Synthesis and photochemical properties of a novel iron-sulfur-nitrosyl cluster derivatized with the pendant chromophore protoporphyrin IX

Article abstract of DOI:10.1021/ic049459a

The novel Roussin red-salt ester (PPIX-RSE) with a pendant porphyrin chromophore was prepared and investigated as a precursor for the photochemical generation of nitric oxide. PPIX-RSE has the general formula Fe ₂(NO)₄{(μ-S,μ-S')P} (where (S,S')P is the bis(2-thiolatoethyl) diester of protoporphyrin IX. The photoexcitation of PPIX-RSE with 436- or 546-nm light in an aerated chloroform solution led to the photodecomposition of the cluster with the respective quantum yields (5.2 ± 0.7) × 10^-4 and (2.5 ± 0.5 × 10 ^-4) and the concomitant release of NO. PPIX-RSE is a significantly more effective NO generator at longer wavelength excitation than are other Fe₂(μ-SR)₂(NO)₄ esters for which R is a simple alkyl group such as CH₃CH₂- because of the much higher absorptivity of the pendant PPIX chromophore at these wavelengths and a modestly higher quantum yield. Fluorescence intensity and lifetime data indicate that the photoexcited porphyrin of PPIX-RSE is largely quenched by the energy transfer to the Fe₂S₂(NO)₄ cluster's core. However, a small fraction of this emission is not quenched, and it is proposed that PPIX-RSE may exist in solution as two conformers.

Full text of DOI:10.1021/ic049459a

Inorg. Chem. 2004, 43, 5543−5549
Synthesis and Photochemical Properties of a Novel Iron−Sulfur−Nitrosyl
1
Cluster Derivatized with the Pendant Chromophore Protoporphyrin IX
,†
Christa L. Conrado,† Stephen Wecksler,† Christian Egler,†,2 Douglas Magde,‡ and Peter C. Ford*
Department of Chemistry & Biochemistry, UniVersity of California,
Santa Barbara, California 93106-9510, and Department of Chemistry & Biochemistry,
UniVersity of California, San Diego, California 92093-0332
Received April 27, 2004
The novel Roussin red-salt ester (PPIX-RSE) with a pendant porphyrin chromophore was prepared and investigated
as a precursor for the photochemical generation of nitric oxide. PPIX-RSE has the general formula Fe2(NO)4{(µ-
S,µ-S′)P} (where (S,S′)P is the bis(2-thiolatoethyl) diester of protoporphyrin IX. The photoexcitation of PPIX-RSE
with 436- or 546-nm light in an aerated chloroform solution led to the photodecomposition of the cluster with the
respective quantum yields (5.2 ± 0.7) × 10-4 and (2.5 ± 0.5 × 10-4) and the concomitant release of NO. PPIX-
RSE is a significantly more effective NO generator at longer wavelength excitation than are other Fe2(µ-SR)2(NO)4
esters for which R is a simple alkyl group such as CH CH − because of the much higher absorptivity of the
3
2
pendant PPIX chromophore at these wavelengths and a modestly higher quantum yield. Fluorescence intensity
and lifetime data indicate that the photoexcited porphyrin of PPIX-RSE is largely quenched by the energy transfer
to the Fe2S2(NO)4 cluster’s core. However, a small fraction of this emission is not quenched, and it is proposed that
PPIX-RSE may exist in solution as two conformers.
Introduction
in treating solid tumors. In these contexts, ongoing studies
in this laboratory have been concerned with the photochemi-
cally activated delivery of NO to specific physiological
targets on demand.5 Earlier work here demonstrated that the
iron-sulfur-nitrosyl cluster anions Fe4S3(NO)-7 (“Rous-
sin’s black salt” anion, RBS) and Fe2(µ-S2)(NO)24- (“Rous-
sin’s red salt” dianion, RRS) release NO with moderate
quantum yields upon photolysis.6 It was shown that visible-
light irradiation of V79 Chinese hamster cell cultures treated
first with Roussin’s red salt enhanced the cell-killing
effectiveness of γ-radiation, and it was concluded that the
NO released photochemically is the sensitizer responsible
for this effect. However, RRS does not have the high optical
absorptivity at longer wavelengths desirable for a photore-
active material to be used in mammalian tissues. This led to
an interest in developing derivatives of RRS with substituents
that incorporate chromophores having desirable spectra and
other tunable properties.
Nitric oxide (nitrogen monoxide) is well known to play
important roles in mammalian biology as a bioregulatory
molecule and as a toxic agent formed in immune response
to pathogen invasion.3 Furthermore, NO has also been shown
to sensitize hypoxic cells to ionizing radiation,4 and thus it
may serve to enhance the cell-killing effect of γ-radiation
* Author to whom correspondence should be addressed. E-mail: ford@
chem.ucsb.edu.
† University of California, Santa Barbara.
‡ University of California, San Diego.
(1) Taken in part from the Ph.D. Dissertation of C. L. Conrado, University
of California, Santa Barbara, 2002.
(2) Undergraduate student from Westfaelische Wilhelms-Universitaet,
Muenster, Germany.
