Water-Soluble Nitroxyl Porphyrin Complexes FeIITPPSHNO and FeIITPPSNO- Obtained from Isolated FeIITPPSNO·

Article abstract of DOI:10.1021/jacs.9b09161

The first biomimetic water-soluble Fe^II-porphyrin nitroxyl complexes were obtained and characterized by UV-vis in protonated and deprotonated forms by reduction of previously isolated and characterized Fe^IITPPSNO^·. The pK_a involved in the Fe^II-HNO → Fe^II-NO^- + H⁺ equilibrium was estimated to be around 9.7. The Fe^IITPPSHNO complex spontaneously reoxidizes to the nitrosyl form following a first-order kinetic decay with a measured kinetic constant of k = 0.017 s^-1. Experiments show that the HNO adduct undergoes unimolecular homolytic cleavage of the H-NO bond. DFT calculations suggest a phlorin radical intermediate for this reaction. The deprotonated NO^- complex resulted to be more stable, with a half-life of about 10 min.

Full text of DOI:10.1021/jacs.9b09161

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Article
II
Water-soluble nitroxyl porphyrin complexes FeTPPSHNO
II
-
II
•
and FeTPPSNO obtained from isolated FeTPPSNO
Agostina María Mazzeo, Juan Pellegrino, and Fabio Doctorovich
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b09161 • Publication Date (Web): 27 Oct 2019
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Water-soluble nitroxyl porphyrin complexes FeIITPPSHNO and
FeIITPPSNO− obtained from isolated FeIITPPSNO•
Agostina Mazzeo, Juan Pellegrino* and Fabio Doctorovich*.
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Departamento de Química Inorgánica, Analítica, y Química Física, Facultad de Ciencias Exactas y Naturales, Universi-
dad de Buenos Aires. INQUIMAE-CONICET, Ciudad Universitaria, Pab. 2, C1428EHA, Buenos Aires, Argentina.
KEYWORDS: Nitroxyl complexes, porphyrin, water-soluble.
ABSTRACT: The first biomimetic water soluble FeII-porphyrin nitroxyl complexes were obtained and characterized by UV-
Vis in protonated and deprotonated forms by reduction of previously isolated and characterized FeIITPPSNO•. The pKa in-
volved in the FeII-HNO ⇄ FeII-NO− + H+ equilibrium was estimated to be around 9.7. The FeIITPPSHNO complex spontaneous-
ly reoxidizes to the nitrosyl form following a first order kinetic decay with a measured kinetic constant of k = 0.017 s-1. Ex-
periments show that the HNO adduct undergoes unimolecular homolytic cleavage of the H-NO bond. DFT calculations sug-
gest a phlorin radical intermediate for this reaction. The deprotonated NO− complex resulted more stable, with a half-life of
about 10 minutes.
Introduction
octaethyl porphyrin), by one-electron UV electrochemical
reductions of the nitrosyl {FeNO}7 analogues, in dichloro-
methane.16,17 Later on, both complexes could be obtained
chemically and electrochemically by Ryan’s group, and
further stabilized in THF solution, which allowed their
characterization by FT-IR spectroscopy.18,19 Evidence for
the protonated [(OEP)Fe(HNO)] complex was also ob-
tained, resulting stable for hours in organic media in the
presence of phenols as proton sources.20 The hexacoordi-
nated derivative [(OEP)Fe(HNO)(5-MeIm)] was prepared
via hydride attack on the corresponding ferric nitrosyl by
Nitric oxide (NO•) and nitroxyl (HNO/NO−) have been
consistently studied during recent years due to their bio-
logical as well as chemical relevance. Previously only re-
garded as an environmental pollutant, nitric oxide’s role as
a signaling agent in living organisms has already been
established, acting in vasodilation and nerve signal trans-
duction at low concentrations.1,2 More recent investiga-
tions have also suggested that endogenous generation of
the reduced nitroxyl species is possible at physiological
conditions, from many biological metabolites,3–6 which
implies that HNO/NO− could have independent signaling
activity of its own, and might also be responsible for some
of the physiological effects initially attributed to NO•, like
protection against ischemia related injuries.7–9 Heme pro-
teins are one of the principal targets for these molecules in
biological systems, as they tend to bind to metal centers
through the nitrogen atom.10
1
Richter-Addo et al. and characterized by H NMR, although
in 11% yield and at -20°C.21 A particularly stable {FeNO}8
complex reported by our group could be isolated and char-
acterized by UV-Vis, IR and 15N NMR, using the perhalo-
genated
porphyrin
TFPPBr8
(β-octabromo-meso-
tetra(pentafluorophenyl porphyrin)) in organic media.22
Nevertheless, attempts to protonate the species to give the
expected {FeHNO}8 complex were unsuccessful, reacting to
give the {FeNO}7 complex (FeII-NO•). Lehnert et al. ob-
served analogous results with other electron-poor porphy-
rins, and more interestingly, reported the first ferrous-
heme HNO complex using a very sterically hindered bis
picket fence porphyrin which resulted of great stability,
decomposing completely only after ~ 20 h. They concluded
that the bulky substituents on the porphyrin ring disfa-
vored a bimolecular reoxidation pathway, proposed for
unhindered porphyrins in organic media.23
In addition, such heme-protein adducts of metal-site co-
ordinated NO•/HNO/NO− species are key intermediates in
the mechanism of action of different enzymes of the nitro-
gen biogeochemical cycle, such as nitrite or nitric oxide
reductases.11–13 These considerations make it vital to de-
velop biomimetic models of such active sites in order to
study their reactivity and structure. Thus, nitrosyl/nitroxyl
porphyrin iron complexes have become particularly inter-
esting to study from a bioinorganic point of view.
