Synthesis, properties, and reactivity of a series of non-heme {FeNO} 7/8 complexes: Implications for Fe-nitroxyl coordination

DOI: 10.1016/j.jinorgbio.2012.08.026

Source and publish data:

Journal of Inorganic Biochemistry p. 115 - 127 (2013)

Update date:2022-08-04

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Authors:

Sanders, Brian C.

Patra, Ashis K.

Harrop, Todd C.

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Article abstract of DOI:10.1016/j.jinorgbio.2012.08.026

The biochemical properties of nitroxyl (HNO/NO^-) are distinct from nitric oxide (NO). Metal centers, particularly Fe, appear as suitable sites of HNO activity, both for generation and targeting. Furthermore, reduced Fe-NO^-/Fe-HNO or

Full text of DOI:10.1016/j.jinorgbio.2012.08.026

Journal of Inorganic Biochemistry 118 (2013) 115 –127

Contents lists available at SciVerse ScienceDirect

Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Synthesis, properties, and reactivity of a series of non-heme {FeNO}^7/8complexes:

Implications for Fe-nitroxyl coordination

⁎

Brian C. Sanders, Ashis K. Patra, Todd C. Harrop

Department of Chemistry, The University of Georgia, 1001 Cedar Street, Athens, GA 30602, USA

Center for Metalloenzyme Studies, The University of Georgia, 1001 Cedar Street, Athens, GA 30602, USA

a r t i c l e i n f o

a b s t r a c t

The biochemical properties of nitroxyl (HNO/NO⁻) are distinct from nitric oxide (NO). Metal centers, particularly

Fe, appear as suitable sites of HNO activity, both for generation and targeting. Furthermore, reduced Fe–NO⁻/Fe–

HNO or {FeNO}⁸(Enemark–Feltham notation) species offer unique bonding proﬁles that are of fundamental

importance. Given the unique chemical properties of {FeNO}⁸systems, we describe herein the synthesis and

properties of {FeNO}⁷and {FeNO}⁸non-heme complexes containing pyrrole donors that display heme-like prop-

erties, namely [Fe(LN₄^R)(NO)] (R = C₆H₄or Ph for 3; and R = 4,5-Cl₂C₆H₂or PhCl for 4) and K[Fe(LN₄^R)(NO)] (R =

Ph for 5; R = PhCl for 6). X-ray crystallography establishes that the Fe–N–O angle is ~155° for 3, which is atypical

for low-spin square-pyramidal {FeNO}⁷species. Both 3 and 4 display ν_NOat ~1700 cm⁻¹in the IR and reversible

diffusion-controlled cyclic voltammograms (CVs) (E_1/2=~−1.20 V vs. Fc/Fc⁺(ferrocene/ferrocenium redox

couple) in MeCN) suggesting that the {FeNO}⁸compounds 5 and 6 are stable on the CV timescale. Reduction

of 3 and 4 with stoichiometric KC₈provided the {FeNO}⁸compounds 5 and 6 in near quantitative yield, which

were characterized by the shift in ν_NOto 1667 and ~1580 cm⁻¹, respectively. While the ν_NOfor 6 is consistent

with FeNO reduction, the ν_NOfor 5 appears more indicative of ligand-based reduction. Additionally, 5 and 6 en-

gage in HNO-like chemistry in their reactions with ferric porphyrins [Fe^III(TPP)X] (TPP = tetraphenylporphyrin;

X = Cl⁻, OTf⁻(triﬂuoromethanesulfonate anion or CF₃SO₃⁻)) to form [Fe(TPP)NO] in stoichiometric yield via

reductive nitrosylation.

Article history:

Received 12 June 2012

Received in revised form 17 August 2012

Accepted 18 August 2012

Available online 6 October 2012

1. Introduction

nitric oxide synthase (NOS) in the absence of its biopterin cofactor

[17]. Additionally, iron-coordinated HNO/NO⁻are proposed inter-

The one-electron reduced analogue of nitric oxide (NO•), termed a

nitroxyl or nitrosyl hydride (HNO or NO⁻(nitroxyl anion) depending

on the pH; pK_a=11.6) [1,2], has received special attention of late

including this thematic issue of the Journal of Inorganic Biochemistry.

Most research on this enigmatic inorganic small molecule stems

from its potential as a therapeutic [3]. For example, HNO has been

shown to increase myocardial contractility, i.e. the strength of heart

muscle tissue, and thus represents a promising drug for heart failure

[4–6]. In fact, HNO is already clinically approved for other disease

treatments. This includes the HNO-donor molecule cyanamide

(N-hydroxycyanamide is the actual HNO-donor), which has shown

to be an effective anti-alcoholism drug by disturbing alcohol metabo-

lism through inhibiting the enzyme aldehyde dehydrogenase [7,8].

The mechanism of action of HNO appears to be through its interac-

tions with thiol-containing biomolecules [9–11] and ferric heme

proteins [1,12–16]. While the endogenous production of HNO has

not been clearly established, there is evidence of its formation from

mediates of the enzymes involved in denitriﬁcation, namely nitrite

reductase (NiR) and nitric oxide reductase (NOR) [12,18–21]. How-

ever, the basic chemistry and biology of this small molecule are

challenging to study. The short half-life of HNO, due to the self-

condensation reaction to form nitrous oxide (N₂O) and water, ne-

cessitates the use of reliable HNO-donor molecules to understand

its fundamental biochemical reactions [1,3,14,17,22,23]. The most

commonly employed HNO-donors are Angeli's salt (Na₂N₂O₃)

[24,25], derivatives of Piloty's acid (sulfohydroxamic acids) [3,26],

and others [27,28] making this quite an active area of research in

the HNO ﬁeld. As such, there still remains much to be answered in

terms of the chemistry and biology of HNO.

First-row transition metals, especially iron, appear to be among

the most likely sites for the potential endogenous generation and

targets of HNO [1,3,12,15,17,18,29]. Metal–NO complexes are typical-

ly deﬁned by the notation of Enemark and Feltham (EF notation) that

describes the total number of electrons in this delocalized bond as a

post-superscript, designated as {MNO}ⁿ[30]. Iron nitroxyl complexes

proposed to form in denitrifying enzymes are thus classiﬁed as

{FeNO}⁸or {FeHNO}⁸depending on the pH. Despite their importance

⁎

Corresponding author. Tel.: +1 706 542 3486; fax: +1 706 542 9454.

E-mail address: tharrop@uga.edu (T.C. Harrop).

http://dx.doi.org/10.1016/j.jinorgbio.2012.08.026

116

B.C. Sanders et al. / Journal of Inorganic Biochemistry 118 (2013) 115–127

Table 1

in deﬁning the fundamental coordination chemistry of HNO with Fe

in biology, there remains a paucity of information on this class of

iron-nitrosyls.

Electrochemical and spectroscopic data of {FeNO}⁸systems.

Complex

E_1/2(V)^aν_NO(cm⁻¹

)

Δν_NO(cm⁻¹

)

Ref.

The pioneering work by Kadish and Ryan on the {FeNO}⁸complexes

of tetraphenylporphyrin (TPP) and octaethylporphyrin (OEP) repre-

sents the ﬁrst foray into this unique EF notation (see Chart 1, Table 1)

[31–33]. Both [Fe(TPP)NO]⁻and [Fe(OEP)NO]⁻were studied in

situ by spectroelectrochemistry where they displayed reversible

{FeNO}⁷/{FeNO}⁸redox couples at relatively low potentials (~−1 V

vs. SCE = saturated calomel reference electrode) in a variety of sol-

vents (Table 1) [31–33]. While these derivatives were never isolated

as discrete solids (even after exhaustive electrolysis), vibrational

data (Table 1) was obtained on these systems, which exhibited NO

stretching frequencies (ν_NO) at ~1500 cm⁻¹[31]. The ﬁrst and

only isolated heme {FeNO}⁸analogue, [Co(Cp)₂][Fe(TFPPBr₈)(NO)]

(Chart 1; where TFPPBr₈is the dianion of 2,3,7,8,12,13,17,18-

octabromo-5,10,15,20-[tetrakis-(pentaﬂuorophenyl)]porphyrin),

was reported just recently in 2010 by Doctorovich and coworkers

[34,35]. Extensive halogenation of the TPP framework resulted in a

signiﬁcant stabilization of this {FeNO}⁸derivative and is represented

by the cathodic shift in E_1/2to −0.20 V (vs. SCE in CH₂Cl₂) and corre-

sponding blue-shift in ν_NOto ~1550 cm⁻¹from the TPP derivative

[34]. Additional theoretical work by Lehnert and Rodgers has also

helped establish the structural and spectroscopic features of heme

{Fe(H)NO}⁸complexes although no small molecule has been charac-

terized by any structural methods [36,37]. Farmer's group, however,

was successful in obtaining structural information on the {FeHNO}⁸

complex with myoglobin, which is apparently very stable [38–40].

The X-ray absorption experiments revealed Fe–N and N–O lengths

of 1.82 and 1.24 Å, respectively, with a severely bent Fe–N–O angle

of 131° [38]. These values were largely in agreement with theoretical

calculations for small molecules with diamagnetic ground states.

Non-heme examples of {FeNO}⁸are even rarer with a total of three

such reported complexes [41,42] to date and only one being isolable

[43]. The extensive spectroscopic studies by Wieghardt on

[Fe(cyclam-ac)(NO)] represent the ﬁrst reported non-heme deriva-

tive [42]. While these studies were performed on in situ generated

material at low temperature, they were the ﬁrst to include

Mössbauer studies on an {FeNO}⁸complex (δ: 0.41 mm/s, ΔE_Q:

1.69 mm/s) and report the lowest ν_NO(1271 cm⁻¹) of all {FeNO}⁸

complexes [42]. These values were consistent with a low-spin (LS)

Fe(II) (S=0) coordinated to a singlet ¹NO⁻(S=0) assignment.

[Fe(LN₄^Ph)(NO)]⁻(5)

[Fe(LN₄^PhCl)(NO)]⁻(6)

[Fe(LN₄^pr)(NO)]⁻(7)

[Fe(TPP)NO]⁻

−0.83^c

−0.76^c

−0.98^c

−0.93^e

−1.08^g

−0.19^e

~1580^d

1667^d

1604^d

1496^f

−140

−43

This work

[43]

[31,33]

[59]

[34]

[42]

[41]

−100

−185

−229

−176

−336

−264

[Fe(OEP)NO]⁻

1441^f

[Fe(TFPPBr₈)(NO)]⁻

~1550^d

1271^c

1384^h

trans-[Fe(NO)(cyclam-ac)] −0.99^c

[Fe(CN)₅(HNO)]³⁻

−1.00^h

Data represents the E_1/2value for the {FeNO}^7/8redox couple employing the isolat-

ed {FeNO}⁷complexes and normalized to the saturated calomel reference electrode

(SCE) based on information found in Geiger [60].