(3) (a) Ignarro, L. J., Ed. Nitric Oxide: Biology and Pathobiology;
Academic Press: San Diego, CA, 2000. (b) Fang, F. C., Ed. Nitric
Oxide and Infection; Kluwer Academic/Plenum Publishers: New York,
1999. (c) Wink, D. A.; Hanbauer, I.; Grisham, M. B.; Laval, F.; Nims,
R. W.; Laval, J.; Cook, J.; Pacelli, R.; Liebmann, J.; Krishna, M.;
Ford, P. C.; Mitchell, J. B. Curr. Top. Cell. Regul. 1996, 34, 159
(4) (a) Howard-Flanders, P. Nature (London) 1957, 180, 1191. (b) Russo,
A.; Mitchell, J. B.; Dinkelstein, E.; DeGraff, W. G.; Spiro, I. J.;
Gamson, J. Radiat. Res. 1985, 103, 232-239. (c) Mitchell , J. B.;
Wink, D. A.; DeGraff, W.; Gamson, J.; Keefer, L. K.; Krishna, M. C.
Cancer Res. 1993, 53, 5845. (d) Chung, P.; Cook, T.; Liu, K.;
Vodovotz, Y.; Zamora, R.; Finkelstein, S.; Billiar, T.; Blumberg, D.
Nitric Oxide 2003, 8, 119-126
(5) Ford, P. C.; Bourassa, J.; Miranda, K.; Lee, B.; Lorkovic, I.; Boggs,
S.; Kudo, S. Laverman, L. Coord. Chem. ReV. 1998, 171, 185.
(6) (a) Bourassa, J.; DeGraff, W.; Kudo, S.; Wink, D. A.; Mitchell, J.;
Ford, P. C. J. Am. Chem. Soc. 1997, 119, 2853. (b) Bourassa, J.; Ford,
P. C. Coord. Chem. ReV. 2000, 200, 887-900.
10.1021/ic049459a CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/04/2004
Inorganic Chemistry, Vol. 43, No. 18, 2004 5543

Conrado et al.
SCH2C6H5)2(NO)4 (II), were prepared as described previously.12
The red esters are moderately air- and light-sensitive and require
storage in the dark under an inert atmosphere.
Roussin’s red salt esters (RSE), Fe2(µ-SR)2(NO)4, can be
prepared from the reaction of RRS with an alkyl halide (eq
1).7
Instruments. UV-vis absorption spectra were recorded for
solutions in 1-cm quartz cells using an HP8572 diode array
spectrophotometer. Infrared spectra were obtained in an IR cell with
CaF2 windows using a BioRad FTS 60 SPC 3200 FTIR spectro-
meter. Solution NO was analyzed electrochemically with a Harvard
Apparatus nitric oxide analyzer.
Emission Studies. Emission studies were carried out on a SPEX
Fluorolog spectrofluorimeter. The samples were dissolved in
chloroform or tetrahydrofuran under aerated conditions such that
the absorbance values were approximately unity at the excitation
wavelength of 404 nm. The emission spectra were fluorescence
scanned from 420 to 750 nm. Deaerating the solutions had little
effect on the emission spectra or intensities.
Continuous wave (CW) photolysis studies in this laboratory
have demonstrated that RSE compounds release NO upon
irradiation (λirr ) 365 nm) in aerated solutions and that ∼4
mol of NO is generated for each mole of RSE that undergoes
photoconversion.8,9 Thus, the RSE complexes would appear
to be promising candidates for photochemical NO precursors;
however, when R is a simple alkyl group, these compounds
are only weak absorbers at the longer visible-range wave-
lengths. To address this issue, one can envision utilizing eq
1 to construct new esters where R- is a chromophore with
a much larger absorptivity in the visible region. Described
here are the synthesis and photochemical/physical properties
of the supramolecular complex PPIX-RSE consisting of the
RSE chromophore covalently linked to a red-absorbing
antenna derived from protoporphyrin IX (PPIX), a strongly
absorbing chromophore at longer wavelengths.10 The pho-
tochemical properties of this species will be compared with
those of related compounds. Additional interest in PPIX
systems is derived from the possibility that porphyrin
derivatives may have biological specificity in terms of
localizing in certain tissues.11
Fluorescence decays were characterized by time-correlated
single-photon counting (TCSPC) at UC San Diego. A neodymium:
vanadate laser at 530 nm (Coherent Verdi) pumped a custom-built
titanium:sapphire laser that generated femtosecond mode-locked
pulses by self-phase locking. Harmonic doubling provided excitation
pulses near 400 nm. A portion of the beam was picked off and
sent to a photodiode to provide “stop” pulses. Emission from
solutions in ordinary luminescence cells was collected and sent
through a half-meter monochromator (Spex 1870) to a microchan-
nelplate photomultiplier (Hamamatsu 1564U-01). After amplifica-
tion (Philips 774), the pulses were recognized by a constant-fraction
discriminator (Tennelec TC454) to provide “start” signals to the
time analyzer (Canberra 2044). The stop pulses came from the
photodiode through a separate discriminator (EGG-Ortec 934). On
this occasion, pulse selection was not used. Electronic gating13 was
employed to avoid a data pile-up at the time analyzer. The histogram
of the delay times between fluorescence and excitation was collected
by a multichannel pulse-height analyzer (Norland 5300) and
transferred to a microcomputer for processing. The instrument
response function to instantaneous emission was measured using a
colloidal suspension. Deconvolution was carried out using iterative
reconvolution within a least-squares routine based on the Marquardt
method14 with an in-house program incorporating insights from
Grinvald and Steinberg.15 It also accommodates an infinite sequence
of excitation pulses producing decays longer than the repetition
period. For exponential decays, this involves summing a simple
geometric series for each fit component. A general introduction to
the methodology was provided previously;16 however, the internal
reference described in that report was not used with the current
laser system.
