1. Synthesis and Characterization of Iron(III) and Iron(II) Complexes to Model Mononuclear and
Binuclear Nonheme Oxygenase Active Sites
Ramya L. Prathuri, Paul Tarves, Josh McNally, John P. Caradonna
Boston University Department of Chemistry, Boston MA 02215
MNOs Introduction Non-heme iron BNOs
are observed in a similar fashion, by • A specific “dioxygen activating” binuclear non-heme oxygenase, methane monooxygenase
• Mononuclear non-heme oxygenases (MNOs) catalyze the decomposition of aromatic oxygenases, or iron core enzymes, synthetically reproducing the dimer active (MMO), has been researched due to its broad “substrate specificity.”4,10 The enzyme can
compounds, several of them pollutants, in the environment.9 catalyze an assortment of reactions by sites.7 The syntheses a few MNO model
catalyze the oxidation of several alkanes, alkenes, ethers, and aromatic compounds among
other substrate groups.4
• It is for their key role in “metabolically important reactions” in organisms that the study of inserting a dioxygen molecule (O2) into complexes are discussed below. The
these enzymes is greatly pursued.5 • Through the examination of MMO’s properties, a reaction mechanism for the enzyme has
inactivated substrates.6 These abundant creation of a ferric complex with similar been proposed and largely accepted (figure A, below).2 The reactive species Q, is currently
• To understand the chemistry of MNO active sites, model complexes have been thought to be the definitive step in the mechanism that catalyzes the oxidative cleavage
synthesized to mimic the chemistry of these elaborate enzymes, providing several insights enzymes can be subdivided into two chemistry and structure as the MNO process.7 The structure of Q has been proposed as the “diamond core” shown below.2
into the MNOs properties.
categories: mononuclear and binuclear 1DLM is presently pursued.9 Attempts to • Using the kinetic studies of a synthesized phenolate model compound,
nonheme oxygenases (MNOs and BNOs). characterize the binuclear oxygenase
FeII2(H2HBamb)2(NMI)2 (figure B), an alternative structure of intermediate Q has been
suggested as a possibility (figure C; intermediates are steps 2 and 3).7
Intradiol Dioxygenase (1DLM)
Models of specific MNOs have been MMO have begun recently and • By creating models of an MMO active site with enzymatic properties, some characteristic
synthesized to imitate enzyme chemistry.5 preliminary research is shown.8 information regarding the mechanism of the enzyme, specifically, the reactive intermediate,
The reaction mechanisms of certain BNOs can be discovered.8
Synthesis and Characterization Characterizing Intermediate Q Figure A (right): The accepted
reaction mechanism for MMO.2
O
O O O O O
• A ferric complex has been synthesized to model the chemistry of Intradiol Dioxygenase, or 1DLM (pictured above). The synthesis and characterization of the ligand and both ferric and ferrous complexes are described.
O
FeII DMANO
FeII FeII FeIV
The ferrous complex was created for comparative purposes and to observe trends in the reactivity of phenolate ligated complexes. Binding with catechol was attempted using the ferric complex and the results are included. • Efforts are currently directed towards successfully O O O k1 = 5x103 s-1 M-1
O O O Figure C (left): Proposed reaction mechanism
using the phenolate complex model. DMANO
characterizing intermediates 2 and 3 from figure C.7 1 S u b stra te O x id a tio n 2 functions as the oxygen atom donor (OAD). The
Various methods including Mössbauer, EPR, X-ray k2 = 0.2 s-1
proposed FeIV=O intermediate 2 undergoes a
Absorption, and Resonance Raman spectroscopies ligand reorganization and becomes 3, which
N2O2-ph Ligand H NMR of N2O2-ph Ligand:
1
will be used to identify the intermediates. Raman
spectroscopy will ideally detect the distinct vibration
O
FeIII
O O O
FeIII
O
O
FeII
O O
O
FeIV
O
has been shown to be capable of hydroxylating
cyclohexane to cyclohexanol in 300 turnovers.6
1
H NMR (D20, 400 MHz) 7.172 (t, 2H), 6.945 (d, 2H), 6.848 (d, 2H), 6.828 (t, frequency of the FeIV=O present in both O O O O k3 = 0.003 s-1
O O O O
2H), 3.703 (s, 4H), 2.671 (s, 4H), 2.287 (s, 6H). intermediates (Figure D). However, Raman study
Yield: 32% 4 3
The slight detection of signal around 7.0ppm indicates that a small amount of results using FeII2(H2HBamb)2(NMI)2 show
reactant still remains in the dissolved ligand solution. This reactant may most
likely be 2-hydroxybenzaldehyde which was detected before the NMR was considerable interference from the phenolate–FeIV NMI
taken and a silicon dioxide column was run to purify the ligand. The amount bond vibrational frequency. The desired FeIV=O O O O O O
of aldehyde remaining was insignificant and did not affect the syntheses of Figure B (right): The synthesis of the phenolate FeII(NMI)2(MeOH)2Cl2
the complexes. frequency lies at the edge of the phenolate complex with which kinetic studies were carried out,
NH HN FeII FeII
frequency, creating an overlap of the two bands
O O O
13
C NMR of N2O2-ph Ligand: leading to the proposed FeIV=O reactive O- -O
intermediate. NMI
13
C NMR (D2O, 400MHz) 157.99, 129.14, (Figure E). To use resonance Raman effectively, a H2HBamb
FeII(H2HBamb)2(NMI)2
128.75, 121.81, 119.39, 116.43, 62.01, new alkoxy ligand, with t-Butoxy groups instead of
54.287, 41.99.
