on the
a-keto group. In either pathway, the resulting peroxy adduct
would then spontaneously undergo oxidative decarboxylation.
In summary, we have characterized two iron(II) complexes
of a-hydroxy acids that undergo oxidative decarboxylation
upon exposure to O2. An iron(III)–superoxo species is
implicated in the reaction mechanism. These model complexes
thus serve as functional mimics of the nonheme iron enzyme
CloR and, by extension, HEPD, both of which oxidatively
cleave aliphatic C–C bonds and highlight the versatility of
nonheme iron in catalyzing various oxidations in biology.
This work was supported by the U.S. National Institutes of
Health (GM-33162 to L.Q.) and the DST, India (Project
SR/S1/IC-10/2006 to T.K.P.). S.P. thanks CSIR, India, for a
fellowship. We thank Dr Victor G. Young, Jr., William W.
Brennessel, and Benjamin Kucera of the University of
Minnesota X-ray Crystallographic Laboratory for determining
the crystal structure of 1.
Scheme 2 Proposed mechanism for the oxidative decarboxylation of
a-hydroxy acids.
initiate the oxidative decarboxylation reaction by abstracting
the hydrogen atom from the a-CH bond of the substrate
(Scheme 2). In support, we observed that the oxidative
decarboxylation of 1 required 10 h for completion when
mandelate-2-d1 was used in place of mandelate, showing
C–H bond cleavage is partially rate determining.
Further evidence for the crucial first step was obtained by
the use of TEMPOH as an intercepting agent. Previously,
TEMPOH was utilized effectively as a hydrogen atom donor
by Karlin and co-workers to convert a structurally characterized
copper(II)–superoxo complex to form a reactive copper(II)–
hydroperoxo complex.15 Thus, when 1 was oxygenated in the
presence of 1 equiv. TEMPOH, TEMPO radical was produced
in 8% yield relative to iron, as determined by integration of its
characteristic g = 2 EPR signal (Fig. S4, ESIw). Furthermore
NMR analysis of the organic products showed the presence of
residual mandelate with a mandelate-to-benzoate ratio of 1 : 9
(Fig. S5, ESIw). Increasing the amount of added TEMPOH
increased the amount of residual mandelate, changing the
mandelate-to-benzoate ratio to 1 : 1 for 10 equiv. TEMPOH
and 9 : 1 for 20 equiv. TEMPOH (Fig. S6, ESIw). The
observed interception by TEMPOH suggests that the initially
formed Fe–O2 adduct has iron(III)–superoxide character and
cleaves the weak O–H bond of TEMPOH in an intermolecular
reaction that is in competition with intramolecular H-atom
abstraction from the a-CH bond of mandelate.
In the C–C bond cleavage reaction catalyzed by HEPD,
Whitteck and co-workers have proposed the abstraction of the
C2–H atom from HEP by an iron(III)–superoxide species
followed by hydroperoxylation and peroxo rearrangement
to convert substrate into products.2 On the basis of our
experimental observations, we propose an analogous mechanism
in Scheme 2 where the nascent superoxide abstracts the a-C–H
atom of the a-hydroxyacid to initiate the oxidative decarboxyl-
ation reaction. In the next step, C–O bond formation between
the a-carbon and the dioxygen-derived moiety occurs either by
Notes and references
1 J. D. Lipscomb and A. M. Orville, in Metal Ions in
Biological Systems, ed. H. Sigel and A. Sigel, Marcel Dekker,
New York, 1992, vol. 28, pp. 243–298.
2 J. T. Whitteck, R. M. Cicchillo and W. A. van der Donk, J. Am.
Chem. Soc., 2009, 131, 16225–16232.
3 R. M. Cicchillo, H. Zhang, J. A. V. Blodgett, J. T. Whitteck, G. Li,
S. K. Nair, W. A. van der Donk and W. W. Metcalf, Nature, 2009,
459, 871–874.
4 E. L. Hegg and L. Que, Jr., Eur. J. Biochem., 1997, 250, 625–629.
5 K. D. Koehntop, J. P. Emerson and L. Que, Jr., J. Biol. Inorg.
Chem., 2005, 10, 87–93.
6 F. Pojer, R. Kahlich, B. Kammerer, S.-M. Li and L. Heide, J. Biol.
Chem., 2003, 278, 30661–30668.
7 S. C. Kampranis, N. A. Gormley, R. Tranter, G. Orphanides and
A. Maxwell, Biochemistry, 1999, 38, 1967–1976.
8 A. Maxwell and D. M. Lawson, Curr. Top. Med. Chem., 2003, 3,
283–303.
9 R. Carballo, B. Covelo, E. M. Vazquez-Lopez, E. Garcıa-Martınez,
´ ´ ´ ´
A. Castineiras and C. Janiak, Z. Anorg. Allg. Chem., 2005, 631,
2006–2010.
10 A. Beghidja, G. Rogez, P. Rabu, R. Welter and M. Drillon,
J. Mater. Chem., 2006, 16, 2715–2728.
11 Y.-M. Chiou and L. Que, Jr., J. Am. Chem. Soc., 1995, 117,
3999–4013.
12 J. Kim, Y. Zang, M. Costas, R. G. Harrison, E. C. Wilkinson and
L. Que, Jr., J. Biol. Inorg. Chem., 2001, 6, 275–284.
13 A. Diebold and K. S. Hagen, Inorg. Chem., 1998, 37, 215–223.
14 T. K. Paine, H. Zheng and L. Que, Jr., Inorg. Chem., 2005, 44,
474–476.
15 D. Maiti, D.-H. Lee, K. Gaoutchenova, C. Wurtele,
¨
M. C. Holthausen, A. A. N. Sarjeant, J. Sundermeyer,
S. Schindler and K. D. Karlin, Angew. Chem., Int. Ed., 2008, 47,
82–85.
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