Experimental Section
Materials. PPIX and its dimethyl ester PPIX-DME were
purchased from Aldrich. The water for the spectroscopic studies
was purified by a Millipore system. Methanol was distilled under
nitrogen from I2 and Mg. THF was distilled under N2 over sodium
and benzophenone. Chromatography-grade argon was used as
received from Air Liquide. All inert-atmosphere manipulations were
performed on a vacuum manifold. Deaeration of the solutions was
achieved by three freeze-pump-thaw (f-p-t) cycles. Nitric oxide
(99.0%) was purchased from Matheson and was passed through a
stainless steel column filled with Ascarite II to remove higher
nitrogen oxides. Stainless steel tubing and fittings were used, and
manifold connections were stainless steel-to-glass fittings. Roussin’s
red ester compounds, Fe2(µ-SCH2CH3)2(NO)4 (I) and Fe2(µ-
Photochemical Experiments. Continuous-wave photolysis stud-
ies were performed using an optical train with a 200-W Hg arc
lamp as the light source. Interference filters were used to isolate
appropriate excitation wavelengths. Light intensities were deter-
mined using ferric oxalate or Reinecke’s salt (>436 nm) as chemical
actinometers. All solutions were stirred continuously and protected
from extraneous light. Known concentrations of the samples were
prepared and irradiated under aerated conditions. The light intensity
for the photolysis experiments ranged from 1.0 × 10-6 to 3.0 ×
(7) (a) Complexes of the structure Fe2(SR)2(NO)4 have been described
as esters in earlier literature. (b) Butler, A. R.; Glidewell, C.; Hyde,
A. R.; McGinnis, J.; Seymore, J. S. Polyhedron 1993, 2, 1045-1052.
(b) Butler, A. R.; Glidewell, C.; Johnson, I. L. Polyhedron 1987, 6,
2091-2094. (c) Glidewell, C.; Lambert, R. J. J. Chem. Soc., Dalton
Tans. 1990, 2685-2690.
(8) Conrado, C. L.; Bourassa, J. L.; Egler, C.; Wecksler, S.; Ford, P. C.
Inorg. Chem. 2003, 42, 2288-2293.
(9) Bourassa, J. L. Photochemical Studies of Iron-Sulfur-Nitrosyls:
Roussin’s Salts and Related Compounds. Ph.D. Dissertation, University
of California, Santa Barbara, CA, 1998.
10-6 Ein L-1 s-1
.
(12) (a) Seyferth, D.; Gallagher, M. K.; Cowie, M. Organometallics 1986,
5, 539-548. (b) Glidewell, C.; Lambert, R. J.; Harman, M. E.;
Hursthouse, M. B. J. Chem. Soc., Dalton Trans. 1990, 2685-2690.
(13) Laws, W. R.; Potter, D. W.; Sutherland, J. C. ReV. Sci. Instrum. 1984,
55, 1564
(10) PPIX ) 7,12-diethenyl-3,8,13,17-tetramethyl-2,18-porphine-dipropi-
onic acid.
(11) (a) Anreoni, A., Cubeddu, R., Eds. Porphyrins in Tumor Therapy;
Plenum Press: New York, 1984. (b) Phillips, D. Pure Appl. Chem.
1995, 67, 117-126.
(14) Marquardt, D. W. J. Soc. Ind. Appl. Math. 1963, 11, 431-441.
(15) Grinvald, A.; Steinberg, I. Z. Anal. Biochem. 1974, 59, 583-598.
(16) Magde, D.; Campbell, B. F. SPIE Proceedings 1989, 1054, 61-68.
