Coupled Oxidation of Heme without Pyridine. Formation of Cyano Complexes of Iron Oxophlorin and 5-Oxaporphyrin (Verdoheme) from Octaethylheme

Article abstract of DOI:10.1021/ic970503e

Oxidative degradation of iron porphyrins under coupled oxidation conditions, which generally has involved pyridine as solvent and source of axial ligands, results in the production of verdoheme, the iron complex of 5-oxaporphyrin in which an oxygen atom replaces a methine group. Under pyridine-free conditions, ClFe^III(OEP) (OEP is the dianion of octaethylporphyrin) and {Fe^III(OEPO)}₂ (OEPO is the trianion of octaethyloxophlorin) have been converted into the 5-oxaporphyrin complex (verdoheme) {(NC)₂Fe^III(OEOP)} (OEOP is the anion of octaethyl-5-oxaporphyrin). Additionally, through control of the cyanide ion concentration, the oxidation of ClFe^III(OEP) can be stopped to produce {Fe^III(OEPO)}₂ or allowed to proceed to give {(NC)₂Fe^III(OEOP)}. A convenient method for the preparation of {Fe^III(OEPO)}₂, and from it the free base H₂OEPOH, is reported. Anionic complexes, [(NC)(py)Fe(OEPO)^- and [(NC)₂Fe(OEPO)]^-, related to these oxidative degradation processes have been obtained through the addition of bis(triphenylphosphine)iminium cyanide to (py)₂Fe(OEPO) in pyridine solution or through cleavage of {Fe^III(OEPO)}₂ with potassium cyanide in methanol or with tetra(n-butyl)ammonium cyanide in chloroform solution. The products have been characterized principally through their characteristic ¹H NMR spectra, which show sizable hyperfine shifts with meso resonances upfield and methylene resonances with both up- and downfield shifts. In the presence of air, [(NC)₂Fe(OEPO)]^2- undergoes oxidation to form the 5-oxaporphyrin complex (verdoheme) {(NC)₂Fe^III(OEOP)}. Treatment of BrFe^III(OEPO) with CN^- produces [(NC)₂Fe(OEPO)]^-, which can also be obtained through oxidation of [(NC)₂Fe(OEPO)]^2- with diiodine.

Full text of DOI:10.1021/ic970503e

3892
Inorg. Chem. 1997, 36, 3892-3897
Coupled Oxidation of Heme without Pyridine. Formation of Cyano Complexes of Iron
Oxophlorin and 5-Oxaporphyrin (Verdoheme) from Octaethylheme
Alan L. Balch,*,† Richard Koerner,† Lechosław Latos-Graz3yn´ski,‡ Jane E. Lewis,†
Tamara N. St. Claire,† and Edward P. Zovinka†
Departments of Chemistry, University of California, Davis, California 95616,
and University of Wrocław, Wrocław, Poland
ReceiVed May 1, 1997X
Oxidative degradation of iron porphyrins under coupled oxidation conditions, which generally has involved pyridine
as solvent and source of axial ligands, results in the production of verdoheme, the iron complex of 5-oxaporphyrin
in which an oxygen atom replaces a methine group. Under pyridine-free conditions, ClFeIII(OEP) (OEP is the
dianion of octaethylporphyrin) and {FeIII(OEPO)}2 (OEPO is the trianion of octaethyloxophlorin) have been
converted into the 5-oxaporphyrin complex (verdoheme) {(NC)2FeIII(OEOP)} (OEOP is the anion of octaethyl-
5-oxaporphyrin). Additionally, through control of the cyanide ion concentration, the oxidation of ClFeIII(OEP)
can be stopped to produce {FeIII(OEPO)}2 or allowed to proceed to give {(NC)2FeIII(OEOP)}. A convenient
method for the preparation of {FeIII(OEPO)}2, and from it the free base H2OEPOH, is reported. Anionic complexes,
[(NC)(py)Fe(OEPO)]- and [(NC)2Fe(OEPO)]2-, related to these oxidative degradation processes have been obtained
through the addition of bis(triphenylphosphine)iminium cyanide to (py)2Fe(OEPO) in pyridine solution or through
cleavage of {FeIII(OEPO)}2 with potassium cyanide in methanol or with tetra(n-butyl)ammonium cyanide in
chloroform solution. The products have been characterized principally through their characteristic 1H NMR spectra,
which show sizable hyperfine shifts with meso resonances upfield and methylene resonances with both up- and
downfield shifts. In the presence of air, [(NC)2Fe(OEPO)]2- undergoes oxidation to form the 5-oxaporphyrin
complex (verdoheme) {(NC)2FeIII(OEOP)}. Treatment of BrFeIII(OEPO•) with CN- produces [(NC)2Fe(OEPO)]-,
which can also be obtained through oxidation of [(NC)2Fe(OEPO)]2- with diiodine.