phenolates has been synthesized (Figure F) and
complex formation using this new ligand set is an O O O O
[Fe (N2O2-ph)Cl2] Ferric Complex
immediate goal.
III Mass Spec. UV-vis
NH HN NH HN
• Currently, efforts are focused Figure F (left): The new t-butoxy ligand will eliminate the
interference and overlap of frequencies in Raman studies
around obtaining X-ray caused by the phenolate ligand.
(MeOH, Neg. mode) (MeOH, graph shown on left)
OH HO OH HO
diffraction quality crystal
structures.
m/z = 424.0 λmax = 277nm (3,800)
• The creation of an oxygen
[Na][Fe (N2O2-ph)Cl2]
III (calculated = 424.04) 320nm (1,700) active complex –catechol
• Two alkoxy ligand syntheses were carried out in order to allow for the synthesis of alkoxy complexes for Raman
Figure E (right): The band around 438nm
represents the overlap of the FeIV=O and
FeIV–phenolate charge transfer bands. To
active site is also being
505nm (1,400) studies. The alkoxy ligands L4alk and L6alk shown below were successfully synthesized and attempts to eliminate this interference, alkoxy
Yield: 26% pursued.
synthesize their respective ferric complexes are discussed as well. Although ferric complexes with the alkoxy
complexes are to be used to perform
Raman studies. λ = 438nm
• A byproduct of rust was removed via vacuum filtration.
ligands are inert, they will provide essential structural information about the produced complex, for crystal growing
has been observed to be more productive using ferric complexes. The aim of these initial experiments is to
• Hexane was added to acetone-complex solution, placed in a freezer, and the resulting precipitate was collected. determine whether the synthesized alkoxy complexes are monomers or the desired dimers.
• Complex was a purple powder. • The synthesis of the compound was carried out in an N atmosphere glove box. The product should, theoretically,
2
be highly reactive with dioxygen because the introduction of O2 to the enzyme the complex is modeling begins the
FeIIIN2O2-ph Concentration = 0.22mmol
catalytic process. However, upon the addition of catechol in the N2 atmosphere, the purple complex solution became
green without O2 involved. When left out of the box in contact with oxygen overnight, no further reaction took place,
L4 and L6 Alkoxy Ligands L6 Alkoxy Complex 5
O O1 O 2O • The figure to the
7
O
and the solution remained green. • A UV-vis spectrum before and after the catechol- O O O
Attempted Synthesis of L6alk Complex: FeIII FeIII left represents the
1750 complex came into contact with oxygen confirms the HO O O O3 4 O O target “complex”
2 + THF NH HN
+ 2 which is our goal.
1500 poor reactivity of the catecholate adduct. However, a TEA
H H
O O
6 8
Extinction Coefficient
OH NH 2 NH2
FeIII Complex + Catechol comparison of other UV-vis spectra before and after L6alk • The deprotonated hydroxide groups on the
1250
FeIII Complex
catechol was added provides proof that the catechol • The L6alk precipitate consisted of off white solid particles.
NH HN CH3COO ligand should bind with iron at places 1,2,3
1000 did bind to the complex. OH HO Yield: 99% diethylether and 4 while the double bonded oxygen atoms
• This is the first time L6alk ligand has been synthesized in this FeCl3 "Complex"
750 lab. should coordinate with iron at sites 5,6,7 and
500
•
The peak around 350 nm lessens significantly, 8.
almost disappearing after catechol is added, • Initial characterization of the produced
OH HO
250 representing the supposed loss of LMCT bands from O
O O
compound has not revealed whether the
the chloride ion(s) functioning as labile ligands. The
[Na] [Fe (N2O2-ph)Cl2] + Catechol
HO O
• The reaction was conducted in a acetone-ether mixed solvent. Both were complex is a monomer or dimer. However,
NH HN
III 0
330 380 430 480 530 580 630 680 730 780 830 loss of the chloride bands might indicate that the
2 +
H 2N NH 2
THF
TEA
+ 2 H H
used in a 1:1 ratio. electrochemical analysis has confirmed that
OH L4alk
Wavelength (nm)
catechol bound to the two open sites deserted binding with iron did occur. Currently,
• Upon addition of ferric chloride solution to the ligand solution, the
by the labile chloride ions. The phenolate LMCT band around 435nm also decreases after the addition of catechol, • The L4alk precipitate was a fine white powder. resulting solution turned milky brown in color. various combinations of solvents are being
OH HO Yield: 85%
• This model mimics intradiol-cleavage – the complex binds to the suggesting that the phenolate may have disassociated along with only one of the chloride ions, providing the two tested in order to grow X-ray diffraction
• The precipitate was a light brown powder.