5544 Inorganic Chemistry, Vol. 43, No. 18, 2004

NoWel Iron-Sulfur-Nitrosyl Cluster
Syntheses. Protoporphyrin IX Bis-(2-iodoethyl) diester) (PPIX-
I). A round-bottom flask equipped with a stir bar was charged with
protoporphyrin IX (0.5643 g; 1.00 mmol) and ∼50 mL of dry,
distilled THF. After dissolution, 1-hydroxybenzotriazole (0.271 g;
2.00 mmol) and 4-(dimethylamino)pyridine (DMAP) (∼0.024 g;
0.1 mmol) were added. The solution was cooled in an ice bath to
0 °C, and 1,3-dicyclocarbodiimide (DCC) (0.413 g; 2.00 mmol)
was added slowly to the reaction flask. The mixture was stirred in
an ice bath for approximately 10 min before 2-iodoethanol (0.18
mL; 2.006 mmol) was added dropwise via syringe. This solution
was stirred in an ice bath for ∼3 h and then was allowed to come
to room temperature and stirring was continued overnight. The
mixture was filtered, and the residue was washed with THF to
dissolve any remaining product of the DCC coupling reaction. The
solvent was removed in vacuo leaving a solid that was washed with
cold chloroform (using gravity filtration). The chloroform dissolves
the PPIX-I product and leaves the urea byproduct as a solid. The
chloroform was removed under vacuum. The material was purified
further using column chromatography (silica gel/CH2Cl2). The
product was collected as a purple solid with a 50% yield after
chromatography (some material sticks to the column) and was
for C38H40N8O8Fe2S2: C, 50.0; H, 4.39; N, 12.3; Fe, 12.3. Found:
C, 49.8; H, 4.53; N, 10.2. The carbon and hydrogen values agree
well with the expected values, but the nitrogen result is low.
However, it should be noted that low N values are a common
problem we have encountered in the analysis of such non-heme
iron nitrosyl complexes, even though the values for other elements
were very close to theoretical values. The iron content in the same
sample was determined using a Shimadzu AA 6200 atomic
absorption flame emission spectrophotometer with iron calibration
curves generated with dried 99.9% Fe2O3 (Aldrich) digested in hot
nitric acid and diluted in Nanopure water to appropriate concentra-
tions (5-75 ppm). The Fe content was found to be 12.7 ((0.7)%
(average of four individual samples), in very good agreement with
the theoretical value.
Results and Discussion
Preparation and Properties of PPIX-RSE. The goals
of the present study were to prepare a supramolecular
compound combining the NO carrying function of Roussin’s
red salt Fe-S-NO cluster with an antenna PPIX chro-
mophore having a strong absorption in the red and to study
the photochemical properties of this material. The synthesis
of PPIX-RSE from PPIX and Roussin’s red salt is outlined
in Scheme 1. A key step is the functionalization of PPIX to
bis(2-iodoethyl) diester using a DCC coupling reaction. The
reaction of the resulting PPIX-I with Roussin’s red salt anion
displaced both iodides to give PPIX-RSE, a relatively stable
reddish-purple solid. Although X-ray quality crystals have
not yet been obtained, the elemental analysis and high-
resolution MS data have confirmed the molecular composi-
tion of PPIX-RSE, and the NMR, IR, and UV-vis spectra
were fully consistent with the formulation represented to be
the Roussin salt ester product of Scheme 1.
The electronic spectrum of PPIX-RSE is dominated by
the porphyrin chromophore and is qualitatively and quanti-
tatively very similar to that of PPIX. This is illustrated by
Figure 1, which displays the spectra of both PPIX-RSE and
PPIX in THF solutions. The lower section of Figure 1
displays the expanded visible-range spectra of PPIX-RSE,
PPIX, and Fe2(SEt)2(NO)4 (I) in THF and illustrates that
the spectrum of PPIX-RSE is close to being the summed
spectra of its two chromophores. In the IR spectra, the νNO
bands of PPIX-RSE appear at nearly the same frequency
as those seen for representative Fe2(SR)2(NO)4 derivatives I
(R ) Et) and II (R ) CH2Ph). This is consistent with earlier
studies8 that showed NO stretching frequencies of Roussin’s
red salt esters to be relatively independent of the nature of
the R- groups but markedly different from those of RRS
or of monoalkylated RSE. Table 1 lists the peak maxima
and extinction coefficients characterizing the optical spectrum
of PPIX-RSE and the spectra of I and II and of PPIX itself.
Also listed are the νNO frequencies for the iron cluster
compounds.
1
characterized by low- and high-resolution mass, UV-vis, and H
NMR spectroscopy and elemental analysis. LRMS-FAB m/z: 871
([M + 1]+), 745 ([M - I]+), 635 ([M - 2I + H2O]+), 619 ([M -
2I + 2H]+). HRMS-FAB: 870.11557, calcd 870.113910 (M)+.
UV-vis (CH2Cl2): 406 nm (ꢀ ) 1.65 × 105 M-1 cm-1), 506 (1.50
× 104), 542 (1.25 × 104), 576 (7.53 × 103), 630 (5.86 × 103). 1H
NMR (CDCl3, δ): -3.8 (s, 2H), 3.16 (t, J ) 6.8, 4H), 3.31 (t, J )
7.4, 4H), 3.63 (s, 3H), 3.64 (s, 3H), 3.71 (s, 3H), 3.72 (s, 3H), 4.33
(t, J ) 7, 4H), 4.42 (t, J ) 7.6, 4H), 6.2 (d, J ) 11.6, 2H), 6.38 (d,
J ) 17.6, 2H), 8.28 (dd, J ) 11.6, 6, 2H), 10.04 (s, 1H), 10.08 (s,
1H), 10.17 (s, 1H), 10.22 (s, 1H). Anal. Calcd for C38H40N4O4I2:
C, 52.4; H, 4.60; N, 6.44. Found: C, 52.3; H, 4.49; N, 6.42.