Denotes the change in N–O stretching frequency upon reduction from {FeNO}⁷to

{FeNO}⁸.

MeCN.

KBr.

CH₂Cl₂.

THF-d₈.

THF.

H₂O.

The IR value is in-line with another non-heme {FeHNO}⁸complex,

[Fe(CN)₅(HNO)]³⁻(Chart 1), which displays a ν_NOof 1380 cm⁻¹

[41]. The only non-heme protein {FeNO}⁸example has been produced

by cryoreduction of the {FeNO}⁷adduct of the taurine:α-ketoglutarate

dioxygenase (TauD) enzyme [44]. This study was the ﬁrst and is still

the only to report a paramagnetic {FeNO}⁸species from an extensive

theoretical calibration of valid bonding models with experimental

Mössbauer parameters (δ: 1.07 mm/s, ΔE_Q: 2.39 mm/s). These results

contrast those obtained for [Fe(cyclam-ac)(NO)] due to the triplet

ground state of TauD-{FeNO}⁸that has been assigned as a high-

spin (HS) Fe(II) (S=2) antiferromagnetically coupled to triplet

³NO⁻(S=1). This small collection of reported {Fe(H)NO}⁸com-

plexes has led to some general properties of {FeNO}⁸: (i) the prefer-

ence of S=0 (diamagnetic) ground states (TauD being the only

outlier) suggestive of a coordinated singlet nitroxyl or nitroxyl anion

(¹HNO or ¹NO⁻); (ii) ν_NOvalues of ~1500 cm⁻¹for heme;

~1300 cm⁻¹for non-heme with the exception of MbHNO (ν_NO

1385 cm⁻¹) [38,45]; and (iii) low E_1/2values for the {FeNO}⁷/{FeNO}⁸

redox couple of ~−1 V (vs. SCE) with one noted exception in

[Fe(TPFFBr₈)(NO)]. Although several theoretical and spectroscopic

Chart 1. Representative structures of reported low molecular weight {Fe(H)NO}⁸coordination complexes.

B.C. Sanders et al. / Journal of Inorganic Biochemistry 118 (2013) 115–127

117

Chart 2. Iron nitrosyl complexes reported in this work.

examinations have shed some insight on the elusive {FeNO}⁸species,

the reactivity of these systems has not been thoroughly explored.

As part of our continuing efforts to establish the deﬁning features of

this unique class of iron–nitrosyls and a means of understanding biolog-

ical Fe–nitroxyl interactions, we describe herein the synthesis and prop-

erties of several iron coordination complexes utilizing a heme-like

diimine/dipyrrolide N₄supporting ligand, namely (Et₄N)[Fe(LN₄^Ph)Cl]

(1), (Et₄N)[Fe(LN₄^PhCl)Cl] (2), [Fe(LN₄^Ph)(NO)] (3), [Fe(LN₄^PhCl)(NO)] (4),

under an inert atmosphere of N₂using standard Schlenk-line techniques

or in an MBraun Labmaster glovebox under an atmosphere of puriﬁed

N₂. All reactions and measurements involving NO(g) and the FeNO

complexes were performed in the dark with minimal light exposure by

wrapping the reaction ﬂasks/vials with aluminum foil to avoid any

photochemical reactions.

K[Fe(LN₄^Ph)(NO)] (5), and K[Fe(LN₄^Ph)(NO)] (6) (see Chart

2 for

2.2. Physical methods

{FeNO}^7/8complexes 3–6). We previously published a detailed account

of the synthesis and properties of the ﬁrst isolable non-heme {FeNO}⁸

complex, [Co(Cp*)₂][Fe(LN₄^pr)(NO)], where we established its nitroxyl-

like reactivity with met-myoglobin under pseudo-physiological condi-

tions [43]. Seeking to add to the list of isolable {FeNO}⁸complexes, we

describe in this paper new {FeNO}⁸complexes based on similar ligand

backbones with subtle electronic/structural differences as a logical exten-

sion of our previous study. The objective of the present contribution was

to deﬁne new synthetic strategies to access Fe–nitroxyls and to under-

stand the fundamental spectroscopic, structural, and reaction chemistry

of isolable {FeNO}⁸systems. The long-term goal is to utilize this knowl-

edge in (i) establishing spectroscopic benchmarks for Fe–(H)NO interme-

diates traversed in biological denitriﬁcation and (ii) the development of

metal-based HNO donors with therapeutic applications.

FTIR spectra were collected with a ThermoNicolet 6700 spectro-

photometer running the OMNIC software. Samples were run as solids

as KBr pellets in a stream of dry N₂. Solution FTIR spectra were

obtained using a demountable airtight liquid IR cell from Graseby-

Specac with CaF₂windows and 0.1 mm spacers. All FTIR samples

were prepared inside a glovebox. The closed liquid cell was taken

out of the box and spectra were acquired immediately. Room temper-

ature (RT) solid-state magnetic susceptibility measurements were

performed with a Johnson Matthey magnetic susceptibility balance.

Solution-state susceptibility measurements were performed in a

solution at 298 K using the Evans method on a Varian Unity Inova

500 MHz NMR spectrometer [48]. X-band (9.60 GHz) EPR spectra

were obtained on a Bruker ESP 300E EPR spectrometer controlled

with a Bruker microwave bridge at 10 K. The EPR was equipped

with a continuous-ﬂow liquid He cryostat and a temperature control-

ler (ESR 9) made by Oxford Instruments, Inc. Electronic absorption

spectra were run at 298 K using a Cary-50 UV-visible (UV-vis) spec-

trophotometer containing a Quantum Northwest TC 125 temperature

control unit. The UV–vis samples were prepared anaerobically in

gas-tight Teﬂon-lined screw cap quartz cells with an optical

pathlength of 1 cm. Electrochemistry measurements were performed

with a PAR Model 273A potentiostat using a Ag/Ag⁺(0.01 M AgNO₃/

0.1 M ⁿBu₄NPF₆in CH₃CN) reference electrode, Pt-wire counter elec-

trode, and a glassy carbon working milli-electrode (diameter=

2 mm) under an Ar atmosphere. Measurements were performed at

ambient temperature using 1.0–10.0 mM analyte in various solvents

containing 0.1 M ⁿBu₄NPF₆as the supporting electrolyte. Ferrocene

(Fc) was used as an internal standard and all potentials are reported

relative to the Fc⁺/Fc couple. ¹H and ¹³C NMR spectra were recorded

in the listed deuterated solvent on a 400 MHz Bruker BZH 400/52

NMR spectrometer or a Varian Unity Inova 500 MHz NMR at 298 K

2. Materials and methods

2.1. General information

All reagents were procured from commercial suppliers and used as

received unless otherwise noted. Research grade nitric oxide gas,

(NO(g), UHP, 99.5%) was obtained from Matheson Tri-Gas. The NO(g)

was puriﬁed by passage through an Ascarite II® column (sodium

hydroxide-coated silica, purchased from Aldrich) and handled under

anaerobic conditions. ¹⁵NO(g)

(

¹⁵N, ≥98%) was purchased from

Cambridge Isotope Laboratories and used without further puriﬁcation.

Acetonitrile (MeCN), methylene chloride (CH₂Cl₂), tetrahydrofuran

(THF), diethyl ether (Et₂O) and pentane were puriﬁed by passage

through activated alumina columns using an MBraun MB-SPS solvent

puriﬁcation system and stored over 3 Å molecular sieves under a nitro-

gen (N₂) atmosphere before use. N,N-dimethylformamide (DMF) was

puriﬁed with a VAC solvent puriﬁer containing 4 Å molecular sieves

and was stored under similar conditions. Anhydrous MeOH and EtOH

were obtained by distilling the alcohol from Mg(OR)₂(R = Me for

MeOH, Et for EtOH) and stored under N₂. Toluene was puriﬁed by stirring

overnight with 3 Å molecular sieves, then distilling from CaH₂, which

was ﬁnally stored over 3 Å molecular sieves. Acetone was dried by stir-

ring over 3 Å molecular sieves for 24 h, which was decanted and stored

under N₂. All solvents were ﬁltered to remove sieve particulate with a

0.45 μm nylon ﬁlter immediately before use. The Fe(II) salt, (Et₄N)₂

[FeCl₄] [46], and {FeNO}⁷complex, [Fe(TPP)NO] [47], were prepared

according to the published procedures. All reactions were performed

with chemical shifts referenced to TMS (tetramethylsilane

Si(CH₃)₄) or residual protio signal of the deuterated solvent as previ-

ously reported [49]. Low resolution ESI-MS (electrospray ionization

mass spectrometry) data were collected on a Perkin Elmer Sciex API

Plus quadrupole mass spectrometer whereas high resolution

ESI-MS data were collected using a Bruker Daltonics 9.4 T APEXQh

FT-ICRM. Elemental microanalyses for C, H, and N were performed by

QTI-Intertek (Whitehouse, NJ) or Columbia Analytical Services (Tucson,

AZ). For some complexes, notably 5–6, residual solvent is present in the

microanalysis and is consistent with what is observed for these com-

pounds in their solid-state IR spectra.

118

B.C. Sanders et al. / Journal of Inorganic Biochemistry 118 (2013) 115–127

2.3. Synthesis of compounds

To ensure complete deprotonation, an occasional vacuum was

applied while stirring for ~15 min. To this reaction mixture was

then added a 10 mL MeCN solution of (Et₄N)₂[FeCl₄] (0.4366 g,

0.9529 mmol) resulting in the formation of a pale gray-white precip-

itate (NaCl) and a solution color change to dark brown-yellow. The

reaction mixture was stirred for another 2 h at RT, which resulted

in no further change. The solution was ﬁltered of insolubles and the

homogeneous dark ﬁltrate was stripped of MeCN and treated with

~20 mL of THF to precipitate any excess Et₄NCl. The THF insolubles

were ﬁltered and washed with THF, and the dark ﬁltrate was concen-

trated and dried under vacuum resulting in 1 as a shiny dark solid

(0.3582 g, 0.7434 mmol, 78%). FTIR (KBr matrix), ν_max(cm⁻¹):

3062 (w), 2975 (w), 2945 (w), 1589 (vs), 1557 (vs), 1481 (w),

1462 (m), 1437 (m), 1383 (s), 1288 (s), 1219 (m), 1183 (m), 1169

(m), 1100 (w), 1077 (w), 1027 (vs), 969 (m), 891 (m), 870 (m),

787 (m), 743 (s), 684 (w), 608 (m), 591 (m), 578 (m), 551 (w), 531

(w), and 480 (w). UV–vis (THF, 298 K), λ_max, nm (ε, M⁻¹cm⁻¹):

313 (12,900), 364 (27,000), and 414 (16,200). Anal. calcd for C₂₄H_32-

N₅ClFe·0.5 H₂O: C, 58.73; H, 6.78; N, 14.27. Found: C, 58.76; H, 6.44;

N, 13.92.