The increasing number of reports on {FeNO}8 (according
to the Enermark-Feltham notation)14 complexes have con-
tributed to the spectroscopic characterization of this ini-
tially elusive species but still much more investigation is
needed to elucidate mechanistic issues.15 The first porphy-
rinate iron-nitroxyl complexes were reported by Kadish et
al., using TPP (meso-tetraphenylporphyrin) and OEP (β-
Regarding aqueous chemistry, the only examples report-
ed consist of the nitroxyl-myoglobin adduct, Mb-HNO, and
other globin adducts studied by Farmer and coworkers. 24,
25
The Mb-HNO adduct could even be characterized by
Raman, NMR and X-ray absorption.26 These complexes did
not decompose for weeks, suggesting that the protein’s
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distal pocket residues could stabilize the HNO moiety by combusted. CO2, H2O and N2 generated were separated by
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hydrogen bonding. No biomimetic water-soluble iron
heme complex has been stabilized or characterized in
aqueous solution.
gas chromatography and quantified after thermal conduc-
tivity detection. Cyclic voltammetry experiments were
carried out using a Princeton Applied Research (PAR)
273A instrument and a PAR303A mercury drop electrode
(area 0.024 cm2), a Ag/AgCl reference electrode (3.0 M
KCl), and a platinum wire counter electrode.
On the other hand, a very stable water-soluble (although
non-heme) {FeNO}8 complex could be obtained upon two-
electron reduction of [Fe(CN)5(NO)]2-, and characterized
spectroscopically, in both its protonated and non-
protonated form.27 The pKa for [Fe(CN)5(HNO)]3- was esti-
Materials
1
mated via H NMR to be about 7.7, giving the first experi-
9
Meso-tetra(4-sulfonatophenyl)porphine in the form of
the tetrasodium, dodecahydrate salt (Na4TPPS•12H2O )
was purchased from Frontier Scientific and used as re-
ceived.
mental value for bound HNO. The special stability reported
for this complex led us to consider that other factors, like
hydrogen bonds, could stabilize the Fe-HNO moiety.
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In this work we isolated for the first time the {FeNO}7
adduct of the water-soluble porphyrin TPPS (meso-
tetra(4-sulfonatophenyl)porphine), which has been widely
used in previous works on NO chemistry (Figure 1).28,29
Moreover, we chemically obtained the corresponding
{FeNO}8 elusive adducts, FeIITPPSHNO and FeIITPPSNO−,
making these complexes the first ferrous-heme water-
soluble nitroxyl models. The products have been charac-
terized by UV-Vis, and cyclic voltammetry experiments.
The pKa for FeIITPPSHNO deprotonation was estimated
using spectrophotometric and electrochemical methods.
The spontaneous reoxidation reaction of the protonated
form to the {FeNO}7 adduct FeIITPPSNO• was kinetically
studied via experimental measurements and DFT calcula-
tions.
Buffers were freshly prepared with recently degassed
water before every experiment. Sodium phosphate mono-
basic and dibasic salts were purchased from Carlo Erba.
Citric acid, sodium carbonate and sodium tetraborate salts
were purchased from Biopack. Sodium hydroxide was
purchased from Dorwil.
Sodium dithionite (82%) was purchased from Sigma Al-
drich and recrystallized following literature procedures30
in order to increase its purity and separate unwanted by-
products. This reagent was stored always in a freezer un-
der argon. Solutions of this compound were always made
basic (pH ≥ 10) in order to minimize decomposition reac-
tions.
Gaseous NO was produced anaerobically from a mixture
of NaNO2, NaBr, FeSO4 (Biopack) and 5 ml purged mQ
water according to literature techniques.31
2,2,6,6-Tetramethylpiperidine 1-oxyl, (TEMPO) was
purchased from Sigma Aldrich and used as received for
qualitative experiments.
Tetrafluoro boric acid 48% wt. water solution was pur-
chased from Sigma Aldrich, and was deoxygenated by
argon bubbling before use.
DFT calculations
All calculations were carried out with the program pack-
age Gaussian 09. Meso-tetraphenyl porphyrin (TPP) was
used as a model. The structures of all molecules were fully
optimized at the DFT level, using the PBE exchange-
correlation functional. LANDL2DZ basis set and pseudopo-
tential were used for the Fe atom. For the remaining atoms
(H, N, O, C) the 6-31G** basis set was used. Energy scans
were performed, and the QST3 method was used as im-
plemented to optimize the geometry of the transition state.
Figure 1. FeIITPPSNO• anionic moiety. Sodium counterions
were omitted for clarity.