[µ-S, µ-S′-Protoporphyrin IX Bis(2-thioethyl) diester]tetrani-
trosyldiiron (PPIX-RSE). A round-bottom flask equipped with a
stir bar was charged with approximately 100 mL of distilled,
deoxygenated THF. The solution was entrained with N2, and the
flask was then charged with Roussin’s red salt, Na2[Fe2(µ-S)2-
(NO)4]‚8H2O, (0.500 g; 1.03 mmol). The flask was sealed with a
rubber septum, and the solution was kept under an inert atmosphere
with a flow of nitrogen. A separate flask was charged with
approximately 50 mL of distilled deoxygenated THF to which 0.897
g (1.03 mmol) of PPIX-I was added under dinitrogen. The RRS
solution was then slowly transferred dropwise to this flask via a
cannula with continuous stirring, and the resulting solution was
allowed to stir overnight under an inert atmosphere. After this, the
reaction mixture was filtered under N2 and then evaporated under
vacuum to dryness. The product was further purified via careful
chromatography (silica gel/CH2Cl2/CH3OH) to give a reddish-purple
solid in 65% yield. This was characterized by low- and high-
resolution mass, 1H NMR, UV-vis, and IR spectroscopy and
elemental analysis IR (CHCl3): νNO bands at 1781 and 1754 cm-1
.
LRMS-FAB m/z: 913 (M + H)+, 822 ([M - 3NO]+. HRMS:
found: 913.120488, 912.114887; theoretical: 913.118758 (M +
H)+, 912.110933 (M)+. UV-vis (CH2Cl2): 406 nm (ꢀ ) 1.49 ×
105 M-1 cm-1), 506 (1.30 × 104), 538 (1.02 × 104), 576 (6.0 ×
1
103), 632 (4.2 × 103). H NMR (CDCl3, δ): -3.8 (s, 2H), 1.1-
Photochemical Studies. The quantitative photochemical
reactions of Roussin’s red salt anion and several red-salt
esters, Fe2(NO)4(SR)2, were previously studied6,8,9 using
changes in the electronic spectra to determine the quantum
1.9 (m, centered at 1.5, 8H closest to Fe), 3.38 (s, 3H), 3.48 (s,
3H), 3.66 (s, 3H), 3.7 (s, 3H), 3.8-4.9 (m, centered at 4.4, 8H
closest to porphyrin ring), 6.21 (d, J ) 7.6, 2H), 6.39 (d, J ) 14,
2H), 8.28 (s, 2H), 10.1 (m, 4 H, porphyrin meso hydrogens).
Elemental analysis (C, H, N) for PPIX-RSE was performed by
the Marine Science Analytical Laboratory at UCSB. Anal. Calcd
(17) Gouterman, M.; Khalil, G. E. J. Mol. Spectrosc. 1974, 53, 88-100.
Inorganic Chemistry, Vol. 43, No. 18, 2004 5545

Conrado et al.
Scheme 1. Pathway for the Preparation of PPIX-RSE
Table 1. UV-Vis and IR Absorption Spectral Data for Various Ester
Complexes in CHCl3
FTIR
UV-vis
max (nm) (log ꢀ)
compound
ν
NO (cm-1) (log ꢀ)
λ
Fe2(µ-SCH2CH3)2(NO)4 (I)
1750 (3.72)
1776 (3.77)
1800 (w)
310 (3.97), 364 (3.93)
430 sh (3.61)
Fe2(µ-CH2Ph)2(NO)4 (II)
1751 (3.70)
1778 (3.74)
1800 (w)
312 (3.97), 364 (3.93)
PPIX
277 (5.49)
406 (Soret, 5.16)
504 (4.16), 538 (4.06)
576 (3.83), 632 (3.75)
277 (5.56)
406 (Soret, 5.17)
506 (4.11), 542 (4.01)
576 (3.78), 630 (3.62)
PPIX-RSE
1754 (3.66)
1781 (3.70)
coupled with the use of a nitric oxide-specific electrode, such
optical spectral changes demonstrated that 0.5 NO was
released per RRS anion decomposed,6 whereas 4 NO was
released by each RSE.8 With PPIX-RSE, the photoreaction
could not be conveniently monitored by changes in the
optical spectrum because this was dominated by the por-
phyrin chromophore and underwent little change upon
photolysis. Instead, these reactions were studied quantita-
tively by monitoring the changes in the IR spectra at
frequencies characteristic of the νNO bands (Figure 2).
Quantum yields (Φdis) determined in this way for the
disappearance of these bands when PPIX-RSE and other
representative RSE were photolyzed at the irradiation
wavelengths (λirr) of 436 and 546 nm are listed in Table 2.
For I and II, the Φdis values that were measured using IR
detection were (within experimental uncertainty) the same
as those determined by monitoring the optical absorbance
Figure 1. (Top) Electronic absorption spectra of PPIX (6.16 µM) and
PPIX-RSE (6.02 µM) solutions in THF. Absorbances at the Soret band
maximum are 0.89. These solutions were used for the steady-state emission
measurements (Figure 3). (Bottom) Quantitative spectra of PPIX-RSE
(blue), PPIX (red), and I (green) in THF in the Q-band region of the visible
spectrum indicating that the spectrum of PPIX-RSE is close to being the
summed spectra of its two chromophores.
yields for photodecomposition of these materials. When
5546 Inorganic Chemistry, Vol. 43, No. 18, 2004

NoWel Iron-Sulfur-Nitrosyl Cluster
Figure 2. Absorbance changes of IR bands in the νNO spectral region
upon 546-nm irradiation of CHCl3 solutions of PPIX-RSE.