2.3.1. Synthesis of (N1E,N2E)-N1,N2-bis((1H-pyrrol-2-yl)methylene)-

benzene-1,2-diamine (LN₄H₂^Ph

)

To an anaerobic solution of 1,2-phenylenediamine (2.161 g,

19.98 mmol) in 25 mL of MeCN was added a 25 mL MeCN solution

containing 3.801 g (39.97 mmol) of pyrrole-2-carboxaldehyde followed

by addition of activated 3 Å molecular sieves (15% w/v). The brown solu-

tion mixture was reﬂuxed for 24 h and allowed to cool to RT before

workup. Once cooled the red mixture was ﬁltered (sieves and insoluble

yellow product) and the insolubles were washed with CHCl₃. The red ﬁl-

trate was concentrated under vacuum to afford a red amorphous paste

that was dried for several hours under reduced pressure prior to tritura-

tion with ~30 mL of Et₂O. The insoluble yellow material was ﬁltered,

washed with cold Et₂O, and dried under vacuum to afford 3.255 g

(12.36 mmol, 62%) of product. Mp: 197–199 °C. ¹H NMR (400 MHz,

CDCl₃, δ from TMS): 12.34 (br, 1H, NH), 7.68 (s, 1H, CH_N), 7.25

(t, 1H, Ar–H), 7.07 (dd, 1H, Ar–H), 6.40 (d, 1H, Ar–H pyrrole), 6.23 (s,

1H, Ar–H pyrrole), and 6.01 (t, 1H, Ar–H pyrrole). ¹³C NMR (100.6 MHz,

CDCl₃): 150.7 (CH_N), 145.9 (Ar–C), 131.0 (Ar–C), 126.8 (Ar–C), 123.9

(Ar–C), 119.1 (Ar–C), 117.3 (Ar–C), and 109.7 (Ar–C). FTIR (KBr matrix),

_max(cm⁻¹): 3445 (w), 3141 (m), 3085 (w), 2970 (w), 2855 (w), 2746

2.3.4. Synthesis of (Et₄N)[Fe(LN₄^PhCl)Cl] (2)

(w), 1617 (vs), 1574 (s), 1549 (m), 1485 (w), 1442 (m), 1412 (s), 1336

(m), 1310 (m), 1275 (w), 1247 (w), 1211 (m), 1189 (w), 1160 (w), 1138

(m), 1094 (s), 1036 (s), 1026 (s), 971 (w), 966 (w), 884 (m), 877 (m),

846 (m), 801 (w), 783 (w), 742 (s), 659 (w), 608 (m), 579 (w), and

551 (m). LRMS-ESI (m/z): [M+H]⁺calcd for C₁₆H₁₅N₄, 263.1; found,

263.2.

To a batch of LN₄H₂^PhCl(0.3001 g, 0.9061 mmol) dispersed in 8 mL of

MeCN was added a 3 mL MeCN slurry of NaH (0.0435 g, 1.813 mmol),

which resulted in H₂(g) evolution and a dark brown-green solution

indicative of ligand deprotonation. To ensure complete deprotonation,

an occasional vacuum was applied while stirring the solution for

~15 min. To this solution was then added a 5 mL MeCN slurry of

(Et₄N)₂[FeCl₄] (0.4151 g, 0.9060 mmol) resulting in the formation of a

pale gray-white precipitate (NaCl) and a solution color change to dark

brown-yellow. The reaction mixture was stirred for another 2 h at RT,

which resulted in no further change. The solution was then ﬁltered to

obtain a homogeneous dark ﬁltrate. The ﬁltrate was stripped to dryness,

and treated with 20 mL of THF to precipitate any free Et₄NCl. The THF

insolubles were ﬁltered and washed with THF, and the dark ﬁltrate

was concentrated and dried under vacuum resulting in a shiny dark

black solid (0.4500 g, 0.8171 mmol, 90%). FTIR (KBr matrix), ν_max

(cm⁻¹): 3080 (w), 2975 (w), 2945 (w), 1578 (vs), 1541 (vs), 1481

(w), 1457 (m), 1444 (m), 1434 (m), 1383 (s), 1292 (s), 1280 (s),

1254 (s), 1184 (m), 1170 (m), 1119 (m), 1076 (w), 1029 (s), 998 (m),

970 (m), 891 (m), 867 (m), 809 (w), 784 (w), 758 (m), 745 (s), 683

(w), 672 (w), 609 (m), 538 (w), 493 (w), and 447 (m). UV–vis (THF,

298 K), λ_max, nm (ε, M⁻¹cm⁻¹): 324 sh (14,000), 370 (33,000), and

430 (23,000). Anal. calcd. for C₂₄H₃₀Cl₃FeN₅·0.75 H₂O: C, 51.09; H,

5.63; N, 12.41. Found: C, 51.19; H, 5.72; N, 12.10.

2.3.2. Synthesis of (N1E,N2E)-N1,N2-bis((1H-pyrrol-2-yl)methylene)-

4,5-dichlorobenzene-1,2-diamine (LN₄H₂^PhCl

)

Under anaerobic conditions, a 5 mL MeCN solution of 1.004 g

(5.671 mmol) of 4,5-dichlorobenzene-1,2-diamine was added to a

5 mL MeCN solution containing 1.074 g (11.29 mmol) of pyrrole-2-

carboxaldehyde in the presence of 3 Å molecular sieves (15% w/v).

This solution was then reﬂuxed overnight at 70 °C for 20 h resulting

in a black-to-deep-red color change over the reﬂux period. Cooling

the resulting mixture to RT resulted in the precipitation of a dark

insoluble material. The insolubles (product and sieves) were ﬁltered

through a glass frit, thoroughly washed with CHCl₃, and the ﬁltrate

was concentrated and dried in vacuo to a dark red paste. The material

was then redissolved in minimal MeCN (~10 mL) and kept at −5 °C

to induce precipitation of the desired product. The procedure of

concentration, dissolution in minimal MeCN, and precipitation was

repeated three times to afford the dark red-brown solid product

(1.110 g, 3.341 mmol, 59%). Mp: 191–194 °C. ¹H NMR (400 MHz,

CDCl₃, δ from residual protio solvent): 11.87 (br, 0.7H, NH, integrates

slightly low due to the exchangeable nature of the pyrrole proton),

7.64 (s, 1H, CH_N), 7.16 (s, 1H, Ar–H), 6.48 (d, 1H, Ar–H pyrrole),

6.38 (d, 1H, Ar–H pyrrole), 6.10 (t, 2H, Ar–H pyrrole). ¹³C NMR

(100.6 MHz, CDCl₃): 151.5 (CH_N), 145.3 (Ar–C), 130.6 (Ar–C),

129.6 (Ar–C), 124.5 (Ar–C), 120.7 (Ar–C), 118.8 (Ar–C) and 110.6

(Ar–C). FTIR (KBr matrix), ν_max(cm⁻¹): 3446 (w), 3140 (w), 2967

(w), 2890 (w), 2847 (w), 2745 (w), 1611 (vs), 1567 (m), 1484 (w),

1471 (w), 1435 (w), 1413 (s), 1374 (w), 1355 (m), 1331 (m), 1312

(m), 1263 (w), 1245 (w), 1223 (w), 1163 (m), 1126 (s), 1092 (m),

1032 (s), 961 (w), 887 (m), 856 (w), 831 (w), 808 (w), 741 (m),

678 (w), 652 (w), 605 (m), 594 (m), 505 (w), 497 (w), 440 (w),

and 429 (w). LRMS-ESI (m/z): [M+H]⁺calcd for C₁₆H₁₃Cl₂N₄,

331.1; found, 331.2.

2.3.5. Synthesis of [Fe(LN₄^Ph)(NO)], {FeNO}⁷(3)

To an 8 mL MeCN solution of 1 (0.5951 g, 1.235 mmol) was purged

NO(g) for 2 min at RT. The resulting solution changed immediately

from brown-yellow to red-brown with concomitant precipitation of a

dark microcrystalline solid. The reaction mixture was then stirred for

30 min at RT under an atmosphere of NO. After this time, excess

NO(g) was removed in vacuo and replaced with N₂and this solution

was then placed in a −20 °C refrigerator for 1 h to precipitate more

material. The microcrystalline product was ﬁltered, washed with 6 mL

of cold MeCN, and dried under vacuum to yield 0.351 g (1.01 mmol,

82%) of product. X-ray quality red crystals were grown by slow diffusion

of pentane into a toluene solution of the complex at −20 °C. FTIR (KBr

matrix), ν_max(cm⁻¹): 3088 (w), 3059 (w), 2999 (w), 1698 (vs, ν_NO),

1583 (m), 1549 (s), 1506 (m), 1460 (w), 1443 (w), 1379 (s), 1323

(m), 1290 (s), 1256 (m), 1193 (m), 1170 (w), 1153 (w), 1077 (w),

1034 (s), 984 (m), 929 (m), 893 (m), 847 (w), 819 (w), 780 (m), 743

(s), 678 (m), 636 (w), 623 (m), 602 (m), 548 (w), 477 (w), 434 (w),

and 419 (w). FTIR (solution); ν_NO(cm⁻¹): 1705 (MeCN); 1716

(2-MeTHF); 1716 (toluene); 1716 (CH₂Cl₂); μ_eff(solution, 298 K): 2.4

BM in DMSO-d₆. UV–vis (THF, 298 K), λ_max, nm (ε, M⁻¹cm⁻¹): 309

2.3.3. Synthesis of (Et₄N)[Fe(LN₄^Ph)Cl] (1)

To a batch of LN₄H₂^Ph(0.2500 g, 0.9531 mmol) dispersed in 5 mL

of dry MeCN was added a 3 mL MeCN slurry of NaH (0.0457 g,

1.904 mmol), resulting in H₂(g) evolution and a color change from

orange-yellow to bright yellow indicative of ligand deprotonation.