Experimental Section
Anoxic atmosphere considerations
Equipment and measurements
All experiments involving FeIITPPSNO• were carried out
under strict anaerobic conditions. A Schlenk argon-vacuum
line and appropriate Schlenk glassware were used
throughout the development of this work. When necessary,
air-sensitive compounds were manipulated inside a nitro-
gen glove box. All solids were purged with various argon-
vacuum cycles, and liquid reagents and solvents were
deoxygenated using techniques varying from argon bub-
bling (acid solutions) to freeze-pump-thaw (organic sol-
All UV-Vis measurements were carried out using a
Hewlett-Packard HP8453 diode array spectrometer with 1
cm or 0.2 cm pathlength cuvettes. All NMR measurements
were carried out in a BRUKER AVANCE NEO 500 MHz
spectrometer. All IR measurements were made using a
Thermo Nicolet Avatar FT-IR spectrophotometer. Ele-
mental analysis was carried out in a Carlo Erba EA 1108
system for C, N and H by INQUIMAE. The sample was fully
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vents, except methanol) or direct vacuum methods (water proved to be quite stable as a solid, and was characterized
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and methanol). Quartz cuvettes of 1 cm and 2 mm path-
lengths topped with new septa were used each time to
avoid oxygen filtration. Gastight Hamilton syringes ranging
from 10 μL to 5 mL were used for the correct manipulation
of the air-sensitive solutions and gases.
by IR in Fluorolube and elemental analysis. N-O stretching
was observed at 1668 cm (Figure S2). This value is in
-1
good agreement with literature data of iron nitrosyl por-
phyrin compounds.6 Elemental analysis: Anal. Calc. for
FeIITPPSNO•Na4 • 5(H2O) • 3(CH3COCH3): %C 46.8 %N
5.1 %H 3.8 Exp: %C 46.4 %N 5.1 %H 3.7 A 3/1 ace-
1
tone/porphyrin molar proportion was verified by H NMR
Preparation and isolation of FeIITPPSNO•·Na4 (1)
spectroscopy in d4-MeOH (Figure S3, Table S1). It is solu-
ble in water and, to a lesser extent, in methanol; it is insol-
uble in other organic solvents.
Na4TPPS•12H2O was metallated according to literature
techniques,32 but replacing the tenfold excess of FeSO4 salt
with less than 3 eq. of FeCl2. This gave chloride salts as
byproducts, which resulted much easier to wash than their
sulphate analogues. Approximately 200 mg (0.16 mmol) of
free porphyrin were mixed with 1.5 equivalents of FeCl2
and 2 eq. NaOH. The reaction was carried out in a water
reflux, under argon flow, in order to avoid early oxidation
of the ferrous reactant. The process was monitored by UV-
Vis spectroscopy until full conversion was achieved. Extra
base or FeCl2 were added when necessary. The resulting
solution was passed through a DOWEX cation exchange
column to remove excess Fe3+ ions and afterwards alka-
linized to pH = 5. The solvent was pumped out and the
resulting solid was repeatedly recrystallized by adding
distilled acetone to a saturated aqueous solution of the
compound until no more traces of chloride were found in
the supernatant (checked by AgCl formation after addition
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Kinetic measurements
Experiments to determine the kinetic parameters of the
FeIITPPSHNO (2) reoxidation reaction were carried out in
the UV-Vis range using a 2 mm pathlength quartz cuvette.
Absorbance traces at 414 and 418 nm (1 and 2 Soret band
peak wavelength respectively) were monitored. Kinetic
measurements were made after 1.5-2 eq. of dithionite
were added to a solution of 1. Cycle time was set to 1 s and
the experiments ran for about 5 minutes. These experi-
ments were carried out in a variety of conditions, of which
the most studied was in 2 mM phosphate buffer at pH = 6.
They were also carried out in phosphate at pH = 7 and
citrate buffers at pH = 5 of the same concentration and in
plain distilled and degassed water, using in this case a non-
buffered, pH neutral dithionite solution prepared immedi-
ately before use.
of
AgNO3).
The
product,
ferric meso-tetra(4-
sulfonatophenyl)porphine (FeIIITPPS) was dried under
vacuum and stored at room temperature. Yield: 124 mg, 62
%.
The kinetic data were analyzed making a deconvolution
of the total absorbance registered at 418 nm. Considering
that initially the only species present is 1, whose absorp-
tion spectra and molar absorptivity were already report-
ed,36 and having calculated the molar absorptivity coeffi-
cient of 2 (See Results section), the molar fraction of the
nitroxyl compound and its absorbance was calculated as a
function of time. Integral and differential kinetic method
plots and fitting were made in order to determine whether
the decay followed an order 0, 1 or 2 behavior. Afterwards,
appropriate non-linear fittings were made with Origin Pro
8 to estimate the associated rate constant. Multiwave-
length analyses were also performed using MatLab and
proved to give according results.
The five coordinated nitrosyl ferrous complex (FeIITPP-
SNO•) was obtained by reaction of the ferric porphyrinate
with an HNO donor, as reported in literature for porphyrin
nitrosyls in organic media.33 Approximately 40 mg of
FeIIITPPS were dissolved in 0.5 ml water along with 1.5
equivalents of 4-fluoro-N-hydroxybenzenesulfonamide
(fluoro-Piloty’s Acid, F-PA, Figure S1) in the presence of
two equivalents of NaOH (See Results, Scheme 1). F-PA
was synthesized according to literature methods34 and
further purified using a silica gel chromatography column
as reported independently.35 The identity and purity of the
resulting compound was monitored by 1H in d6-DMSO
(Sigma Aldrich). The nitrosylation reaction was carried out
in water at room temperature, under argon atmosphere
and constant stirring. A different Piloty’s acid derivative,
nitro-PA (synthesized analogously) also proved useful for
this reaction, and other variants may be tried, as long as
they are at least partially water soluble. Conversion was
followed by monitoring the Soret bands corresponding to
the reactant and the product in the UV-vis region (395 and
414 nm respectively). Additional base and Piloty’s acid
equivalents were added when needed. The product was
precipitated by addition of freshly degassed acetone. The
remaining solid was recrystallized twice analogously
(H2O/acetone) and washed with acetone until Piloty’s acid
byproducts were no longer detectable by spectrophotome-
try in the UV region. The resulting red solid, FeIITPP-
SNO•·Na4, 1, was stored under argon in a Schlenk tube.