Introduction
The coupled oxidation process, in which heme degradation
is brought about by dioxygen in the presence of a reducing agent
(ascorbic acid or hydrazine),4,5 has been widely used as a model
for biological heme catabolism.1 This process has generally
involved pyridine as both solvent and source of axial ligands.
In early work, Lemberg found that protoheme underwent
negligible coupled oxidation in the absence of pyridine, but
found that certain proteins when complexed with protoheme
promoted coupled oxidation.6,7 Since proteins, as well as
pyridine, ligate to the iron atom of the heme molecule at one
or both axial positions, binding of such axial ligands may
activate the heme group and render the methine positions
susceptible to oxidative cleavage. We have recently shown that
coupled oxidation of (py)2FeII(OEP) (py, pyridine; OEP, dianion
of octaethylporphyrin) produces 1,8 the iron(III) complex of
Heme degradation is an important process for the disposal
of unwanted heme,1 for neurotransmission (through carbon
monoxide production),2 and for catalysis (where catalyst lifetime
is dependent on its oxidative stability).3 Until recently, however,
the coordination chemistry (axial ligation, metal ion oxidation,
and spin states) of the process of oxidative heme degradation
has received scant attention.
† University of California.
‡ University of Wrocław.
X Abstract published in AdVance ACS Abstracts, July 15, 1997.
(1) (a) Maines, M. D. Heme Oxygenase: Clinical Applications and
Functions. CRC Press Boca Raton, FL, 1992. (b) Bissell, D. M. In
LiVer: Normal Function and Disease. Bile Pigments and Jaundice;
Ostrow, J. D., Ed.; Marcel Decker, Inc.: New York, 1986; Vol. 4, p
133. (c) Brown, S. B. In Bilirubin, Heirwegh, K. P. M., Brown, S. B.,
Eds.; CRC Press, Inc.: Boca Raton, FL, 1982; Vol. 2, p 1. (d) Torpey,
J.; Ortiz de Montellano, P. R. J. Biol. Chem. 1996, 271, 26067. (e)
Hawkins, B. K.; Wilks, A.; Powers, L. S.; Ortiz de Montellano, P. R.
Biochim. Biophys. Acta, Protein Struct. 1996, 1295, 165. (f) Takahashi,
S.; Ishikawa, K.; Takeuchi, N.; Ikeda-Saito, M.; Yoshida, T.; Rousseau,
D. L. J. Am. Chem. Soc. 1995, 117, 6002. (g) Sun, J.; Wilks, A.; Ortiz
de Montellano, P. R.; Loehr, T. M. Biochemistry 1993, 32, 14151.
(h) Takahashi, S.; Wang, J.; Rousseau, D. L.; Ishikawa, K.; Yoshida,
T.; Host, J. R.; Ikeda-Saito, M. J. Biol. Chem. 1994, 269, 1010. (i)
Takahashi, S.; Wang, J.; Rousseau, D. L.; Ishikawa, K.; Yoshida,
Takeuchi, N.; Ikeda-Saito, M. Biochemistry 1994, 33, 5531. (j) Wilks,
A.;Ortiz de Montellano, P. R. J. Biol. Chem. 1993, 268, 22357. (k)
Wilks, A.; Sun, J.; Loehr, T. M.; Ortiz de Montellano, P. R. J. Am.
Chem. Soc. 1995, 117, 2925. (l) Hernandez, G.; Wilks, A.; Paolesse,
R.; Smith, K. M.; Ortiz de Montellano, P. R.; La Mar, G. N.
Biochemistry, 1994, 33, 6631. (m) Schmid, R.; McDonagh, A. F. In
The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1979;
Vol. 6, p 258. (n) O’Carra, P. In Porphyrins and Metalloporphyrins;
Smith, K. M., Ed.; Elsevier: New York, 1975; p 123.
octaethylbilindione (a biliverdin analog) along with the long-
known verdoheme, 2, which is a diamagnetic iron(II) complex
(4) Warburg, O.; Negelein, E. Chem. Ber. 1930, 63, 1816.
(5) Lagarias, J. C. Biochim. Biophys. Acta 1982, 717, 12.
(6) Lemberg, R. Biochem. J. 1935, 29, 1322.
(7) Lemberg, R. ReV. Pure Appl. Chem. 1956, 6, 1.
(8) Balch, A. L.; Latos-Graz˘yn´ski, L.; Noll, B. C.; Olmstead, M. M.;
Safari, N. J. Am. Chem. Soc. 1993, 115, 9056.
(2) Verma, A.; Hirsch, D. J.; Glatt, G. E.; Ronnett, G. V.; Snyder, S. H.
Science 1993, 259, 381.
(3) Traylor, P. S.; Dolphin, D.; Traylor, T. G. J. Chem. Soc., Chem.