catechol, activating the cleavage of the catechol C–C bond. A dioxygen coordination sites for the catechol. There is no evidence acquired yet to verify the true structure of complex-catechol quality crystals of the compound.
molecule is inserted, successfully catalyzing the initial step of created.
decomposition.9
References
• The catechol occupies two
[Fe (N2O2-ph)] Ferrous Complex
II •
A carboxylate ligand set was initially used to prepare
model complexes of MNOs. When a phenolate ligand set
was first proposed, ferric as well as ferrous complexes
N2O2
N O
N2O2-ph Conclusion The N2O2-ph ligand has been successfully
(1) Costas, M; Mehn, M.P.; Jensen, M.P.; Que, L. Chem. Rev. 2004, 104,
939-986.
(2) Wallar, B.J.; Libscomb. J.D. Chem. Rev. 1996, 96, 2625-2657.
of the six coordination sites were synthesized. The development of such “families” of iron Complex HO OH
HO N synthesized, characterized, and utilized in creating iron complexes. The ferric and ferrous (3) Bruijnincx, P.C.A.; Lutz, M.; Spek, A.L.; Hagen, W.R.; Koten, G.;
Gebbink, R.J.M.K. Inorganic Chemistry 2007, 46 (20), 8391-8402.
when bound to the ferric
complexes are used to observe trends in the chemistry and reactivity O N N OH complexes were also characterized, and the former was observed to be structurally similar (4) Stassinopoulos, A.; Caradonna J.P. Journal of the American Chemical
center.9
[Fe (N2O2-ph)]
II
such electrochemical trends. The chart to the right compares the to 1DLM. Electrochemical data for the ferric and ferrous phenolate complexes compared to
Society 2009, 112 (19), 7071-7073.
(5) Velusamy, M.; Mayilmurugan, R.; Palaniandavar, M. Inorganic
reduction potentials of previously used carboxylate complexes to E1/2 = -15 mV E1/2 = -270 mV that of carboxylate ligated complexes shows that phenolate complexes are more reactive Chemistry 2004, 43 (20), 6284-6293.
(6) Foster, T.L.; Caradonna J.P. Journal of the American Chemical Society
• This catecholate adduct appeared to be Yield: 73% those of newly developed phenolate complexes. The lower the
potential, the more reactive the complex is with dioxygen. An
Fe II
(ΔEp = 140 mV) (ΔEp = 95 mV)
with dioxygen, a notable quality in MNOs decompose aromatics. The ferric–catechol
compound was observed to be inert in oxygen.
2003, 125 (13), 3678-3679.
(7) Rowe, G.T.; Rybak-Akimova, E.V.; Caradonna, J.P. Inorganic
Chemistry 2007, 46 (25), 10594-10606.
stable upon exposure to O2.
• The entire reaction was carried out in an N2 atmosphere.
increased reactivity is desired in order to fulfill the catalytic E1/2 = -10 mV E1/2 = -270 mV Regarding BNOs, alkoxy ligands have been successfully synthesized and characterized (8) Mukerjee, S.; Stassinopoulos, A.; Caradonna J.P. Journal of the
American Chemical Society 1997, 119 (34), 8097-8098.
• The enzyme mechanism proposes loss of requirement in modeling the actual enzyme. Both phenolate and syntheses of alkoxy complexes are being tested. The L6-FeIII complex obtained is (9) Wang, C.H.; Lu, J.W.; Wei, H.H; Takeda, M. Inorganica Chimica Acta
Tyrosine residue to initiate chemistry. • The produced complex was an off-white, gray color. complexes are significantly more reactive with dioxygen than the
FeIII (ΔEp = 155 mV) (ΔEp = 110 mV) 2007, 360, 2944-2952.
being used to grow crystals to determine whether the alkoxy ligands bind to iron to create (10) Cappillino, P.J.; Tarves, P.C.; Rowe, G.T.; Lewis, A.J.; Harvey, M.;
• This complex was charge neutral (therefore a mass spec was not taken). carboxylate complexes, making the former group better models of dimers. Rogge, C.; Stassinopoulos, A.; Lo, W.; Armstrong, W.H.; Caradonna, J.P.
Inorganica Chimica Acta 2008.
intradiol-cleaving MNOs.