B.C. Sanders et al. / Journal of Inorganic Biochemistry 118 (2013) 115–127

119

sh (16,000), 359 (24,000), and 469 (10,000). Anal. calcd for

₁₆H₁₂FeN₅O: C, 55.52; H, 3.49; N, 20.23. Found: C, 55.27; H, 3.08; N,

(0.2261 mmol, 87%). FTIR, ν_NO(cm⁻¹): 1629 (KBr matrix, Δν_NO

38 cm⁻¹).

20.24.

2.3.11. Synthesis of K[Fe(LN₄^PhCl)(NO)], {FeNO}⁸(6)

2.3.6. Synthesis of [Fe(LN₄^Ph)(¹⁵NO)], {Fe¹⁵NO}⁷(3–¹⁵NO)

To a dark red heterogeneous mixture of 4 (0.1200 g, 0.2891 mmol)

The isotopically-labeled complex was prepared in a similar proce-

dure as 3 except for using ¹⁵NO(g) and 0.561 g (1.164 mmol) of 1.

Yield: 0.3550 g (1.025 mmol, 88%). FTIR, ν_ΝΟ(cm⁻¹): 1667 (KBr

matrix, Δν_NOfrom natural abundant isotope in 3: 31 cm⁻¹); 1680

(MeCN, Δν_NO: 25 cm⁻¹); 1681 (2-MeTHF, Δν_NO: 35 cm⁻¹); 1684

(toluene, Δν_NO: 32 cm⁻¹); 1685 (CH₂Cl₂, Δν_NO: 31 cm⁻¹).

in 3 mL of acetone was added KC₈(0.0430 g, 0.3180 mmol), which

immediately formed

a homogeneous (apart from C₈) darkened

solution. This mixture was stirred for an additional 5 min at which

point the insoluble C₈was removed by vacuum ﬁltration and the

acetone soluble ﬁltrate was concentrated to dryness. The dark residue

was charged with 3 mL of Et₂O and stirred for 5 min followed by decan-

tation. This procedure was repeated three times at which point the ma-

terial was dried under vacuum to afford 0.1150 g (0.2631 mmol, 91%)

of dark solid product. FTIR (KBr matrix), ν_max(cm⁻¹): 3084 (w), 2971

(w) 2923 (w), 2853 (w), 1705 (m, ν_CO), 1579 (s, ν_NO, ν_{C_N}), 1545 (s),

1460 (m), 1436 (m), 1382 (s), 1354 (m), 1292 (s), 1271 (s), 1228

(w), 1181 (m), 1115 (m), 1080 (w), 1034 (s), 978 (m), 913 (w), 894

(m), 864 (w), 819 (w), 794 (w), 749 (s), 678 (m), 657 (w), 613 (m),

2.3.7. Synthesis of [Fe(LN₄^PhCl)NO], {FeNO}⁷(4)

To a 10 mL MeCN solution containing 0.1401 g (0.2544 mmol) of

2 was purged a stream of puriﬁed NO(g) for 2 min at RT under dark

conditions. Immediately upon introduction of NO(g), a dark burgun-

dy powdered solid appeared and the solution became red-burgundy

in color. The reaction mixture was then stirred for 30 min at RT

under an atmosphere of NO in the headspace of the ﬂask. After this

time, excess NO(g) was removed in vacuo and replaced with N₂.

This solution was then placed in a −20 °C refrigerator for 1 h to

precipitate more material. Finally, the powdered solid was ﬁltered,

washed with 3 mL of cold MeCN, and dried under vacuum to afford

0.0852 g (0.2048 mmol, 81%) of product. FTIR (KBr matrix), ν_max

(cm⁻¹): 2922 (w), 1772 (w), 1720 (s, ν_NO), 1570 (s), 1542 (s),

1520 (s), 1500 (m), 1462 (w), 1449 (m), 1379 (s), 1327 (w), 1289

(m), 1272 (s), 1258 (m), 1193 (w), 1116 (w), 1039 (s), 982 (w),

924 (w), 912 (w), 881 (w), 854 (w), 810 (w), 752 (m), 679 (w),

653 (w), 600 (w), 549 (w), 522 (w), 470 (w), and 424 (w). FTIR

(solution); ν_NO(cm⁻¹): 1722 (CH₂Cl₂); 1716 (THF); 1702 (DMSO).

531 (w), 492 (w), 475 (w), and 448 (w). UV–vis (THF, 298 K), λ_max

nm (ε, M⁻¹cm⁻¹): 371 (24,000), 419 (16,300), and 471 sh (10,300).

Anal. calcd for C₁₆H₁₀Cl₂FeKN₅O·1.25 acetone·0.5 H₂O: C, 44.28; H,

3.48; N, 13.07. Found: C, 44.05; H, 3.25; N, 12.83.

2.3.12. Synthesis of K[Fe(LN₄^PhCl)(NO)] (6–¹⁵NO)

The isotopically-labeled complex 6–¹⁵NO was prepared analo-

gously to 6 except for using 0.0982 g (0.2366 mmol) of 4–¹⁵NO

dissolved in 3 mL of acetone and 0.0351 g (0.2597 mmol) of KC₈.

Yield: 0.0951 g (0.2094 mmol, 89%). FTIR, ν_NO(cm⁻¹): obscured

due to overlap with ν_{C_N}

_eff(solution-state, 298 K): 2.1 BM in DMSO-d₆. UV–vis (DMF, 298 K),

2.4. Reactivity studies

_max, nm (ε, M⁻¹cm⁻¹): 317 sh (22,000), 361 (31,000), and 478 sh

(12,000); (THF, 298 K), λ_max, nm (ε, M⁻¹cm⁻¹): 319 sh (14,000),

361 (21,000), and 480 (8600). Anal. calcd for C₁₆H₁₀Cl₂FeN₅O: C,

46.30; H, 2.43; N, 16.87. Found: C, 46.43; H, 2.57; N, 16.53.

2.4.1. Chemical oxidation of K[Fe(LN₄^Ph)(NO)] (5)

To a 1 mL MeCN solution of complex 5 (0.0225 g, 0.0584 mmol)

was added a 1 mL MeCN solution containing 0.0192 g (0.0580 mmol)

of ferrocenium hexaﬂuorophosphate (FcPF₆). Addition of the oxidant

resulted in immediate precipitation of a dark material and the solution

color became paler. This heterogeneous solution was stirred for 30 min

at RT, which resulted in no further change. The solution was concen-

trated to dryness and treated with 2 mL of MeOH to separate and

yield 0.0178 g (0.0514 mmol, 89%) of the {FeNO}⁷complex 3. The

FTIR spectrum (KBr) of this solid is consistent with authentic 3

(ν_NO: 1698 cm⁻¹).

2.3.8. Synthesis of [Fe(LN₄^PhCl)(¹⁵NO)], {Fe¹⁵NO}⁷(4–¹⁵NO)

The isotopically-labeled complex 4–¹⁵NO was prepared analo-

gously to 4 except for using 0.1660 g (0.3014 mmol) of 4 dissolved

in 5 mL of MeCN and ¹⁵NO(g). Yield: 0.0951 g (0.2291 mmol, 76%).

FTIR, ν_NO(cm⁻¹): 1686 (KBr matrix, Δν_NO: 34 cm⁻¹); 1684 (THF,

Δν_NO: 32 cm⁻¹); 1663 (DMSO, Δν_NO: 39 cm⁻¹).

2.3.9. Synthesis of K[Fe(LN₄^Ph)(NO)], {FeNO⁸} (5)

To a dark mixture of compound 3 (0.2000 g, 0.5778 mmol) in

5 mL of acetone was added KC₈(0.0850 g, 0.6288 mmol), which

immediately formed a dark-red homogeneous solution (apart

from the insoluble graphite). his mixture was allowed to stir for

an additional 5 min prior to removal of the graphite by ﬁltration.

The ﬁltrate was concentrated and treated with 2×5 mL of Et₂O, which

was decanted from the solid. The material was collected and dried to af-

ford 0.2001 g (0.5194 mmol, 90%) of a dark-red solid product. FTIR (KBr

matrix), ν_max(cm⁻¹): 3435 (w), 3069 (w), 2958 (w), 2917 (m), 2849

(m), 1705 (m, ν_CO), 1667 (m, ν_NO), 1589 (s), 1553 (s), 1463 (m), 1442

(w), 1382 (s), 1355 (m), 1285 (s), 1256 (m), 1223 (w), 1175 (w), 1092

(w), 1032 (s), 972 (w), 892 (w), 872 (w), 801 (w), 743 (s), 679 (w),

603 (w), 579 (w), 532 (w), and 481 (w). UV–vis (THF, 298 K), λ_max, nm

(ε, M⁻¹cm⁻¹): 364 (21,300), 409 (12,700), and 455 sh (8000). Anal.

calcd for C₁₆H₁₂FeKN₅O·acetone: C, 51.48; H, 4.09; N, 15.80. Found: C,

52.54; H, 3.75; N, 15.41. The compound may contain trace graphite (C₈)

from workup resulting in the higher than expected percent C.

2.4.2. Chemical oxidation of K[Fe(LN₄^PhCl)(NO)] (6)

This reaction and workup was performed analogously to the

oxidation of 5 except for using 0.0409 g (0.0901 mmol) of 6 and

0.0298 g (0.0900 mmol) of FcPF₆. The addition of FcPF₆resulted in

a burgundy-red insoluble material, which after workup yielded

0.0322 g (0.0776 mmol, 86%) of {FeNO}⁷complex 4. The FTIR spec-

trum (KBr) of this solid is consistent with authentic 4 (ν_NO

1720 cm⁻¹).

2.4.3. UV–vis monitoring of the reaction of {FeNO}⁷and {FeNO}⁸com-

pounds with [Fe(TPP)Cl]

A 1 mM stock solution of [Fe(TPP)Cl] in THF was prepared anaer-

obically and in the dark. Addition of a 0.025 mL aliquot of the

[Fe(TPP)Cl] stock to 2.975 mL of THF resulted in an 8.33 μM working

solution of [Fe(TPP)Cl]. After a THF blank was recorded at 298 K, the

spectrum of the working solution was recorded. The UV–vis spectrum

thus obtained was consistent with literature [50] and was monitored

over 15 min resulting in no observable change. To this cuvette was

then added a 0.025 mL aliquot of a 1 mM stock of the {FeNO}⁷(3 or

4 in THF) or {FeNO}⁸(5 or 6 in acetone) compounds (1:1 ratio of

[Fe(TPP)Cl]/{FeNO}^7/8) to initiate the reaction. The reaction mixture

was allowed to equilibrate at 298 K for 1 min and the spectrum was

2.3.10. Synthesis of K[Fe(LN₄^Ph)(¹⁵NO)], {Fe¹⁵NO}⁸(5–¹⁵NO)

The isotopically-labeled complex 5–¹⁵NO was prepared analogously

to 5 except for using 0.0901 g (0.2603 mmol) of 3–¹⁵NO dissolved in

3 mL of acetone and 0.0385 g (0.2848 mmol) of KC₈. Yield: 0.0871 g

120

B.C. Sanders et al. / Journal of Inorganic Biochemistry 118 (2013) 115–127

recorded. Subsequent UV–vis spectra were recorded at 298 K until no

further change was observed.