Commun. 1984, 279.
S0020-1669(97)00503-X CCC: $14.00 © 1997 American Chemical Society

Coupled Oxidation of Heme without Pyridine
Inorganic Chemistry, Vol. 36, No. 18, 1997 3893
Chart 1
of octaethyl-5-oxaporphyrin.9,10 Additionally, a number of iron
complexes that involve the meso-hydroxyoctaethylporphyrin, H2-
OEPOH (or octaethyloxophlorin), and octaethyl-5-oxaporphyrin,
HOEOP, ligand systems have been thoroughly characterized.11-17
Recent work on the coupled oxidation of cobalt porphyrins
has revealed that successful conversion of CoII(OEP) into
[CoII(OEOP)]+ (OEOP, anion of octaethyl-5-oxaporphyrin) and
Cl2CoIII(OEOP) depends critically on the solvent and its ability
to function as an axial ligand.18,19 Thus, coupled oxidation of
CoII(OEP) does not proceed in pyridine or pyridine/dichlo-
romethane solutions but does work in a mixture of dichlo-
romethane and tetrahydrofuran.
Here we describe some iron porphyrin chemistry relevant to
heme degradation that occurs under pyridine-free conditions.
We have found that iron complexes of octaethylporphyrin
and octaethyloxophlorin air oxidize in the presence of ascorbic
acid and cyanide ion to form either the dimeric complex
11
{FeIII(OEPO)}2 or the octaethyl-5-oxaporphyrin complex
{(NC)2FeIII(OEOP)}16 (verdoheme) under varying reaction
conditions. Some intermediates in this process have also been
detected through the use of paramagnetic NMR studies.
(9) Balch, A. L.; Koerner, R.; Olmstead, M. M. J. Chem. Soc. Chem.
Commun. 1995, 873.
(10) Balch, A. L.; Latos-Graz˘yn´ski, L.; Noll, B. C.; Olmstead, M. M.;
Sz˘terenberg, L.; Safari, N. J. Am. Chem. Soc. 1993, 115, 1422.
(11) Balch, A. L.; Latos-Graz˘yn´ski L.; Noll, B. C.; Olmstead, M. M.;
Zovinka, E. P. Inorg. Chem. 1992, 31, 2248.
(12) Morishima, I.; Fujii, H.; Shiro, Y.; Sano, S. J. Am. Chem. Soc. 1986,
108, 3858.
(13) Balch, A. L.; Noll, B. C.; Reid, S. M.; Zovinka, E. P. Inorg. Chem.
1993, 32, 2610.
Results
Synthetic Studies. The chemical transformations uncovered
in this work are summarized in Chart 1. Four of the speciess
{FeIII(OEPO)}2,11 {(py)2Fe(OEPO)} (OEPO, trianion of octa-
ethyloxophlorin),12-14 {BrFeIII(OEPO•)},15 and {(NC)2FeIII-
(OEOP)}16sshown in the chart have been characterized previ-
1
(14) Morishima, I.; Fujii, H.; Shiro, Y.; Sano, S. Inorg Chem. 1995, 34,
1528.
ously by H NMR and UV/visible spectroscopy, and three of
(15) Balch, A. L.; Latos-Graz˘yn´ski, L.; Noll, B. C.; Szterenberg, L.;
Zovinka, E. P. J. Am. Chem. Soc. 1993, 115, 11846.
(16) Balch, A. L.; Noll, B. C.; Safari, N. Inorg. Chem. 1993, 32, 2901.
(17) Balch, A. L.; Koerner, R.; Latos-Graz˘yn´ski, L.; Noll, B. C. J. Am.
Chem. Soc., 1996, 118, 2760.
(18) Balch, A. L.; Mazzanti, M.; Olmstead, M. M. J. Chem. Soc., Chem.
Commun. 1994, 269.
(19) Balch, A. L.; Mazzanti, M.; St. Claire, T. N.; Olmstead, M. M. Inorg.
Chem. 1995, 34, 2194.

3894 Inorganic Chemistry, Vol. 36, No. 18, 1997
Balch et al.
theses{(py)2Fe(OEPO)},17 {BrFeIII(OEPO•)},15 and {(NC)2FeIII-
(OEOP)}16swere isolated and subjected to single-crystal X-ray
diffraction studies. However, all of the chemical transformations
shown in Chart 1 are new except for the conversions of dimeric
{FeIII(OEPO)}2 into {(py)2Fe(OEPO)}13 and into {BrFeIII-
(OEPO•)},15 which were described earlier.