Table 2. Quantum Yields for the Decomposition of Various RSEs in
CHCl3 Solutions
compound
λ
irr (nm)
ꢀ (λirr)
Φdisa (× 104)
Fe2(µ-SCH2Ph)2(NO)4] (II)
436
546
436
546
436
546
6.9 × 103
9.3 × 102
4.7 × 103
7.6 × 102
3.2 × 104
1.2 × 104
3.2 ( 0.1
1.9 ( 0.3
not determined
1.7 ( 0.2
5.2 ( 0.7
2.5 ( 0.2
Figure 3. Fluorescence spectra of PPIX (6.16 µM) and PPIX-RSE (6.02
µM) in THF solutions with matched absorbances at the excitation
wavelength (404 nm) under ambient conditions (22 ( 1 °C, aerated
solutions).
Fe2(µ-SEt)2(NO)4 (I)
PPIX-RSE
PPIX-RSE is much more efficient at absorbing red light and
converting it to the net photochemistry.
a The Φdis values represent quantum yields for the disappearance of the
noted complexes from at least three independent measurements by monitor-
ing absorbance changes of the IR nitrosyl stretching bands at 1780 and
The greater photochemical efficiency of PPIX-RSE in the
longer-wavelength photochemistry can be illustrated as
follows. A 10 µM solution of Fe2(µ-SEt)2(NO)4 would have
an absorbance (Abs) of 0.008 at an excitation wavelength
of 546 nm (1-cm path length), whereas a 10 µM solution of
PPIX-RSE would have an Abs of 0.12. The rate of NO
photoproduction can be defined in terms of the intensity of
the light absorbed, Ia, according to eq 2, where Ia ) Io(1 -
10-Abs). Therefore, for a fixed incident light intensity Io, the
rate of NO photogeneration by PPIX-RSE at an initial
concentration of 10 µM would be about 19-fold faster than
for I at the same concentration.
1754 cm-1. In each case, the concentrations were defined so that at λirr
)
436 nm the intensity of absorbed light Ia was 3.0 × 10-6 Ein L-1 s-1 (Io )
7.2 × 10-6 Ein L-1 s-1). At 546 nm, Ia ) 1.0 × 10-6 Ein L-1 s-1 (Io ) 2.5
× 10-6 Ein L-1 s-1).
changes. Qualitative studies using a nitric oxide-specific
electrode also demonstrated the concomitant photochemical-
induced release of NO, and on the basis of earlier studies
with other RSEs, it can be assumed that ΦNO ≈ 4Φdis.
As summarized in Table 2, Φdis is 2-fold larger at λirr
)
436 nm than at 546 nm. (As shown before, photolysis at
365 nm gave considerably larger values of Φdis for other
RSEs.)8 Of special interest is the observation that the
measured Φdis values for PPIX-RSE were ∼50% higher at
the respective λirr than those observed for both I and II. This
is particularly significant because the porphyrin chromophore
of PPIX-RSE dominates the optical spectrum of that
compound and most of the incident light is absorbed by the
porphyrin. PPIX-RSE displays photochemistry that is both
qualitatively and quantitatively characteristic of the Fe/S/
NO cluster core; therefore, initial excitation must be followed
by nonradiative processes leading to the net reaction at the
cluster core. In other words, there is no “inner filter” effect
because of the unproductive absorption of light by the PPIX
moiety; instead, the photochemical behavior is nearly the
same as that seen by the direct excitation of closely related
esters not having the pendant chromophore. Thus, there must
be a high-efficiency energy transfer to the reactive state(s)
because Φdis values are not diminished when the porphyrin
serves as the primary chromophore. Because the porphyrin
is the much stronger chromophore at longer wavelengths,
∂[NO]
∂t
) ΦNO Ia
(2)
Emission Studies. The fluorescence spectra for PPIX-
RSE and PPIX were recorded under ambient conditions in
tetrahydrofuran solutions with concentrations (∼6 µM) that
gave matched absorbances (∼1.0) at the 404-nm excitation
wavelength. Figure 1 was generated from such a pair of
solutions. Both solutions displayed fluorescence in the 630-
740-nm range (largest peak at λmax ) 636 nm) characteristic
of the porphyrin chromophore (Figure 3). However, the
relative emission intensity from PPIX-RSE was only 1/6 that
from PPIX. Not surprisingly, red-salt esters I and II did not
display any emission under comparable conditions. Equimo-
lar (∼6.8 × 10-6 M) solutions of Fe2(NO)4(µ-SEt)2 and PPIX
together showed the same emission properties as the por-
phyrin alone; thus, there was little or no bimolecular
quenching at these concentrations. Solutions of PPIX and
Inorganic Chemistry, Vol. 43, No. 18, 2004 5547

Conrado et al.