Tables 1 and S1 (see the Supporting information). Selected bond

distances and angles for one unique molecule of complex 3 are

given in Table 1. Perspective views of the complexes were obtained

using ORTEP [58]. An ORTEP view of complex 3 showing the disor-

dered O atom is illustrated in Fig. S1.

2.4.4. UV–vis monitoring of the reaction of K[Fe(LN₄^PhCl)NO] (6) with

[Fe(TPP)OTf]

The UV–vis reactions of 6 with [Fe(TPP)OTf] were performed anal-

ogously to those with [Fe(TPP)Cl] from Section 2.4.3. The [Fe(TPP)

OTf] was obtained by literature methods [51] and compared well to

known spectroscopic data including UV–vis (see Fig. S16 in the

Supporting information) and FTIR [51,52].

3. Results and discussion

3.1. Synthesis of {FeNO}⁷complexes

Coordination of HNO or its anion to Fe has been proposed in

several heme and non-heme systems as described previously

[31,32,34,35,41–44,59]. Although the list is sparse, the collective

use of porphyrin and cyclam N-ligands has been a popular choice in

the construction of {FeNO}⁸complexes. In this regard, we chose to

utilize non-macrocyclic but pseudo-planar N₄-ligand platforms

with electronically variable peripheral atoms, denoted as LN₄^RH₂

(H represents dissociable pyrrole protons; R is deﬁned above), for

the synthesis of FeNO complexes in this work. The presence of the

pyrrolide N-donors in these ligand frames replicates the electronic

properties provided by porphyrins/hemes but in a non-macrocyclic

i.e. non-heme environment. These ligands thus represent hybrid

heme/non-heme constructs that would allow one to achieve

{FeNO}⁸with the relatively weak π-basic pyrrolide-N, but with the

coordination ﬂexibility of a non-heme system. Indeed, such ligands

have been used in the construction of high-valent iron-oxos [60,61]

and vanadium-N₂O [62] coordination complexes. Collectively, our

strategy was to synthesize {FeNO}⁷complexes by introduction of

NO(g) to Fe(II) precursor complexes, namely (Et₄N)[Fe(LN₄^R)Cl]

(1 and 2), and then reduce the {FeNO}⁷species (3 and 4) chemically

or electrochemically to obtain the {FeNO}⁸complexes (5 and 6, see

Scheme 1).

2.4.5. Reaction of {FeNO}⁷and {FeNO}⁸compounds with [Fe(TPP)Cl]

The bulk reactivity studies were performed using 0.0200 g of

[Fe(TPP)Cl] (0.0284 mmol) and a stoichiometric equivalent of the

respective {FeNO}⁷(3/3–¹⁵NO or 4/4–¹⁵NO) and {FeNO}⁸(5/5–¹⁵NO

or 6/6–¹⁵NO) species. A solution of [Fe(TPP)Cl] was prepared either in

2 mL of THF (for {FeNO}⁷complexes 3 or 4) or 2 mL of THF/acetone

(1%) (for {FeNO}⁸5 or 6) to mimic the UV–vis conditions. To these

[Fe(TPP)Cl] solutions was added a 1 mL THF (3 or 4) or THF/acetone

(1%) (5 or 6) solution containing the {FeNO}⁷or {FeNO}⁸species,

respectively. The reaction was stirred at RT and in the dark for 2 h at

which point the solvent was removed in vacuo to afford a dark

brown-purple residue, which was characterized by FTIR. Pure [Fe(TPP)

NO] was isolated by treating the brown-purple mixture with ~2 mL of

MeOH, which resulted in an isolable product. Example yields: 0.0171 g

(0.0243 mmol, 86%) and 0.0186 g (0.0264 mmol, 93%) of purple solid

corresponding to [Fe(TPP)NO] and [Fe(TPP)¹⁵NO] from the reaction of

[Fe(TPP)Cl] with 6 and 6–¹⁵NO, respectively.

2.5. X-ray crystallographic data collection and structure solution and

reﬁnement

The {FeNO}⁷complexes were synthesized by direct NO(g) purge

into MeCN solutions of the precursor molecules, (Et₄N)[Fe(LN₄^Ph)Cl]

(1) and (Et₄N)[Fe(LN₄^PhCl)Cl] (2), to afford [Fe(LN₄^Ph)(NO)] (3) and

[Fe(LN₄^PhCl)(NO)] (4), respectively, as red-brown solids in ~80% yield

at RT (Scheme 1). Complexes 3 and 4 readily precipitated from

MeCN upon formation, which allowed for straightforward isolation

and puriﬁcation of the {FeNO}⁷product. Particularly, 3 precipitated

as a ﬁne microcrystalline solid due to its partial solubility in MeCN,

whereas 4 came out of the solution as an amorphous powder with

limited MeCN solubility. Complexes 3 and 4 appear to be relatively

stable with respect to air or excess NO(g) purge and thus can be

handled safely under ambient conditions.

Dark-red crystals of [Fe(LN₄^Ph)(NO)] (3) were grown under anaer-

obic conditions by slow diffusion of pentane into a toluene solution

of 3 at −20 °C. Suitable crystals were mounted on a glass ﬁber. All

geometric and intensity data were measured at 100 K on a Bruker

SMART APEX II CCD X-ray diffractometer system equipped with

graphite-monochromatic Mo Kα radiation (λ=0.71073 Å) with

increasing ω (width 0.5° per frame) at a scan speed of 10 s/frame

controlled by the SMART software package [53]. The intensity data

were corrected for Lorentz-polarization effects and for absorption

[54] and integrated with the SAINT software. Empirical absorption

corrections were applied to structures using the SADABS program

[55]. The structures were solved by direct methods with reﬁnement

by full-matrix least-squares based on F²using the SHELXTL-97

software [56] incorporated in the SHELXTL 6.1 software package

[57]. The atom of O(2) bonded to N(10) on the terminal NO group

was found disordered in two sets labeled as O(2) (one set) and

O(2′) (another set), respectively (see Fig. S1 in the Supporting infor-

mation). Each of these two sets is divided using the PART commands

and proper restraints. The set of O(2) has 60% occupancy while the

other O(2′) has 40% occupancy. The hydrogen atoms were ﬁxed in

their calculated positions and reﬁned using a riding model. All

non-hydrogen atoms were reﬁned anisotropically. Selected crystal

data and metric parameters for complex 3 are summarized in

3.2. X-ray crystal structure of [Fe(LN₄^Ph)(NO)] (3)

Single crystals of {FeNO}⁷complex 3 were grown from slow diffu-

sion of pentane into a toluene solution of 3 at −20 °C over the course

of 3 days. There are two unique molecules in the asymmetric unit of 3

which are nearly isostructural (Figs. 1 and S1) and their metric param-

eters are reported below (Table 2) and in the Supporting information

(Table S2). No signiﬁcant close contacts are observed between the

molecules; however, there appears to be a weak interaction between

the nitrosyl N (N5) of one molecule and the phenyl carbon (C23) of

Scheme 1. Synthetic routes for {FeNO}^7/8complexes. (i) NO (g), MeCN, RT; (ii) KC₈, acetone, RT or [Co(Cp*)₂], toluene, RT; (iii) FcPF₆, MeCN, RT. R groups are deﬁned in Chart 2.

B.C. Sanders et al. / Journal of Inorganic Biochemistry 118 (2013) 115–127

121

bond length due to the relaxed and non-conjugated coordination

i.e. the _imine–Fe–N_imine(avg.: 90.10°) and _pyrrole–Fe–N_pyrrole

(avg.: 95.39°) angles are more ideal. The Fe center in 3 is situated

slightly above the plane deﬁned by the four N-ligands of LN₄by

0.41 Å and is reﬂected in the bond angles of trans N-donors (in the

basal plane) e.g. N2–Fe1–N4: 152.05°, which are signiﬁcantly less

than the 180° expected for a perfect square-pyramid.

The metric parameters associated with the Fe–N–O unit are often

indicative of the nature of the Fe and NO in this highly delocalized

bond. Complex 3 displays features that are mostly characteristic of

other 5C Sq-Py {FeNO}⁷complexes (vide supra); however, some

differences are noted. Analogous to other {FeNO}⁷complexes

[29,65–67], the Fe–N (avg.: 1.696 Å) and N–O (avg.: 1.150 Å)

distances are short and generally consistent with a LS-Fe(II)–NO•

assignment for this complex (vide infra). The Fe–N–O angle in 3

(avg.: 154.4°) is bent from linearity, which is somewhat representa-

tive for this class of nitrosyls and reﬂective of a radical localized on

the nitrogen atom of NO and veriﬁed by the N-hyperﬁne coupling to

g_zin the EPR spectrum (vide infra). However, the average Fe–N–O

angles in similar LS 5C {FeNO}⁷complexes are more bent (Fe–N–O

~140°) [29,66], ~15° less than the angle found in complex 3. Thus,

complex 3 is certainly an outlier with respect to the Fe–N–O angle.

This deviation should be reﬂected in the ν_NOstretching frequency in

the IR; however, the IR spectra of 3 and 4 are quite in-line for {FeNO}⁷

complexes (vide infra). Indeed, computations by Ghosh [68] and

Lehnert [36] for a variety of LS (S=1/2) heme and non-heme 5C

systems suggest that this linearity is due to signiﬁcant d_z–p_zmixing

which alleviates repulsion between the σ lone pair on NO and the

Fe d_z²-based HOMO (highest occupied molecular orbital). The DFT

(density functional theory) results by Ghosh revealed a positive

correlation between the Fe–N–O angle and the percentage of Fe p_z

in the HOMO and a negative correlation with the Fe–N distance in

several theoretical 5C Sq-Py LS {FeNO}⁷derivatives. That is, the larger

Fe–N–O angle leads to greater %p, less electron repulsion, and greater

Fe–N(O) overlap (or contracted Fe\N bond). In using these theoretical

results with those experimentally obtained for complex 3 i.e. its Fe–N–O

angle of ~155°, one would estimate an 8–9% Fe p_zcharacter in its HOMO

and an Fe–N distance of ~1.68–1.69 Å. The Fe–N distance obtained from

the theoretical correlation line ﬁts well with the experimental value.