Addition of ClFeIII(OEP) to a methanolic solution of ascorbic
acid and potassium cyanide leads to oxidative degradation of
the porphyrin ring to form either the oxophlorin complex
{FeIII(OEPO)}2 or the verdoheme {(NC)2FeIII(OEOP)}, depend-
ing upon reaction conditions. When ClFeIII(OEP) is dissolved
in a methanolic solution of ascorbic acid (46 equiv) and
potassium cyanide (10 equiv) and is allowed to stand overnight
under air, dimeric {FeIII(OEPO)}2 precipitates from the solution
and may be collected by filtration. The identity of the precipitate
was determined by comparison of the 1H NMR and UV/visible
spectra with those from an authentic sample.11 This reaction
represents a convenient method of preparation of not only
{FeIII(OEPO)}2 but also of H2OEPOH,20 which may be obtained
by treatment of {FeIII(OEPO)}2 with hydrogen chloride in 92%
yield. We have found that the reaction solution used for the
preparation of {FeIII(OEPO)}2 can be recycled. In a second
use of the same reaction mixture, a 91% yield of {FeIII(OEPO)}2
was obtained. In a third use of the same reaction mixture,
however, the yield dropped to 48%. Adding more ascorbic acid
or more cyanide to the medium does not restore its ability to
oxidize ClFeIII(OEP) to {FeIII(OEPO)}2. It appears that a
product is generated in the reaction mixture that retards the
desired reaction. The nature of the inhibition has been briefly
explored, and it is likely that cyanate formation is involved.
Addition of an equivalent molar amount of potassium cyanate
to a freshly prepared reaction mixture reduces the yield of {FeIII-
(OEPO)}2 to 80%, while addition of a 10-fold excess of
potassium cyanate to the freshly prepared reaction mixture
results in no precipitation of {FeIII(OEPO)}2.
1
Figure 1. The 300 MHz H NMR spectra of pyridine-d5 solutions of
(A) {(py)2Fe(OEPO)}; (B) {(py)2Fe(OEPO)} and 0.5 molar equiv of
bis(triphenylphosphine)iminium cyanide, and (C) {(py)2Fe(OEPO)} and
1.0 molar equiv of bis(triphenylphosphine)iminium cyanide at 25 °C.
Resonances of {(py)2
Fe(OEPO)} are labeled 4 while those of
[(NC)(py)Fe(OEPO)]- are labeled 5 with subscripts m and m' denoting
meso resonances.
5-oxaporphyrin macrocycle via an oxidative process in which
pyridine or another nitrogen donor from a protein is not
involved.
Formation of [(NC)(py)Fe(OEPO)]-. The reaction of
{(py)2Fe(OEPO)} with bis(triphenylphosphine)iminium cyanide
in dioxygen-free pyridine-d5 solution has been monitored by
1H NMR spectroscopy. Relevant data are shown in Figure 1.
Trace A shows the familiar spectrum of {(py)2Fe(OEPO)}12-14
with two, upfield-shifted meso resonances and two of the four
methylene resonances, one with an upfield shift and the other
with a downfield shift. The other two methylene resonances
along with the methyl resonances occur in the 1-10 ppm region.
Trace B shows the spectrum of the solution after the addition
of 0.5 equivalent of cyanide ion. Along with resonances from
{(py)2Fe(OEPO)}, new meso and methylene resonances are
present which are assigned to the formation of [(NC)(py)-
Fe(OEPO)]-. Trace C shows the spectrum of a solution to
which 1 equiv of cyanide ion has been added. The resonances
of the starting material, {(py)2Fe(OEPO)}, are no longer present.
The methylene resonances of the product, [(NC)(py)Fe(OEPO)]-,
that are visible in trace C, appear as diastereotopic pairs.
Addition of up to 10 equiv of cyanide ion to the sample does
not produce further changes in the spectrum. Hence, the
equilibrium constant for the second replacement of pyridine by
cyanide is substantially smaller than that of the first pyridine.
Figure 2 shows a magnitude COSY spectrum that was
obtained from a pyridine solution of [(NC)(py)Fe(OEPO)]-.
Cross peaks are useful for identifying individual ethyl groups
since they are expected between the methylene and methyl
protons with ∼14 Hz coupling for geminal and 7 Hz coupling
for vicinal protons.21 Cross peaks from geminal coupling are
seen in the four pairs of diastereotopic methylene resonances.
Additionally these pairs of resonances have cross peaks that
reveal coupling to the four methyl resonances that occur in the
1-3 ppm region. These spectral results indicate that the
methylene protons in each ethyl group are inequivalent, a
situation that is consistent with the presence of two different
axial ligands, pyridine and cyanide, in the complex.