Table 3. TCSPC Fluorescence Lifetime Data for PPIX, PPIX-DME,
and PPIX-RSE under Various Conditions (Ambient Temperature)
τ (ns) (( 0.5 ns) τ (ns) (( 0.02 ns)
complex
PPIX-DME
solvent
CHCl3
slower process
faster process
0.23
10.4
8.6
PPIX-RSE
CHCl3
mixture of PPIX-DME THF
12.6
(2.5 µM) and I (4.2µM)
PPIX-DME
PPIX
THF
THF
12.6
12.4
10.7
5.4
PPIX-RSE
PPIX-DME
PPIX-RSE
THF
THF (1 atm O2)
THF (1 atm Ar)
0.22
0.20
14.6
PPIX-DME (the PPIX dimethyl ester) at equivalent con-
centrations displayed the same fluorescence intensity, so the
reduced emission intensity from PPIX-RSE is not due to
esterifying the PPIX carboxylic acid groups. Thus, the
diminished porphyrin emission in PPIX-RSE must be
specifically due to energy transfer from the pendant chro-
mophore to the covalently linked iron-nitrosyl-sulfur
cluster. Such intramolecular energy transfer must also explain
the cluster photodecomposition resulting from direct excita-
tion into the strongly absorbing porphyrin antenna.
The relative emission intensities from PPIX-RSE (If′) and
PPIX (If) can be used to estimate the lower limit for the
rate constant of intramolecular quenching (kq) according to
the relationship If/If′ ) 1 + kqτ, where τ is the lifetime
characteristic of PPIX (13 ns in ambient THF).17 This would
give kq ) ∼5 × 108 s-1. However, although such emission
decreases have often been used to evaluate intramolecular
quenching rates, the procedure is flawed by the assumption
that intramolecular quenching is the only change in the PPIX
photophysical properties upon linking this luminophore to
the Fe2S2(NO)4 cluster.
This assumption was tested by directly evaluating the
emission lifetimes of PPIX-RSE and various PPIX deriva-
tives using time-correlated single-photon counting (TCSPC)
techniques. The excitation wavelength was 403 nm, and the
absorbances at this λirr were 0.6 to 0.8. The temporal
fluorescence behaviors of PPIX, PPIX dimethylester (PPIX-
DME), and PPIX-RSE were measured under deaerated and
aerated conditions in THF and CHCl3 solutions. Although
there were but modest differences in the emission lifetimes
between ambient and deaerated solutions, the latter demon-
strated longer-term stability. As will be noted below, the use
of a pure O2 atmosphere had a substantially larger effect on
certain emission lifetimes. Selected data from the lifetime
experiments are summarized in Table 3.
Fluorescence from PPIX (6.0 µM) in ambient THF
demonstrated single-exponential decay curves from which
the lifetime was determined to be 12.4 ns, in reasonable
agreement with the reported value (13 ns, Φ ≈ 0.1).17 The
dimethyl ester PPIX-DME displayed a nearly identical
lifetime (12.6 ns) in THF (4.46 µM) and a somewhat shorter
one (10.4 ns) in CHCl3 (4.26 µM). Thus, the lifetime results
confirm the conclusion based on integrated emission intensi-
ties that esterification has little effect on the emission
properties of the PPIX chromophore.
Figure 4. Fluorescence lifetime trace for PPIX-RSE (4.86 µM) in aerated
THF showing dual emission behavior with τ1 ) 0.22 ns and τ2 ) 10.7 ns.
one quite short (∼200 ps) and the other nearly as long as
that of free PPIX (Figure 4). The dual lifetimes were
obtained by good-quality fits of these decays to the biexpo-
nential function given by eq 3, where τ1 and τ2 are lifetimes,
F1 and F2 are fractions of initial amplitude with a sum of
1.00, and A is a scaling factor. For PPIX-RSE in deaerated
THF, this treatment gave τ1 ) 0.20 ( 0.02 ns (F1 ) 0.82)
and τ2 ) 14.4 ( 1 ns (F2 ) 0.18). Exposing the solution to
air had an insignificant effect on τ1, but τ2 decreased to ∼11
ns. There appeared to be no time-dependent differences in
the emission spectra, thus the porphyrin chromophore appears
to be responsible for the emission under all circumstances.
-t/τ2
I(t) ) A[F1e-t/τ + F2e
]
(3)
1
A control sample consisting of PPIX-DME (2.51 µM) and
Fe2(µ-SCH2CH3)2(NO)4 (4.2 µM) in argon-flushed THF
displayed fluorescence that decayed exponentially, giving a
single lifetime (12.6 ns) identical to that seen for PPIX-
DME alone. Thus, consistent with the steady-state emission
intensity experiments described above, there appears to be
no bimolecular quenching of the PPIX-DME excited state
by the RSE cluster at these concentrations. However, the
emission lifetime of PPIX-DME (2.24 µM) in a THF
solution equilibrated with O2 at 1.0 atm was found to be
only 5.4 ns, and bimolecular quenching by dioxygen would
be appear to be playing a role. Under these conditions [O2]
≈10 mM, so the quenching rate constant kq(O2) can be
roughly estimated to be 1 × 1010 M-1s-1, nearly diffusion-
limited.