This product, although very air-sensitive when in solution,
Results and Discussion
Preparation and isolation of FeIITPPSNO•·Na4
The product was successfully prepared and isolated as a
solid as shown in the reactions shown in Scheme 1, follow-
ing reaction path A using an HNO donor, or reaction path
B, involving reduction followed by NO gas bubbling. In
order to isolate relatively large amounts of compound (20-
30 mg), path A was chosen over path B, since the HNO
donor byproduct is easily washed off the product by ace-
tone, unlike dithionite byproducts. However, path B, either
for in situ or isolation purposes resulted useful for UV
experiments and particular bulk experiments. Other reduc-
ing agents such as CrIIEDTA and BH4─ also proved useful
for reduction of FeIIITPPS.
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Scheme 1. Preparation and isolation of FeIITPP-
퐍퐚 퐒 퐎
ퟐ
ퟐ
FeIIITPPS
ퟒ FeIITPPS
SNO•·Na4. Sodium counterions were omitted for clarity.
R=4-F-C6H4
1
2
3
FeIITPPS 퐍퐎 퐠 ,퐞퐱퐜. FeIITPPSNO•
4
5
6
퐍퐚
퐒 퐎 퐩퐇=ퟔ
ퟐ ퟒ,
ퟐ
FeIITPPSNO•
FeIITPPSHNO
퐫퐞퐨퐱퐢퐝퐚퐭퐢퐨퐧
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8
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Once precipitated by acetone addition, the product was
vacuum-dried and stored in a Schlenk tube under argon.
This allowed us to manipulate the nitrosyl compound in a
relatively simple manner by directly weighing the solid
inside the glovebox before the experiments. Furthermore,
it allowed us to characterize the compound through IR and
elemental analysis techniques for the first time, and carry
out cyclic voltammetry and UV-vis experiments using solid
1 as the starting material instead of having to generate the
species in situ through chemically unclean paths.
Figure 2. UV-Vis Spectral changes observed upon reduction of
FeIITPPSNO• at pH = 6.
The same was observed when 1-2 equivalents of dithio-
nite were added directly to a freshly prepared 5-6 μM
solution of isolated FeIITPPSNO•, in a more controlled,
cleaner manner. In both cases, the resulting 2 complex
spontaneously reoxidizes to the {FeNO}7 nitrosyl com-
pound, 1 (Figure 3).
UV scale experiments - Obtention and characterization of
FeIITPPSHNO4- (2) and FeIITPPSNO5− (3) in aqueous media
A deoxygenized solution (approx. 5-6 μM) of FeIIITPPS in
2 mM phosphate buffer solution at pH = 6 was treated with
1.5-2 equivalents of sodium dithionite to give FeIITPPS.
Excess NO (g) was then added to the mixture to produce 1,
and finally 1-2 more equivalents of dithionite were added
to give the desired product, 2 (Scheme 2). The observed
spectrum peak of the Soret band shifted from 414 to 418
nm and the corresponding molar absorption coefficient
increased from 1.55 105 to 2.17 105 M-1 cm-1 (Figure 2).
Although this product rapidly reoxidized to 1, in presence
of excess dithionite and NO (g) a steady-state regime was
reached, allowing us to determine its molar absorptivity
coefficient. Seki et al.36 transiently obtained the same UV
spectrum through flash photolysis techniques using ben-
zophenone ketyl radicals and reported the adduct’s insta-
bility, with a half-life time of 2 seconds. Note that here, we
established that under these conditions, the adduct exists
in the protonated form, FeIITPPSHNO (2), and not as FeIIT-
PPSNO− (3): see further experiments below.
Figure 3. Observed reoxidation of FeIITPPSHNO to FeIITPP-
SNO• at pH = 6. The time elapsed between the maximum and
minimum peak is ca. 200 s.
Scheme 2. Indirect formation and subsequent reoxida-
tion of FeIITPPSHNO in phosphate buffer at pH = 6.
The same experiments were carried out at pH = 12
phosphate buffer, and in this case a different spectrum
emerged for the {FeNO}8 adduct, which we tentatively
assigned to the deprotonated complex, FeIITPPSNO− (3). In
this case, a subtle decrease in absorbance was observed for
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the Soret band, at 413 nm, as well as a slight widening of
the band. The most important change was observed in the
λmax/nm (ε/M-1cm-1)
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2
3
4
5
6
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Q band zone, resulting in a shift from ≈ 541 nm to ≈ 533
nm, and a noticeable absorbance increase and shape
change of the most intense band (Figure 4). Both spectral
changes (especially in the Q band zone) are surprisingly
consistent with previously reported spectra of other iron-
nitroxyl heme complexes in organic media.23 3 also even-
tually oxidizes to 1, with a half-life of about 10 minutes,
although further analysis and reactivity studies will be
pursued in a different work.