If the coupled oxidation is run with a higher concentration
of potassium cyanide, then the reaction produces the verdoheme
[(NC)2FeII(OEOP)]- rather than {FeIII(OEPO)}2. Thus, when
the potassium cyanide concentration is increased to 127 equiv
and the reaction solution is stirred vigorously under air
for 3 h, a dark green solution is produced. The UV/visible
spectrum of the reaction solution is similar to that of iron(II)
verdoheme complexes.16 Under these conditions, no precipitate
of {FeIII(OEPO)}2 forms. Upon workup of the reaction solution
in air (see Experimental Section), the oxaporphyrin complex
1
{(NC)2FeIII(OEOP)} is isolated in 48% yield. The H NMR
and UV/visible spectra of the product were identical to those
from an authentic sample.
Treatment of a chloroform solution of {FeIII(OEPO)}2 with
10 molar equiv of bis(triphenylphosphine)iminium cyanide in
air produces a deep blue solution from which the oxaporphyrin
complex {(NC)2FeIII(OEOP)} can be isolated in 95% yield. The
identity of the product has been established by comparison of
its UV/visible and 1H NMR spectra with those of an authentic
sample.16 Previously {(NC)2FeIII(OEOP)} had been prepared
from the verdoheme {(py)2FeII(OEOP)} by oxidation with
dioxygen in the presence of hydrogen chloride to form {Cl2-
FeIII(OEOP)} and subsequent treatment of {Cl2FeIII(OEOP)}
with bis(triphenylphosphine)iminium cyanide.16 The conversion
of ClFeIII(OEP) or {FeIII(OEPO)}2 into {(NC)2FeIII(OEOP)}
described here represents the first example of production of a
(20) For other methods of preparation of meso-hydroxyporphyrins (ox-
ophlorins) see: (a) Barnett, G. H.; Hudson, M. F.; Smith, K. M. J.
Chem. Soc., Perkin Trans. 1 1973, 691. (b) Crossley, M. J.; King, L.
G. Pyke, S. M. Tetrahedron 1987, 43, 4569.
(21) Keating, K. A.; de Ropp, J. S.; La Mar, G. N.; Balch, A. L.; Shiau,
F.-Y.; Smith, K. M. Inorg. Chem. 1991, 30, 3258.

Coupled Oxidation of Heme without Pyridine
Inorganic Chemistry, Vol. 36, No. 18, 1997 3895
Figure 3. The 300 MHz 1H NMR spectrum of a methanol-d4 solution
of [(NC)2Fe(OEPO)]2- that was obtained from treating {FeIII(OEPO)}2
with potassium cyanide.
Table 1. Solvent Dependence of Chemical Shifts for
[(NC)2Fe(OEPO)]2-
cyanide
salt
methylene (ppm)
meso (ppm)
solvent
dimethyl
KCN
12.2 2.6 0.76 -3.4
-65.8 -92.5
-65.0 -90.9
sulfoxide-d6 BPCNa
chloroform-d BPCN
12.1 c
14.4 c
c
c
-3.4
-0.015 -68.1 -90.9
methanol-d4
KCN
10.4 2.5 1.80 -1.2
-86.2 -114.7
-59.6 -79.8
acetonitrile-d3 TBA-CNb 14.6 c
c
-3.2
a Bis(triphenylphosphine)iminium cyanide. b tetra(n-butyl)ammonium
cyanide. c Unable to determine chemical shift.
are observed, the axial ligation on both sides of the macrocycle
is the same. Similarly, {FeIII(OEPO)}2 reacts with a 50-fold
molar excess of bis(triphenylphosphine)iminium cyanide in
dioxygen-free chloroform to form [(NC)2Fe(OEPO)]2-. The 1H
NMR spectrum of the complex anion shows a significant solvent
dependence as summarized in Table 1, but the overall pattern
is similar to that seen in Figure 3. The solvent dependance of
the NMR spectra of related iron(III) porphyrin bis(cyano)
adducts is well-known.22,23 In particular, the large difference
seen for the methanol solution when compared with the other
solvents is readily explained by the presence of hydrogen
bonding between the axial ligands and methanol.
The UV/visible absorption spectrum of a chloroform solution
of [(NC)2Fe(OEPO)]2- is shown in Figure 4. The spectrum
displays a Soret peak at 428 nm and broad absorptions at 634
and 678 nm which resemble corresponding features seen in the
UV/visible spectrum of {(py)2Fe(OEPO)}.12,24
Solutions of [(NC)2Fe(OEPO)]2- are air sensitive. Upon
exposure to air, the UV/visible and 1H NMR spectra of solutions
of [(NC)2Fe(OEPO)]2- show changes that indicate that {(NC)2-
FeIII(OEOP)} is eventually formed.
Figure 2. Magnitude COSY spectra for [(NC)(py)Fe(OEPO)]- in
pyridine-d5 at 23.6 °C. (A) and (B) show the full spectra, while (C)
and (D) focus on the crowded 4-1 ppm region.