Thus, the independently reported theoretical results have been

somewhat validated by our experiments. Accordingly, the more

bent angle typically observed in 5C {FeNO}⁷hemes (avg.: 144°)

correlates with a ~3–4% Fe p_z-character in the HOMO and an Fe–N

distance of ~1.73 Å (compare with avg.: 1.73 Å from experiment

[29,65–67,69]). Taken together, our experiments in combination

Fig. 1. ORTEP diagram of [Fe(LN₄^Ph)(NO)] (3) (one unique molecule) at 50% thermal

probability ellipsoids for all non-hydrogen atoms with the atom labeling scheme. Se-

lected bond distances and angles are given in Table 2.

another (N5–C23: 3.125 Å; sum of the van der Waals radii for N and C:

3.25 Å [63]; see Fig. S2) suggestive of N_π–C_πoverlap. The Fe center in 3

is coordinated in a square-pyramidal (Sq-Py) geometry from the planar

[LN₄^{Ph 2−}

ligand comprising the basal plane and N-coordinated NO in

]

the axial position (Fig. 1). This polyhedral assignment is demonstrated

by a τ value of nearly 0 (τ_avg.[average of two independent mole-

cules]=0.025 for 3), where τ is the trigonal distortion parameter as

deﬁned by Addison and Reedijk [64]. The metric parameters (Table 2)

of the complex are consistent with a LS conﬁguration about the Fe center,

especially the Fe–L distances that are all less than 2 Å. The Fe\N_imine

bonds (avg.: 1.926 Å) are slightly shorter than the corresponding

Fe\N_pyrrolebonds (avg.: 1.966 Å). These distances appear to be a

bit contracted, but mostly consistent with other ﬁve-coordinate

(5C) Sq-Py {FeNO}⁷complexes such as [Fe(TPP)(NO)] and

[Fe(OEP)(NO)], which display average Fe–N_pyrroledistances of

~2.00–2.01 Å [29,65]. Although the N_pyrroleis generally considered

as a stronger-ﬁeld ligand than N_imine, the resulting distances in the

structure of 3 presumably arise from the signiﬁcantly acute bite angle

of the phenylenediimine portion of LN₄(avg. N_imine–Fe–N_imine: 81.89°)

versus the more open angle of the N_pyrrole–Fe–N_pyrrole(avg.: 104.22°). In

contrast, the similarly disposed {FeNO}⁷complex [Fe(LN₄^pr)(NO)] (7),

containing a propyl linker between the diimine donors, displays shorter

Fe\N_pyrrole(avg.: 1.945 Å) versus Fe\N_imine(avg.: 1.984 Å) bonds [43].

It is possible that the greater ﬂexibility in the ligand frame of 7 allows

for electronic inﬂuences of the N-donor to be more representative in the

Table 2

Selected bond distances (Å) and bond angles (deg) for one of the two unique molecules

of [Fe(LN₄^Ph)(NO)] (3) as depicted in Fig. 1. See Table S2 and Fig. S1 for metric param-

eters of the other unique molecule.

[Fe(LN₄^Ph)(NO)] (3)

Fe1–N1

Fe1–N2

1.960(3)

1.918(3)

Fe1–N3

1.928(3)

Fe1–N4

1.986(3)

Fe1–N5

1.694(3)

-1

N5–O1

1.150(4)

155.6(3)

ν_{NO: 1720 cm}

O1–N5–Fe1

N1–Fe1–N2

N1–Fe1–N3

N1–Fe1–N4

N1–Fe1–N5

N2–Fe1–N3

N2–Fe1–N4

N2–Fe1–N5

N3–Fe1–N4

N3–Fe1–N5

N4–Fe1–N5

-1

ν_{15NO: 1686 cm}

81.97(12)

153.72(12)

104.40(12)

95.90(13)

81.84(12)

152.05(12)

101.41(13)

81.36(12)

107.51(13)

104.87(13)

Fig. 2. FTIR spectra of [Fe(LN₄^PhCl)(NO)] (4) (solid line) and [Fe(LN₄^Ph)(¹⁵NO)] (4–¹⁵NO)

(dashed line; inset) in a KBr matrix.

122

B.C. Sanders et al. / Journal of Inorganic Biochemistry 118 (2013) 115–127

with theoretical results by others [36,68] suggest that 5C {FeNO}⁷

complexes are better described as LS-Fe(I)–NO⁺, likely in resonance

with LS-Fe(II)–NO• owing to the highly delocalized nature of this

bond.

E : -1.23V

3.3. Spectroscopic and reactive properties of {FeNO}⁷complexes

3.3.1. {FeNO}⁷spectroscopic properties

In addition to the structural characterization of 3, various spectro-

scopic measurements on {FeNO}⁷complexes 3 and 4 were performed.

Generally, FTIR spectroscopy is employed to gauge the strength of the

N\O and M\N(O) bonding in metal–nitrosyls. In particular, the

intense N–O stretching frequency (ν_NO) can be particularly informative

in MNO systems, but should be interpreted with caution [29]. For

example, the solid-state ν_NOvalues for 3 and 4 are observed at 1698

and 1720 cm⁻¹(KBr), respectively (Fig. 2 for 4). These values do not

appear to shift signiﬁcantly in the solution-state for a variety of

weakly-/non-coordinating solvents (~1720 cm⁻¹), but a red-shift for

4 at ~15 cm⁻¹(1705 cm⁻¹) in donor solvents such as MeCN and

DMSO, indicative of potential solvent coordination in these solutions.

Predictably, the ν_NOof 4 is blue-shifted from 3 due to the electron

withdrawing nature of the peripheral Cl groups on the ligand resulting

in decreased Fe π-back-donation into the NO π* MO [29,36,70]. The

average ν_NOvalues for the 5C Sq-Py complexes also appear in this

range (1630–1690 cm⁻¹) [29,66]. When ¹⁵NO(g) is used in the prepa-

ration of 3 and 4, the ν_NOvalues shift in agreement with the classic

harmonic oscillator model, ν_NO: 1667 cm⁻¹(KBr, Δν_NO: 31 cm⁻¹) for

3 and ν_NO: 1686 cm⁻¹(KBr, Δν_NO: 34 cm⁻¹) for 4 (Fig. 2 for 4–¹⁵NO).

The 5C {FeNO}⁷complexes 3 and 4 are paramagnetic and display

magnetic properties that are consistent with a doublet electronic

ground state (S=1/2), which is typical for this class of iron–nitrosyls

[29,65]. This assignment has been conﬁrmed by magnetic moment

measurements (see Materials and methods) and X-band EPR spec-

troscopy. For example, EPR measurements (10 K, 3:1 toluene/MeCN)

on 3 and 4 reveal an axial feature at g~2.07–2.01 consistent with the

high symmetry of these Fe–N₄–NO complexes (see Supporting

information, Fig. S3 for complex 4). Additionally, hyperﬁne coupling

to the I=1 nucleus of the NO nitrogen with A~16 G is observed in

the g_zcomponent. DFT calculations [36,43] have shown that this hyper-

ﬁne coupling is actually representative of a minority spin-density local-

ized on N with the majority spin localized on Fe and consistent with an

LS-Fe(I)–NO⁺description (vide supra). Complexes 3 and 4 are red-

brown in color in organic solvents such as DMF and THF displaying sim-

ilar UV–vis spectral features with an intense λ_max~350 nm and a visible

shoulder at ~470 nm. The electronic transitions at 350 and 470 nm

have been tentatively assigned as π–π* from the ligand frame and

MLCT (metal-to-ligand charge-transfer), respectively. There appears

to be little difference in the UV–vis spectrum when the solvent dielec-

tric is changed to DMF indicating that the electronic structure is similar

in coordinating and weak-/non-coordinating solvents at least at 298 K.

Fig. 3. Cyclic voltammogram (solid line) and differential pulse voltammogram (dashed

line) of a 10 mM MeCN solution of [Fe(LN₄^Ph)(NO)] (3) (0.1 M ⁿBu₄NPF₆supporting elec-

trolyte, glassy carbon working electrode, Pt-wire counter electrode, 100 mV/s scan speed,

RT). The arrow displays the direction of the scan.

thus more difﬁcult to reduce than 4 due to the electron-withdrawing

nature of the Cl substituents on the ligand. These values compare

favorably with the {FeNO}⁷/{FeNO}⁸redox couples for [Fe(TPP)(NO)]

(−0.93 V vs. SCE in CH₂Cl₂) and [Fe(OEP)(NO)] (−1.08 V vs. SCE in

THF, see Table 1), but are signiﬁcantly and expectedly more negative

than the halogenated FeNO complex [Fe(TFPPBr₈)(NO)] (−0.19 V vs.

SCE in CH₂Cl₂) (Table 1). The electrochemical results thus conﬁrm

that the {FeNO}⁸oxidation state is accessible with minimal structural

rearrangement within these ligand architectures.