FeIITPPSNO∙
(1) 414 (155000)a, 543 (11300)a
FeIITPPSHNO
pH = 6
(2)
418 (217000), 541(12600)
FeIITPPSNO−
pH = 12
(3)
413 (146000), 533 (12000)
9
Table 1. UV-vis spectroscopic data for FeIITPPS nitrosyl and
nitroxyl compounds in aqueous phosphate buffer solutions. a:
Reference 35.
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pKa estimation
In order to estimate the pKa value associated to the
FeIITPPSHNO/FeIITPPSNO− acid-base pair, experiments
were carried out in different buffers ranging from pH 5 to
11. On one hand, chemical reductions of 1 with dithionite
were followed by UV-Vis spectrophotometry in order to
stablish at which point the obtained spectra for the
{FeNO}8 adduct changed. UV-Vis spectral changes were
monitored at pH range 5-11 (See Figures S6-S12 in Sup-
porting Information). Spectral evidence for the protonated
adduct could be observed at pH = 9, but not at pH = 10. On
the other hand, evidence for the deprotonated species was
observed at pH = 10, but not below. This suggests that the
pKa value for 2 would be between 9 and 10, as reported for
a ruthenium nitroxyl complex. 37
Figure 4. UV-Vis Spectral changes observed upon reduction of
FeIITPPSNO• at pH = 12.
On the other hand, cyclic voltammetry experiments were
made to study the pH dependence of the reduction poten-
tial involved. A FeIITPPSNO• to FeIITPPSHNO reduction
process would depend on H+ concentration, whereas an
FeIITPPSNO•/ FeIITPPSNO− reduction would not. At pH = 6,
an irreversible strong reduction peak assignable to the
FeIITPPSNO•, H+/FeIITPPSHNO couple was observed at -
0.665 V vs. Ag/AgCl, in concordance with previous re-
ports.38 At pH = 11.3, the irreversible reductive current
wave had effectively shifted to more negative potentials,
In order to confirm the identity of 2 and 3, NaOH was
added to the cuvette once 2 was formed at pH 6, rapidly
rising the pH to 12. The spectrum assigned to 3 was in-
stantly observed as a result (Figure S5, Scheme 3A). Corre-
spondingly, the formation of 2 was observed after lower-
ing the pH from 11 to 6 by adding HBF4 to a cuvette con-
taining initially 3. (Figure S5, Scheme 3B) Therefore, the
UV-vis spectra could be correctly assigned to both FeIIT-
PPSHNO and FeIITPPSNO− species. Spectroscopic data for
the Soret and the main Q band for compounds 1-3 can be
found in Table 1.
reaching the peak at -0.885
V
for the FeIITPP-
SNO•/FeIITPPSNO− couple vs. Ag/AgCl. The complete set of
reductive waves at different pHs values are shown in Fig-
ure 5.
Scheme 3. FeIITPPSHNO and FeIITPPSNO− formation
and deprotonation/protonation reactions.
The peak current potential shifts as a function of pH can
also be used to estimate the pKa of 2. Nernst’s equation for
the FeIITPPSNO•, H+/FeIITPPSHNO redox process involving
one electron and one proton at 298K, predicts a shift of -59
mV per pH unit until the pKa is reached. This behavior is
clearly observed for pHs 6-9 (Figure 6), with an experi-
mental slope of -52 mV (see Figure S13). The curve shown
in Figure 6 represents the expected behavior for the reduc-
+ 퐍퐚 퐒 퐎 ,
ퟐ ퟒ
ퟐ
A)
B)
FeIITPPSNO•
FeIITPPSHNO + 퐍퐚퐎퐇 퐭퐨 퐩퐇=ퟏퟐ FeIITPPSNO−
퐩퐇=ퟔ FeIITPPSHNO
+ 퐍퐚 퐒 퐎 ,
ퟐ ퟒ
퐩퐇=ퟏퟐ FeIITPPSNO−
ퟐ
FeIITPPSNO•
+ 퐇퐁퐅 퐭퐨 퐩퐇=ퟔ
ퟒ
FeIITPPSNO−
FeIITPPSHNO
FeIITPPSHNO
tion potential assuming an acidity constant, Ka, of 2x10-10
,
estimating a pKa of about 9.7 for FeIITPPSHNO, consistent
with observations done in the UV-vis experiments previ-
ously described. It is important to note that irreversible
current peaks were used for this estimation. Previous
reports show the use of irreversible peak data to obtain
related useful thermochemical data.39 Therefore, we be-
FeIITPPSNO•
Table 1. UV-Vis spectroscopic data for FeIITPPS-NO
compounds.
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lieve the method can be used as a semiquantitative estima- cepted pKa value for free HNO was 4.7, obtained by pulse
radiolysis experiments.41 However, this was later corrected
to 7.2, and it has been settled at 11.5 since 2001. 42, 40 It is
also important to note that the studies in Meyer’s work
were carried out in conditions very different from ours,
forming the nitrosyl complex in situ in presence of excess
nitrite instead of using the isolated solid in a clean manner.
Moreover, the species’ behavior at such acidic pHs may
affect the nitrosyl’s stability and even imply a second pro-
tonation process at the HNO ligand and at the sulfonate
groups of the porphyrin substituents.