Formation of [(NC)2Fe(OEPO)]-. Air-sensitive solutions
of [(NC)2Fe(OEPO)]- can be obtained in two ways: through
oxidation of [(NC)2Fe(OEPO)]2- with diiodine or through
treatment of {BrFeIII(OEPO•)}15 with excess tetra(n-butyl)-
ammonium cyanide. Figure 5 shows the 1H NMR spectrum of
a sample of [(NC)2Fe(OEPO)]- that was obtained through the
Cleavage of {FeIII(OEPO)}2 by Cyanide Ion. In the
presence of a large excess of cyanide ion, {FeIII(OEPO)}2
undergoes cleavage slowly to form air-sensitive [(NC)2Fe-
(OEPO)]2-. Thus, treatment of a dioxygen-free methanol sus-
pension of {FeIII(OEPO)}2 with a 100-fold molar excess of
potassium cyanide results in the gradual dissolution of the
iron complex and the eventual formation of a homogenous
green solution. The 1H NMR spectrum of the resulting solution
is shown in Figure 3. The overall pattern of resonances,
with two upfield-shifted meso resonances and methylene
resonances in the 12 to -2 ppm region, is similar to that of
{(py)2Fe(OEPO)}.12-14 Since only four methylene resonances
(22) La Mar, G. N.; Del Gaudio, J.; Frye, J. S. Biochim. Biophys. Acta
1977, 498, 422.
(23) Walker, F. A.; Simonis, U. Biological Magnetic Resonance. NMR of
Paramagnetic Molecules; Berliner, L. J., Reuben, J., Eds.; Plenum
Press: New York, 1993; Vol. 2, p 133.
(24) Bonnett, R.; Dimsdale, M. J. J. Chem. Soc., Perkin Trans 1 1972,
2540.

3896 Inorganic Chemistry, Vol. 36, No. 18, 1997
Balch et al.
Figure 4. UV/visible spectrum of [(NC)2Fe(OEPO)]2- in dichlo-
romethane solution: λmax, nm (ꢀ, M-1 cm-1; 408 (4.4 × 104), 428 (5.6
× 104), 634 (4.1 × 103), 678 (4.7 × 103).
Figure 6. Chemical shift vs 1/T for the meso resonances of
[(NC)2Fe(OEPO)]- in chloroform-d solution.
cobalt18,19 porphyrins yields a similar set of products, nickel
porphyrin behaves differently. Recently this laboratory reported
that in pyridine solution the nickel(II) complex NiII(OEPOH)
(OEPOH, dianion of meso-hydroxyoctaethylporphyrin), under-
goes aerial oxidation to form the stable radical (py)2NiII(OEPO•),
which subsequently undergoes a slower oxidation at the other
meso positions to yield the dioxoporphyrin complexes 10 and
11.25,26 Further work is clearly needed to elucidate how iron
1
Figure 5. The 300 MHz H NMR spectrum of [(NC)2Fe(OEPO)]-
obtained by treating {BrFeIII(OEPO•)} with 3 equiv of tetra(n-butyl)-
ammonium cyanide in chloroform-d at 25 °C. Resonances of the tetra-
(n-butyl)ammonium ion are identified by the label Bu, while the
methylene and meso resonances of [(NC)2Fe(OEPO)]- are identified
by the labels CH2 and m and m'. The resonance labeled * is from an
impurity.
directs the oxidative reactivity away from the unoxygenated
meso bridges and toward the oxygenated site so that C-C bond
cleavage occurs.
addition of 3 molar equiv of tetra(n-butyl)ammonium cyanide
to a chloroform solution of {BrFeIII(OEPO•)}. The spectrum
shows two resonances at -9.8 and -5 ppm from the meso
protons, four quartets in the 4.5-2.0 ppm region that are due
to the methylene protons, and four resonances in the 2-0 ppm
region that are due to the methyl protons. The overall pattern,
with the slight upfield shift of the meso resonances, is similar
to that observed for [(py)2Fe(OEPO)]+, the one-electron oxida-
tion product of {(py)2Fe(OEPO)}.17 These meso resonances
show a marked, non-Curie dependence on temperature which
is shown in Figure 6. Upon cooling, these meso resonances
shift downfield. The unusual temperature dependence of the
1H NMR spectrum is readily understood if the even-electron
complex, [(NC)2Fe(OEPO)]-, has an S ) 0 ground state with
a nearby S ) 1 state that is significantly populated at 25 °C.
Similar non-Curie behavior has been previously observed in the
1H NMR spectra of the related complex, [(py)2Fe(OEPO)]+.17
The formation of {FeIII(OEPO)}2 during the modified coupled
oxidation process developed here offers evidence that the initial
step in heme oxidation involves oxygenation of a meso carbon.