3.4. Synthesis and spectroscopic properties of {FeNO}⁸complexes

3.4.1. Synthesis of {FeNO}⁸complexes

The {FeNO}⁸complexes were synthesized via chemical reduction of

the {FeNO}⁷complexes 3 and 4 to generate [Fe(LN₄^Ph)(NO)]⁻(anion

of 5) and [Fe(LN₄^PhCl)(NO)]⁻(anion of 6), respectively, where the cation

depends on the reductant used in the synthesis (Scheme 1). In previous

accounts by our lab [43] and others [34], this reduction has been ac-

complished using cobaltocene [Co(Cp)₂] or decamethylcobaltocene

[Co(Cp*)₂] as reducing agents with the selection being dependent

on the potential of the {FeNO}⁷/{FeNO}⁸redox couple. These

metallocene reductants work reasonably well, but the necessity to

often purify [Co(Cp*)₂] and the insolubility of the {FeNO}⁸as the

[Co(Cp*)₂]⁺salt complicated the synthesis. For example, Geiger

and Connelly recommend that [Co(Cp)₂] be sublimed before use,

stored in the dark at −10 °C, and used within 10 days [71]. Regard-

less, reaction of complexes 3 or 4 with [Co(Cp*)₂] in toluene at RT

resulted in respectable yields of the {FeNO}⁸complexes, [Co(Cp*)₂]

[Fe(LN₄^Ph)(NO)]

( (

^Cp⁎5) and [Co(Cp*)₂][Fe(LN₄^PhCl)(NO)] ^Cp⁎6),

3.3.2. Electrochemical properties

which precipitated from the reaction medium. The non-methylated

[Co(Cp)₂] would presumably afford a more polar aprotic soluble

cobaltocenium salt, but could not be used since it is not strong

enough of a reducing agent. Although this synthesis was successful

in our hands, the need for constant puriﬁcation of the metallocene

led us to select other chemical reductants. Thus, inspired by classic

reagents used in organometallic reactions, we employed potassium

graphite (KC₈) as the reducing agent. Similarly, the {FeNO}⁸

complexes K[Fe(LN₄^Ph)(NO)] (5) and K[Fe(LN₄^PhCl)(NO)] (6) were

obtained by reaction of stoichiometric KC₈with acetone solutions of

3 and 4, respectively. It is important to point out that stoichiometric

KC₈should be employed in the synthesis as excess leads to disparate

reactivity and multiple reduced species probably associated with the

imine functionality, which is well documented for this reductant

[72,73]. The yields are nearly quantitative and the resulting {FeNO}⁸

The electrochemical properties of MNO systems are of principal

importance since the general goal of our research is to utilize the

changes in the MNO redox levels to access underexplored EF

notations in metal–nitrosyls and, more speciﬁcally, the {FeNO}⁸

state in this contribution. It is therefore a general requirement to con-

struct molecules that display reversible/diffusion-controlled MNO

redox couples in order to synthesize an {FeNO}⁸complex from an

{FeNO}⁷precursor. Indeed, the cyclic voltammograms (CVs) of 3

and 4 revealed this property. For example, the CV of 3 and 4 in

MeCN/RT displayed a reversible E_1/2at −1.23 V (ΔE_p: 0.110 V) and

−1.16 V (ΔE_p: 0.078 V) (both vs. Fc⁺/Fc; or −0.83 and −0.76 V,

respectively vs. SCE [71]), respectively (see Figs. 3–4). Differential

pulse voltammetry (DPV) further conﬁrmed the CV results (see

Figs. 3–4). As expected, the E_1/2for 3 is 70 mV more negative and

B.C. Sanders et al. / Journal of Inorganic Biochemistry 118 (2013) 115–127

123

disparate C\C bond distances in the X-ray structure especially in the

pyrrolide ring (see Table S1). The ν_NOband of 6; however, shifted signif-

icantly from parent 4 (1720 cm⁻¹) to ~1580 cm⁻¹(approximate due

to overlap with LN₄ν_{C_N}bands, see Fig. 5). Both 5 and 6 also display

E : -1.16V

_COvalues at 1705 cm⁻¹that are similar to metal-coordinated acetone

complexes (ν_CO: 1660–1700 cm⁻¹) [74]. The presence of acetone was

also conﬁrmed by elemental analysis (see Materials and methods)

and mass spectrometry measurements (vide infra). Analogous to other

{FeNO}⁸complexes [34], the ν_NOis obstructed from ligand vibrational

bands. The ν_NOvalue for 6 thus supports reduction in the FeNO unit

due to increased occupancy of π* orbitals on NO from its {FeNO}⁷precur-

sor 4. These values are also consistent with other heme {FeNO}⁸

complexes that have been synthesized or studied by high-level

DFT calculations (see Table 1). They are, however, quite higher

than the corresponding values for non-heme {FeNO}⁸systems (avg.:

1300 cm⁻¹) suggesting that the electronic nature of the LN₄frame in

6 is more heme-like.

High-resolution FTMS (HRMS) studies were also performed to

conﬁrm the identity of the {FeNO}⁸complexes. Single crystals of such

systems have been difﬁcult to grow due to the reactive nature of these

complexes (vide infra). As such, no small molecule {FeNO}⁸complex

to date (2012) has been characterized by X-ray crystallography or

X-ray absorption (XAS) studies although the {FeHNO}⁸analogue of

myoglobin has been reported based on XAS [38,40]. Thus, HRMS

would provide further conﬁrmation of these elusive metal nitrosyls.

The HRMS (negative ion mode) experiments do provide such evidence,

as the molecular ion peak [M]⁻for 5 (m/z: 346.0385; calcd: 346.0386)

does appear with the appropriate isotope pattern (see Supporting

information). Complex 6, on the other hand, only displayed peaks con-

sistent with [M+acetone]⁻(m/z: 472.0000; calcd: 472.0026) and

[M+acetone−H]⁻(m/z: 470.9967; calcd: 470.9947) with the latter

being the predominant species. The ¹⁵N isotopomers were also exam-

ined and support the formulation predicted. To the best of our knowl-

edge, the MS studies on the Fe–LN₄^R–NO systems [43] represent the

only MS measurements performed on such FeNO complexes.

Other spectroscopic methods were employed to characterize

complexes 5 and 6. Both 5 and 6 demonstrate excellent solubility in

a variety of organic solvents such as acetone, MeCN, and DMF affording

red-brown colored solutions; moderate solubility is observed in

non-polar solvents such as THF. The UV–vis spectrum of the {FeNO}⁸

complex 6 is similar in shape to {FeNO}⁷complex 4 with some minor

changes (Fig. 6). To date (2012), all reported non-heme {FeNO}⁸

complexes have been reported as diamagnetic; however, this descrip-

tion represents a small sample size (N=2) as the majority of these

systems have only been characterized in situ. This property suggests

that, if we assign the coordinated NO as a nitroxyl anion, the

LS-Fe(II)–¹NO⁻would be the favorable assignment for {FeNO}⁸. Com-

plex 6 displays a complex ¹H NMR spectrum that is consistent with a

diamagnetic system (see Supporting information). The complexity

presumably arises from ligation/deligation of solvent (vide infra) or

structural rearrangement of the LN₄ligand around the Fe center. In con-

trast, the ¹H NMR of 5 is observable, but appears broad in nature and

consistent with a paramagnetic material. The NMR broadness is attrib-

uted to formation of an unknown paramagnetic species upon complex

dissolution and/or a thermally accessible higher spin-state (i.e. ³NO⁻

versus ¹NO⁻or ligand reduction). We favor this description since the

ν_NOof 5 matches quite well with the calculated ν_NOvalues for 6C

{FeNO}⁸complexes, which range from 1612 to 1777 cm⁻¹for

[Fe(P)(Im)(NO)]⁻complexes in the S=1 state [36,37]. Additionally, a

paramagnetic triplet ground state has been assigned to the {FeNO}⁸

adduct of the non-heme TauD system suggesting that higher spin mani-

folds may be accessible for 5 [44]. Although the extra stability of the

{FeNO}⁸in 6 is not quite signiﬁcant from an electrochemical vantage

point (~100 mV), perhaps the presence of the Cl substituents lowers

the energy of the ligand π-orbitals just enough to prevent parallel

spin occupation and/or ligand reduction. Collectively, it appears that

Fig. 4. Cyclic voltammogram (solid line) and differential pulse voltammogram (dashed

line) of a 1 mM MeCN solution of [Fe(LN₄^PhCl)(NO)] (4) (0.1 M ⁿBu₄NPF₆supporting elec-

trolyte, glassy carbon working electrode, Pt-wire counter electrode, 100 mV/s scan speed,

RT). The arrow displays the direction of the scan.

complexes display reasonable solubility in most organic solvents such

as MeCN. Additional beneﬁts of this synthetic route include the relative

insolubility of the graphite by-product, which is easily separated from

the FeNO complex by simple ﬁltration. However, unlike 3 and 4, com-

pounds 5 and 6 are air-sensitive materials that require anaerobic condi-

tions for workup, storage, and characterization.

3.4.2. {FeNO}⁸spectroscopic properties

As described above, vibrational spectroscopy is one of the principal

methods of MNO complex characterization, and vibrational measure-

ments on {FeNO}⁸complexes are rare (see Table 2). Additionally, one

would expect signiﬁcant changes in ν_NOamong {MNO}ⁿredox isomers

depending on the extent of NO-based redox. Indeed, complexes 5 and 6

display shifted ν_NObands from their parent oxidized complexes 3 and 4,

albeit to a different extent. For example, the solid-state IR spectrum of 5

displayed ν_NOat 1667 cm⁻¹(identiﬁed by ¹⁵N isotopic labeling), which

is only 43 cm⁻¹away from the parent {FeNO}⁷complex 3. This value

appears to be more consistent with a ligand-based reduction instead

of {FeNO} unit reduction although this result may be more indicative

of a thermally accessible triplet excited state (vide infra). A ligand

based reduction would not be too surprising given the highly

delocalized nature of the LN₄scaffold, which is evidenced by the

^-1(4)

ν_{NO: 1720 cm}

^-1(6)

ν_{NO: 1580 cm}

Fig. 5. FTIR spectra of K[Fe(LN₄^PhCl)(NO)] (solid line) (6) and [Fe(LN₄^PhCl)(NO)] (dashed

line; inset) (4) in a KBr matrix. The peak at 1705 cm⁻¹in 6 represents coordinated

acetone ν_CO

124

B.C. Sanders et al. / Journal of Inorganic Biochemistry 118 (2013) 115–127

λ: 371 nm

λ: 419 nm

Fig. 6. UV–vis spectral monitor of an 8.33 μM THF solution of K[Fe(LN₄^PhCl)(NO)] (6) at 298 K. Left: UV–vis monitor of 6 upon initial dissolution (dashed line) and subsequent traces

(solid lines) for 15, 30, 45, and 60 min thereafter. Right: continuation of spectral monitor on the left only at 1 h intervals (total time=12 h); inset depicts the ﬁrst (dashed line) and

last (solid line) scans. Arrows illustrate the direction of change.

heme-based {FeNO}⁸complexes and 6 display ν_NO: 1500–1600 cm⁻¹

and generally prefer to be ground state singlets. However, non-heme

{FeNO}⁸systems (excluding the examples reported here and elsewhere

[43]) display ν_NO~1300 cm⁻¹. This data suggests an oxidation state

assignment of LS-Fe(II)–¹NO⁻for the non-heme {FeNO}⁸and more of a

resonance LS-Fe(II)–¹NO⁻↔LS–Fe(I)–NO• description for the heme

{FeNO}⁸.