1
2
3
4
5
6
7
8
tion alongside the UV-measurements.
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On the other hand, the pKa value of 9.7 for coordinated
HNO is in excellent agreement with the one reported for
[Ru(Me3[9]aneN3)(bpy)(NO)]+1, which displayed a pKa of
9.8 37, and falls within the 8-10 range for the pKa value for
FeIIOEP(HNO) estimated by FTIR spectroscopy.20
Reoxidation reaction studies: kinetic measurements
Many hypotheses were considered in order to elucidate
why spontaneous reoxidation took place for the protonat-
ed 2 adduct. Although complete absence of oxygen traces
cannot be guaranteed, the reproducibility in the experi-
ments led us to think that something other than adventi-
tious oxygen was driving this process. Besides, the expect-
edly more air-sensitive deprotonated adduct, 3, seemed to
last longer in the cuvette.
Figure 5. Reductive electrochemical wave assigned to the
{FeTPPSNO}7/{FeTPPSNO}8 couple for pHs 6 – 11. Current
intensities are normalized.
Another hypothesis contemplated the possibility of ex-
cess NO (g) present in the cuvette displacing HNO as a
ligand. However, this was disregarded when the same
results were observed using isolated 1 as the starting ma-
terial, NO gas not being present in excess anymore. In oth-
er experiments, when excess NO (g) was purposely added
to 2 formed in situ, 1 was formed instantly, indicating a
probable ligand exchange reaction.
A third hypothesis included the bimolecular decomposi-
tion:
(1)
In order to test this last case, the system was properly
optimized in order to make robust kinetic measurements,
since processes of this kind have been previously proposed
in literature in organic media, but not tested directly.23
Figure 6. Reduction potential for {FeTPPSNO}7/{FeTPPSNO}8
as a function of pH. Experimental measurements are shown
along with expected fitting for pKa = 9.7. Linear fitting for pH
6-9 and extra information about CV experiments are shown in
Supplementary Information.
Kinetic measurements of the reoxidation reaction were
carried out under different conditions, being the most
robust those experiments carried out in 2 mM phosphate
buffer at pH = 6, using a 2 mm pathlength quartz cuvette at
room temperature. Integral and differential method plots
showed that the decomposition reaction followed a first
order decay and not a second order decay, as expected for
the reaction shown in Eq. 1. (See Figures S14-S17). Further
fitting gave an exponential decay as shown in Figure 7.
The pKa value of 9.7 for FeIITPPSHNO resulted two units
lower than the revised value of 11.5 reported for free HNO,
40
in agreement with the expected stabilization of the NO─
moiety upon coordination. The obtained estimate, howev-
er, strongly differs from the results previously obtained
from electrochemical experiments by Meyer and cowork-
ers, where they report a pH dependent reduction for FeIIT-
PPSNO• at pH < 2.6, implying a pKa close to this value for
the HNO complex.38 Such value probably seemed reasona-
ble for the authors at that time, considering that the ac-
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The reaction was also carried out in presence of excess
1
2
3
4
imidazole at pH 7 to determine if coordination of an axial
sixth ligand would relatively stabilize 2. The obtained
kinetic constant was fairly smaller than in its absence (Ta-
ble 2, row 7), suggesting a somewhat although not sub-
stantial stabilizing effect of the axial ligand.
5
6
7
8
9
10
11
12
13
14
15
Finally, when the experiment was carried out in phos-
phate buffer dissolved in D2O, the rate constant was signif-
icantly smaller than in H2O (Table 2, row 3), kH/kD being
2.7, which seems to correspond to a primary kinetic iso-
tope effect. Therefore, the H-NO bond breaking is involved
in the limiting step of the reoxidation reaction. However, it
could also implicate that water attacks 2 to give 1 and
byproducts through a pseudo first order mechanism.
The observed results suggest that the reoxidation of 2 to
1 occurs by loss of an H atom through a rate-determining
homolytic rupture of the N-H bond. The significantly dif-
ferent value obtained for the reduction with CrIIEDTA
could be explained by a possible hydrogen atom abstrac-
tion activity carried out by this compound or its byprod-
ucts.
Figure 7. FeIITPPSHNO absorbance time traces at 418 nm for
16
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one selected experiment, which fit a monoexponential decay.
See SI (Figures S18-S25) for more representative fittings
under different conditions.
The observed first order kinetic constant under these
conditions is k= 0.017 ± 0.003 s-1, which accounts for a
half-life time of approximately 41 s, considerably longer
that the previously reported lifetime of 2 s.36 It is im-
portant to point out that in those conditions, 2 was gener-
ated in the presence of glucose and benzophenone radicals,
which possibly interfered in the stability of the HNO moie-
ty resulting in a shorter lifespan.
Table 2. Measured kinetic constants for FeIITPPSHNO
reoxidation under different contidions
Row n°
1
Experimental conditions
Phosphate buffer pH 6, 2 mM
Reducing agent: Na2S2O4
Phosphate buffer pH 6 2 mM
Reducing agent: Na2S2O4
Sulphite excess
k (s-1)
N
0.017 ± 0.003
13
Temperature studies of the reaction under these condi-
tions in the 8 - 48°C range were also performed, and an
activation energy of 13 ± 8 kcal mol-1 could be estimated.
The corresponding Arrhenius plot is shown in Figure S26.