Although it has been widely assumed that oxidative heme
degradation involves what is effectively hydroxylation at a meso
position, direct evidence in the form of isolation of an
intermediate from the reaction has been lacking. The ability
to stop aerobic conversion of ClFeIII(OEP) to {FeIII(OEPO)}2
(i.e., without further oxidation to from {(NC)2FeIII(OEOP)})
through control of the cyanide concentration, is largely due to
the poor solubility of {FeIII(OEPO)}2 in methanol. Further
oxidation of {FeIII(OEPO)}2 only occurs when a sufficient
concentration of cyanide ion cleaves {FeIII(OEPO)}2 to form
[(NC)2Fe(OEPO)]2-. Additionally, cleavage of {FeIII(OEPO)}2
by cyanide is a slow process, so that once precipitated, this
dimeric complex is effectively protected from further reaction.
However, it is possible to convert {FeIII(OEPO)}2 into the free
ligand, H2OEPOH, by treatment with acid, and this route from
ClFeIII(OEP) to H2OEPOH is a convenient method for the
preparation of H2OEPOH.
Discussion
The results described here show that cyanide ion and pyridine
behave similarly as axial ligands in oxidative heme degradation.
Thus, an axial amine is not the only ligand that can promote
methine bridge oxidation of iron porphyrins.
(25) Balch, A. L.; Noll, B. C.; Phillips, S. L.; Reid, S. M.; Zovinka, E. P.
Inorg. Chem. 1993, 32, 4730.
(26) Balch, A. L.; Olmstead, M. M.; Phillips, S. L. Inorg. Chem. 1993,
32, 3931.
The metal plays a crucial role in directing the site of oxidation
in such complexes. While oxygenation of both iron8-10 and

Coupled Oxidation of Heme without Pyridine
Inorganic Chemistry, Vol. 36, No. 18, 1997 3897
The fact that the conversion of ClFeIII(OEP) or
{FeIII(OEPO)}2 to {(NC)2FeIII(OEOP)} occurs in the presence
of an excess of such a strongly bound ligand as cyanide suggests
that the process does not require coordination of dioxygen by
iron. Rather it is likely that dioxygen or a dioxygen-derived
species (peroxide, superoxide, or hydroxyl radical) reacts directly
at the porphyrin periphery to induce C-C bond cleavage in
the coupled oxidation processes outlined here.
product was collected by filtration, washed with methanol, and air-
dried. Yield: 52.2 mg (0.0432 mmol), 87.7% . The product was
identified by comparison of its 1H NMR spectrum with that of an
authentic sample.
Preparation of {(NC)2FeIII(OEOP)} Directly from ClFeIII(OEP).
ClFeIII(OEP) (113 mg, 0.181 mmol) was added to a solution of ascorbic
acid (1 g, 5.68 mmol) and potassium cyanide (1.5 g, 23.0 mmol) in
200 mL of methanol and stirred vigorously in air for 3 h. The dark
green solution of [FeII(OEOP)(CN)2]- was evaporated to dryness under
reduced pressure, redissolved in 100 mL of dichloromethane, and
washed with water (4 × 200 mL) in the presence of air. The resulting
blue dichloromethane solution was dried over anhydrous sodium sulfate,
filtered, and evaporated to dryness under reduced pressure. The solid
was redissolved in a minimum volume of dichloromethane, and diethyl
ether was slowly added. The recrystallized material was collected by
filtration, washed with diethyl ether (20 mL), water (5 mL), methanol
(10 mL), and diethyl ether (50 mL), and placed under a vacuum.
Yield: 56.4 mg, 48.3%. The identity of the product was confirmed
by comparison of its UV/visible and 1H NMR spectra with those of an
authentic sample of {(NC)2FeIII(OEOP)}.
The new cyano complexes [(NC)(py)Fe(OEPO)]- and
[(NC)2Fe(OEPO)]2- have spectral characteristics that are similar
to those of {(py)2Fe(OEPO)}. The electronic structure of this
compound has received considerable discussion.12-14,17 The
overall oxidation level is consistent with resonance structures
4a, 4b, and 4c for {(py)2Fe(OEPO)}. However, the properties
Preparation of {(NC)2FeIII(OEOP)} from {FeIII(OEPO)}2. Bis-
(triphenylphosphine)iminium cyanide (42 mg, 0.075 mmol) was added
to a solution of 9 mg (0.0075 mmol) of {FeIII(OEPO)}2 in 15 mL of
chloroform. The solution was stirred with exposure to air for 5 min.
The resulting green solution was evaporated to dryness under vacuum.
The colored solid was dissolved in a minimum volume of dichlo-
romethane. This solution was subjected to chromatography on a 2.5
× 5 cm silica gel column with elution with chloroform. The blue band
that eluted was collected and evaporated to dryness. The solid was
purified by recrystallization from dichloromethane/diethyl ether. Yield
9.1 mg, 95%.
of {(py)2Fe(OEPO)} suggest a complex electronic structure with
strong mixing of ligand and metal orbitals that is not readily
explained by any one conventional oxidation and spin state
assignment. Similar considerations also pertain to the electronic
structures of [(NC)(py)Fe(OEPO)]- and [(NC)2Fe(OEPO)]2-
.