The dual emission seen for PPIX-RSE might be explained
by the presence of two emitting species, the short-lived
component being PPIX-RSE and the longer-lived one being
a metal-free PPIX impurity. However, materials with identi-
cal temporal emission profiles and intensities were the result
of several independent syntheses and repeated recrystalliza-
tions, so this is considered an unlikely explanation. Alter-
natively, progressive PPIX-RSE photodecomposition during
the spectroscopic analytical procedures might release a more
luminactive PPIX derivative from the iron cluster. Although
enhanced emission from a longer-lived component was
identified for an aerated THF solution of PPIX-RSE (4-8
µM) after continuous 365-nm photolysis, the dual emission
property was also seen with fresh solutions. Nonetheless, to
In marked contrast, the emission from PPIX-RSE was not
a simple exponential but displayed two lifetime components,
5548 Inorganic Chemistry, Vol. 43, No. 18, 2004

NoWel Iron-Sulfur-Nitrosyl Cluster
minimize the release of a PPIX derivative during temporal
fluorescence measurements, we restricted data collection to
short periods with weak excitation beams and argon-flushed
solutions, and we repeated lifetime determinations with fresh
solutions. Such experiments gave excellent reproducibility
of the dual lifetimes and amplitudes measured with τ1 at
0.19-0.23 ns, τ2 giving the value 14.4 ( 1 ns, and F1/F2
consistently in the 4.5-5 range. Furthermore, there was little
change when the pump-laser intensity was increased by an
order of magnitude. Thus, we conclude that the dual emission
behavior is characteristic of the PPIX-RSE samples inves-
tigated.
The steady-state and flash-photolysis emission measure-
ments are self-consistent. In the former, the emission intensity
from PPIX-RSE was only about 17% of that seen for PPIX
itself under nearly identical conditions. The temporal fluo-
rescence behavior of analogous solutions of PPIX and PPIX-
DME both display single-exponential decays with compa-
rable relatively long lifetimes. In contrast, emission from the
PPIX-RSE samples is bimodal; the largest component is
much shorter lived than the smaller component, which has
a lifetime comparable to that of PPIX. If one assumes that
the radiative rate constant kr for the porphyrin luminophore
is the same in each case, then the relative fluorescence
intensities under steady-state excitation are proportional to
the relative lifetimes and the amplitude of their relative
contribution (Ii R Fikrτi). Accordingly, the short-lived com-
ponent would contribute little to the integrated fluorescence
spectrum of PPIX-RSE, and one would predict from the
relative amplitudes of the two components that steady-state
emission from PPIX-RSE would be diminished by at least
80% relative to that of PPIX. This is close to that what was
seen given that modest changes in kr values could impact
this estimate.
RSE is dominated by the PPIX residue, both should have
very similar absorption spectra, and the preexponential terms
F1 and F2 would represent the fractions of the two species
present. However, for the unlikely possibility that the
absorption spectra of the two conformers differ significantly,
this will not be true. Another possible explanation would be
a sequential mechanism whereby all PPIX-RSE molecules
have the same ground-state conformation, but after initial
excitation, there is a very fast isomerization or reaction to
give a second species with a lifetime comparable to that of
free PPIX. This would almost have to be a conformational
change or reversible bond cleavage given that (under Ar)
there is little evidence for permanent chemical reactions
under continuous photolysis conditions.
Summary
A porphyrin-derivatized Roussin red-salt ester, PPIX-
RSE, was synthesized, characterized, and the photochemical
and photophysical properties of this novel supramolecular
compound were investigated. The quantum yield data
revealed that PPIX-RSE is efficient at absorbing light and
transferring energy from the excited porphyrin to the Fe2S2-
(NO)4 cluster. The net result is the quenching of the
porphyrin emission coupled to the photodecomposition of
the cluster and concomitant NO dissociation. The emission
studies support the concept that the porphyrin chromophore
acts as a light-absorbing/transferring antenna molecule. The
fluorescence intensity of PPIX-RSE is quenched by ∼85%,
and the temporal fluorescence displays two components, the
larger one with a lifetime of 0.20 ns and other with a τ of
14.4 ns in deaerated THF. The dual lifetime feature may be
due to the presence of two ground-state conformers in
solution with similar absorption spectra that, when excited,
are too short lived to come to conformational equilibrium
before undergoing decay.
The above data clearly indicate that the ∼80-85%
reduction of the steady-state emission intensity and the dual-
lifetime behavior relative to the simpler porphyrins are
reproducible properties of PPIX-RSE. One explanation
would be that the system is heterogeneous in the sense that
this supramolecular species exists in two conformations,
which interchange on a time scale longer than the fluores-
cence lifetime(s). Because the absorption spectrum of PPIX-
Acknowledgment. These studies were supported by the
National Science Foundation (CHE 9726889 and CHE
0095144). We are particularly indebted to Dr. James
Bourassa, who provided guidance and inspiration at the
initiation of this project.
IC049459A
Inorganic Chemistry, Vol. 43, No. 18, 2004 5549