2
3
0.014 ± 0.003
3
3
These results ruled the bimolecular reaction hypothesis
out and left us with two possibilities: either the rate-
limiting step of the HNO complex reoxidation was an in-
trinsic first order decomposition of 2, or a pseudo first
order decay, involving water or buffer species available in
the system. However, no significant differences were ob-
served when buffer was excluded and pure water was used
as a solvent, or different buffer compositions were used
(Table 2).
Phosphate buffer pH 6, 2 mM
in D2O
0.0064 ± 0.0003
Reducing agent: Na2S2O4
Phosphate buffer pH 6, 2 mM
Reducing agent: CrII(EDTA)
Citrate buffer pH 5, 2 mM
Reducing agent: Na2S2O4
Phosphate buffer pH 7, 2 mM
Reducing agent: Na2S2O4
Phosphate buffer pH 7, 2 mM
Reducing agent: Na2S2O4
Imidazole excess
4
5
6
0.17 ± 0.06
0.019 ± 0.009
0.017 ± 0.002
10
3
The rate constant was determined under different ex-
perimental conditions in order to find possible reoxidation
paths (Table 2). The pHs = 5, 6 and 7 gave out virtually the
same reoxidation rate. Surprisingly, the only significantly
different rate, greater by an order of magnitude, was ob-
served when using CrIIEDTA as a reducing agent (Table 2,
row 4). This led us to think that either the metallic traces
in CrIIEDTA or the chromium complex itself catalyzed the
oxidation reaction, or that, inversely, when dithionite was
used as a reducing agent, the reaction proceeded more
slowly. This could be a consequence of dithionite byprod-
ucts, such as sulfite, coordinating to 2 in the sixth position.
However, dithionite experiments in presence of metallic
traces (Cr3+, Fe3+) did not show significant differences; the
same was observed when excess sulfite was added to the
cuvette before reduction.
4
7
0.010 ± 0.002
3
Deionized water
8
0.025 ± 0.007
7
Reducing agent: Na2S2O4
Table 2. Kinetic constants for the spontaneous reoxidation
of FeIITPPSHNO to FeIITPPSNO• measured under different
experimental conditions, along with their standard deviation.
N is the number of experiments considered for each experi-
mental condition.
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In order to indirectly test this hypothesis, UV reduction
experiments were carried out in the presence of the free-
radical TEMPO, which has the ability to abstract H• radicals
and form TEMPOH. Previous studies show that the H-N
bond energy in HNO (49.9 kcal/mol) is substantially small-
er than the average H-N bond (90 kcal/mol), making it
relatively easy to break.43 The reaction of free HNO with
TEMPOL has already been studied, resulting in a hydrogen
atom abstraction to give NO· and TEMPOL-H.44 With this in
mind, 2 eq. of TEMPO were added to a cuvette containing
freshly formed 2 at pH = 6 (by dithionite reduction of 1).
Complete and instantaneous formation of 1 was observed
instead of the progressive oxidation witnessed in analo-
gous experiments in the absence of TEMPO. This qualita-
tive experiment shows that the H• in the Fe-HNO moiety
can readily abstracted, evidencing the relative weakness of
the H-N bond in the complex, supporting the suggested
homolytic rupture as the rate-limiting step.
(2)
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2
3
4
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Figure 8. Optimized structures and calculated energy differ-
ences for FeIITPPHNO, the final phlorin radical intermediate
and transition state.
In order to evaluate the feasibility of the proposed
mechanism, the reoxidation reaction was further explored
through DFT calculations.
The performed calculations suggest a decomposition
mechanism in which the rate-determining step is first
order in the FeIITPPSHNO complex, in agreement with the
experimental measurements, and consists in the formation
of an unstable phlorin radical intermediate (Scheme 4).
This species, which was not detected via UV-vis measure-
ments, would rapidly react re-donating the H• entity to give
unspecified products and the FeIINO• complex, reestablish-
ing the aromaticity of the porphyrin ring. Even if we can-
not specify the fate of the H atom, we are aware that hy-
drogen gas would be the most reasonable product. Prelim-
inary hydrogen measurement experiments were intended,
although they resulted inconclusive and should be pursued
more carefully in a different work. However, previous
work by Abucayon et al. shows H2 formation following the
decomposition of Fe(OEP)(HNO)(MeIm).45
DFT calculations – formation of a phlorin radical interme-
diate
DFT calculations were performed using the FeTPP model
in order to gain more insight into the reoxidation mecha-
nism of 2. The structure for FeIITPPHNO was optimized,
and energy scans along the H-N bond of the HNO moiety
were performed with increasing bond distance. The final
structure showed that the hydrogen atom from the HNO
moiety had migrated to the most proximate meso carbon of
the porphyrin ring. This unexpected result led us to opti-
mize the structures of the product and the transition state
to see if it was a plausible rate-limiting step for a uni-
molecular reaction. Surprisingly, the final phlorin radical
-1
intermediate resulted only 2.4 kcal mol higher in energy
Scheme 4. Proposed reoxidation pathway for FeIIT-
PPSHNO.
than the initial FeIITPPHNO structure, while the calculated
activation energy for the interconversion of these tauto-
mers resulted in 19.4 kcal mol -1, in good agreement with
the experimentally measured value of 13 ± 8 kcal mol-1
(Figure 8). Intramolecular proton-coupled electron trans-
fers of this kind have been recently reported for other
porphyrins, where similar bent phlorin intermediates
were theoretically proposed and detected.46–49 This phlorin
radical can be best interpreted as a 1 electron oxidized
phlorin, rather than a 1 electron reduced porphyrin, since
it bears two substituents on the sp3 hybridized carbon.