Preparation of H2OEPOH from {FeIII(OEPO)}2. A 12.7 mg
(0.0105 mmol) sample of {FeIII(OEPO)}2 was dissolved in 50 mL of
chloroform. Hydrogen chloride was bubbled through the solution for
1 min. The solution was then stirred for 12-18 h. The color of the
reaction solution changed from brown to purple. The solution was
washed with aqueous sodium bicarbonate, and then the organic layer
was separated and dried over sodium sulfate. After decanting the
organic solution and removing the solvent on a rotary evaporator, the
remaining residue was dissolved in a minimum of chloroform and
subjected to chromatography on a silica gel column with chloroform
as the eluant. The blue fraction was collected to yield 10.4 mg (92.4%)
of H2OEPOH. The product was characterized by comparison of the
Additionally [(NC)2Fe(OEPO)]2- and {(py)2Fe(OEPO)} are air
sensitive, and both undergo conversion to oxaporphyrin (ver-
doheme) complexes in the presence of dioxygen.
The one-electron oxidation of [(NC)2Fe(OEPO)]2- parallels
the behavior of {(py)2Fe(OEPO)}, whose redox properties have
been recently described.17 The two oxidation products,
[(NC)2Fe(OEPO)]- and [(py)2Fe(OEPO)]+, share common
physical properties. Both have similar 1H NMR spectral patterns
in which the meso protons have an upfield bias at room
temperature. The temperature dependence of these meso
resonances for both species is non-Curie and indicative of a
thermal equilibrium between a diamagnetic ground state and a
paramagnetic excited state for these even-electron species.
In conclusion, this work demonstrates considerable parallels
in the species present in the coupled oxidation process when
either cyanide ion or pyridine is present as axial ligand. Since
cyanide ion is frequently added to heme proteins to produce
the low spin state of the iron and thus to facilitate their study
by 1H NMR techniques,27 the results reported here have added
relevance to the reactivity that may be seen with such cyanide-
treated proteins.
1
absorption and H NMR spectra to those of an authentic sample.
Instrumentation 1H NMR spectra were recorded on a General
Electric QE-300 FT spectrometer operating in the quadrature mode (1H
frequency is 300 MHz). The spectra of paramagnetic complexes were
collected over a 50-100 kHz bandwidth with 8K complex data points.
For a typical spectrum, between 500 and 2000 transients were
accumulated with a 50 ms delay time. The signal-to-noise ratio was
improved by apodization of the free induction decay. The residual 1H
spectra of chloroform-d or dichloromethane-d2 were used as secondary
references. To obtain unambiguous methyl and methylene assignments
in the diamagnetic region, an inversion recovery sequence was used
with τ values of ∼100 ms.
The MCOSY spectrum21 was obtained after collecting a standard
1D reference spectrum. The 2D spectrum was collected by use of 512
t1 increments that consisted of 400 scans each with a block size of
1024 complex points. A spectral bandwidth of 10 kHz in both
dimensions was utilized as the minimum necessary to observe the
resonances of interest. A preacquisition delay of 150 ms was used.
Prior to the collection of the first block, eight dummy scans were
performed. The data were apodized in both dimensions by an unshifted
squared sine bell function, and zero filled to form a 1K × 1K matrix
prior to Fourier transformation. A magnitude calculation was performed
on the resulting spectrum; this was followed by symmetrization.
Experimental Section
Preparation of Compounds. Authentic samples of {FeIII(OE-
PO)}2,11 {(py)2Fe(OEPO)},13,17 and {BrFeIII(OEPO•)}15 were prepared
as described previously except that {FeIII(OEPO)}2 was recrystallized
from dichloromethane/methanol before use.
Preparation of {FeIII(OEPO)}2. Potassium cyanide (65.5 mg, 1.01
mmol) and ascorbic acid (0.7973 g, 4.53 mmol) were dissolved in 50
mL of methanol. ClFeIII(OEP) (61.4 mg, 0.0984 mmol) was added to
the methanol solution, and the reaction was stirred in air for 12-18 h.
During this time, the product precipitated as a dark black powder. The
Acknowledgment. We thank the National Institutes of
Health (Grant GM26226) for support.
(27) For examples, see: La Mar, G. N.; Chen, Z.; Vyas, K.; McPherson,
A. D. J. Am. Chem. Soc. 1995, 117, 411. Sette, M.; de Ropp, J. S.;
Herna´ndez, G.; La Mar, G. N. J. Am. Chem. Soc. 1993, 115, 5237.
IC970503E