Previous literature reports show that free phlorin macro-
cycles can undergo oxidative processes of up to 3 electrons
at modest potentials, which makes the postulation of the
proposed intermediate reasonable.50, 51
The proposed mechanism, suggested for the first time
for the decomposition of HNO complexes, could be sup-
ported by evaluating if the stability of Fe-HNO complexes
is enhanced by the use of porphyrinoid platforms that
already bear substituted sp3 carbons, such as phlorins,
porphodimethenes and calixpirroles, which are reported
to prevent the oxidation to the corresponding porphy-
rins.52,53 If this turns out to be the case, the obtained com-
plexes might provide a stable platform for performing
interesting reactivity studies of coordinated HNO/NO─,
even if the heme reminiscence is somewhat lost.
The energy associated with reaction (2) was also calcu-
lated, in order to dismiss a pseudo first order mechanism
involving water. As expected for the formation of a reactive
OH• radical, the reaction resulted thermodynamically up-
hill by 93.1 kcal mol -1
.
In addition, the newly proposed mechanism allows for
the re-interpretation of previous results. For instance,
although the presented results do not support the previ-
ously suggested bimolecular decomposition pathway in
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organic media (1), the greater stability of the sterically In addition, other water-soluble platforms will be stud-
1
2
3
4
5
6
7
8
hindered bis picket fence porphyrin could also be due to
the fact that the bulky substituents prevent the structure
from bending and transferring the H• atom to the meso
carbon in an effective manner. Nevertheless, we are aware
that aqueous and organic media chemistry are different
and therefore decomposition mechanisms might not be the
same.
ied. In particular, experiments with cationic porphyrins
like meso-tetra(N-methylpyridyl)porphyrin (TMPyP4+)
could provide valuable insight on the influence of the
charge of the ligand on the stability of nitroxyl compounds.
ASSOCIATED CONTENT
Supporting Information. Complementary IR, NMR and UV
spectra, cyclic voltammetry considerations, additional kinetic
fittings and useful structures are shown in Supporting infor-
mation. This material is available free of charge via the Inter-
net at http://pubs.acs.org.
Additional investigations should be carried out to fur-
ther study these systems, and more importantly, evaluate
the implications of similar reaction pathways and in bio-
logically relevant conditions for enzymatic mechanisms.
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AUTHOR INFORMATION
Corresponding Author
Conclusions and perspectives
A water-soluble iron nitrosyl porphyrin complex, FeIIT-
PPSNO• was obtained through two different routes and
successfully isolated for the first time as a and character-
ized by FT-IR, cyclic voltammetry, elemental analysis and
UV-visible techniques. This compound turned out to be
stable for many months if stored under argon.
Reduction of previously isolated FeIITPPSNO• at different
pHs provided UV-Vis spectroscopic evidence for the for-
mation of the first water-soluble ferrous heme nitroxyl
complexes FeIITPPSHNO and FeIITPPSNO−. This infor-
mation, along with data from electrochemical measure-
ments allowed us to estimate the pKa value for the FeIIT-
PPSHNO/FeIITPPSNO− equilibrium to be around 9.7.
* Juan Pellegrino: pellegrino@qi.fcen.uba.ar
* Fabio Doctorovich: doctorovich@qi.fcen.uba.ar
Funding Sources
We thank CONICET, UBA (grant UBACYT 2018) and ANPCYT
(PICT-2017-1930) for the necessary funding for this work.
ACKNOWLEDGMENT
We thank Dr. Nicolás Neumann for helping with the MatLab
analysis; Dr. Gabriel Gordillo for the electrochemical meas-
urements and Federico Davia for the provided assistance. F.D.
and J.P. are CONICET members, and A. M. M. is a CONICET
doctoral fellow.
Both complexes eventually reoxidize to the initial nitro-
syl species, with an estimated half-time life of 10 min for
FeIITPPSNO− and less than a minute for FeIITPPSHNO.
ABBREVIATIONS
TPPS = Meso-tetra(4-sulfonatophenyl)porphine
Plausible causes for FeIITPPSHNO instability were evalu-
ated, and most of them were ruled out. The reoxidation
reaction is an order 1 decay in pH = 6 phosphate buffer,
with an estimated k = 0.017 s-1 (t1/2 = 41 s, considerably
greater than the one previously reported)14. Our results
strongly indicate that the FeIITPPSHNO moiety undergoes
a rate-limiting homolytic bond break, resulting in the ob-
served FeIITPPSNO• and H• which due to its high reactivity
further reacts with solvent and/or other species (such as
buffer and/or the byproducts of the reducing agents). This
hypothesis is supported by experiments carried out in the
presence of H• withdrawing compounds (TEMPO) and DFT
calculations that predict homolytic H-NO bond cleavage
and intramolecular addition to the porphyrin meso carbon
to give a phlorin radical intermediate. Despite this appar-
ently rapid reoxidation, the protonated nitroxyl species
turned out to be substantially more stable than its organic-
media analogues previously reported. This observation
agrees with the proposed hypothesis that hydrogen bonds
with water help to stabilize the Fe-HNO moiety.
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