reaction takes nearly 13 h to go to completion [43]. Thus, complex 7

could still be used as a potential nitroxyl donor provided that release

of nitroxyl is faster than the disproportionation. We hypothesized

that this reaction may be a common feature for {FeNO}⁸species at

least with the LN₄type of ligand platform. As expected, when a THF

solution of 6 was monitored by UV–vis spectroscopy (298 K), distinct

changes in the visible region were observed with a decrease in the

λ_maxat 371 and 419 nm and new bands appearing at λ_maxof 361

and 481 nm over the course of 9 h (Fig. 6). Complex 6 is thus more

reactive than 7. There appears to be little change in the UV–vis over

the course of ~2 h indicating modest solution stability of the

{FeNO}⁸complex (Fig. 6). Additionally, several isosbestic points are

present in the spectrum during this process at 359, 384, 403 and

490 nm supporting a clean transformation with the ﬁnal trace

displaying bands at 361 and 481 nm, which appear to be indicative

of the {FeNO}⁷complex 4. FTIR analysis of this reaction also revealed

the ν_NObands of 4, which further conﬁrmed the formation of the

{FeNO}⁷complex. Further examination of the UV–vis experiments

indicates that {FeNO}⁷4 is obtained in a quantitative yield, which

eliminates a disproportionation reaction. This result suggests that a

simple oxidation is taking place over prolonged storage of 6 in solu-

tion to result in 4 and reduction of the solvent. Another path to obtain

3.5. Reactivity of {FeNO}⁷and {FeNO}⁸complexes

The reactivity of {FeNO}⁸complexes have received little attention

with the exception of the protonation studies by Doctorovich on

[Fe(TFPPBr₈)(NO)]⁻[34], which was shown to liberate H₂(g) and

reform the {FeNO}⁷complex via a transient {FeHNO}⁸complex. As

the reactivity of {FeNO}⁸systems is underexplored and of fundamen-

tal importance with respect to the biological fate of HNO/NO⁻, we

have initiated such reactivity studies in the present contribution.

Due to principal reduction of the {FeNO} unit in 6, the majority of

our reactivity discussion is focused on this system; however, reac-

tions with 5 (a complex more consistent with ligand-based reduction

or paramagnetic spin-state) were also performed and resulted in

nearly parallel outcomes.

In the absence of structural characterization of 5 or 6 and to support

the electrochemical results, the conversion of the {FeNO}⁸complexes

back to their oxidized {FeNO}⁷counterparts was performed. For

instance, treatment of a MeCN solution of the {FeNO}⁸complexes 5 or

6 with a stoichiometric amount of ferrocenium hexaﬂuorophosphate

resulted in quantitative formation of the corresponding {FeNO}⁷

complexes 3 and 4 as monitored by FTIR spectroscopy. The reversibility

of the FeNO redox states is depicted in Scheme 1. Thus, the

{FeNO}^7/8complexes (3/5 or 4/6) are chemically and electrochemically

interconvertable.

407 nm

537 nm 565 nm

425 nm

606 nm

540 nm

564 nm

The difﬁculty in the isolation of single crystals of {FeNO}⁸deriva-

tives is likely derived from the inherent reactivity of these systems.

For example, Kadish and Olson reported that exhaustive electrolysis

of the {FeNO}⁸complex of TPP simply resulted in isolation of the

starting {FeNO}⁷species [33]. This outcome is likely due to the

presence of some protic material in the non-polar CH₂Cl₂solvent

that resulted in the reaction of H⁺with the formed {FeNO}⁸complex

in an analogous fashion to what is observed by Doctorovich [34]. The

inherent reactivity of this class of iron–nitrosyls at least provides

some explanation for the lack of isolable {FeNO}⁸complexes in the

literature. Previously, our group reported that the structurally similar

{FeNO}⁸complex 7 undergoes disproportionation reactions in MeCN

or THF to yield the {FeNO}⁷complex and an Fe(I)–N₂species. This

609 nm

Fig. 7. UV–vis spectrum of an 8.33 μM THF solution of [Fe(TPP)Cl] (dashed line),

1 min after (dotted line), and 1 h after (solid line) the addition of 1 mol-equiv of

K[Fe(LN₄^PhCl)(NO)] (6) at 298 K. Inset: expansion of the Q-band region of the spec-

trum. Arrows are coded according to the spectrum they represent.

B.C. Sanders et al. / Journal of Inorganic Biochemistry 118 (2013) 115–127

125

{FeNO}⁷from {FeNO}⁸would be via protonation as demonstrated by

Doctorovich [34] as described above; however, this reaction seems

unlikely under the anhydrous/anaerobic conditions employed.

Complex 5 also undergoes a similar reactivity proﬁle in THF but

appears to form the {FeNO}⁷species more rapidly (6 h).

NO] complex (Scheme 2, path i). Alternatively, this spectral change to

425 nm may suggest the formation of other transient 6C {FeNO}⁷–

porphyrin complexes. In fact, the 6C complex, [Fe(TPP)(NO)(MI)],

prepared by Lehnert's group [69] has a Soret absorption band at

425 nm and Q-bands at 538 and 600 nm in CH₂Cl₂. The intermediate

formed here displays λ_maxat 425, 537, and 606 nm, which is slightly

red-shifted and more intense from the ﬁnal trace with λ_maxat 407,

The established reductive nitrosylation [75] reaction of HNO with

ferric hemes is among the most sensitive chemical tests for HNO-

releasing molecules where HNO provides both the electron and NO

to form an {FeNO}⁷ferrous-NO heme [39,76,77]. Additionally,

M(III)-porphyrin complexes have been demonstrated as efﬁcient

traps for HNO [76,78,79]. To test whether complexes such as 6 could

react in a similar manner, we explored the interaction of these

{FeNO}⁸complexes with the ferric heme analogue [Fe(TPP)Cl] under

dark conditions to prevent any ill-deﬁned photochemistry. The reaction

of a THF solution of [Fe(TPP)Cl] at 298 K with stoichiometric amounts of

the {FeNO}⁸complexes was monitored by UV–vis and IR spectros-

copies. Addition of the {FeNO}⁸complex 6 to [Fe(TPP)Cl] (1:1 ratio)

resulted in several changes in the UV–vis spectrum, which are ultimate-

ly consistent with the formation of the {FeNO}⁷porphyrin complex,

[Fe(TPP)NO], after ~1 h (Fig. 7). The trace after 1 min addition of 6

was a red-shift in the Soret band of [Fe(TPP)Cl] from 412 nm to

425 nm with a signiﬁcant increase in absorbance. Spectral changes

such as this have been attributed to several transformations of

[Fe(TPP)Cl]. For example, a similar shift has been observed in the

photo-reduction of [Fe(TPP)Cl] in MeOH to form what is assigned as

the 6C bis-solvato complex, [Fe(TPP)(MeOH)₂] [80,81]. While a reduced

solvato species is possible in the reported reaction, we must note that

the 6C complex described above was formed in MeOH and is only stable

on the millisecond timescale. In contrast, these reactions were

performed in weakly coordinating THF and the λ_max: 425 nm species

is observed on the minute timescale. Authentic [Fe^II(TPP)] has been

prepared in THF by Darensbourg and coworkers where they report a

Soret λ_maxof 412 nm [82]; however, Scheidt and coworkers report

the Soret of [Fe^II(TPP)(THF)₂] at 425 nm in THF [83]. Therefore, a possi-

ble mechanism involving electron transfer from 6 to transiently form a

6C [Fe^II(TPP)(THF)(Cl)]⁻or [Fe^II(TPP)(THF)₂] species cannot be exclud-

ed. This step would be followed by NO• transfer from the resulting

{FeNO}⁷complex 4 to the ferrous porphyrin to furnish the [Fe(TPP)

540, and 609 nm and in-line with authentic [Fe(TPP)NO] (λ_max

408, 536, 607 nm in THF [31]). Support for the 6C-NO porphyrin

path is found in the Q-band region (Fig. 7). Accordingly, if initial

one-electron reduction of [Fe(TPP)Cl] to [Fe(TPP)(THF)(Cl)]⁻or

[Fe(TPP)(THF)₂] is to occur, then this region would change notice-

ably compared to that of [Fe(TPP)Cl] [84]. The UV–vis experiments

show that the Q-band region is essentially unchanged after the ﬁrst

scan (1 min after mixing), whereas the Soret continues to shift

over 1 h toward the 407 nm band indicative of [Fe(TPP)NO]. This

observation further implicates a 6C-NO porphyrin as the ﬁrst step

in the reductive nitrosylation. When [Fe(TPP)OTf] was prepared

[51] and subjected to the same conditions used for [Fe(TPP)Cl] and

6, a similar 426 nm band is observed in the ﬁrst spectral trace.

Though the OTf⁻anion is considered to be a weaker ﬁeld ligand

than Cl⁻, previous reports describe [Fe(TPP)OTf] as a 5C species

instead of an ion pair [51,52]. Since the resulting UV–vis spectral

changes were nearly identical to that of compound 6 with [Fe(TPP)

Cl], we propose that the net NO⁻transfer is occurring through a similar

mechanism with both 5C ferric porphyrins. Thus, a potential mecha-

nism could involve the formation of a binary complex where the NO

ligand of 6 bridges the two reactants to form a [P–Fe(μNO)Fe–L]⁻inter-

mediate (Scheme 2, path ii). This species is comparable to the interme-

diate observed in the transnitrosation of thiolates with nitrosothiols

(RSNO) where attack of the nucleophilic thiolate anion on the electro-

philic RSNO produces the anionic RSN(O)SR⁻intermediate [85,86].

This type of NO-transfer has been postulated to be involved in safe

NO-trafﬁcking, which never involves the release of free NO. By analogy,

the process described here would be deﬁned as a “transnitroxylation”

where the coordinated NO⁻is transferred from one molecule to anoth-

er without involving free NO⁻. Electron transfer and nitrosylation take

place through the bridge i.e. an inner-sphere mechanism and the 6C

Scheme 2. Proposed reaction path (i = electron transfer vs. ii transnitroxylation) intermediates (bracketed and bolded) in the reaction of {FeNO}⁸complex with [Fe^III(TPP)Cl]. The

oval represents porphyrin in the intermediate complex. A similar reaction path is proposed with [Fe^III(TPP)OTf] (see Fig. S17).

126

B.C. Sanders et al. / Journal of Inorganic Biochemistry 118 (2013) 115–127

intermediate decays to yield the more stable 5C [Fe(TPP)NO] complex

and 2 perhaps via a monomeric [Fe^II(TPP)(NO)Cl] complex after split-

ting the bridge (see Scheme 2). The identity of the 425 nm band could

be any of these 6C FeNO intermediates. Therefore, the net reaction

would be: [Fe(LN₄^R)(NO)]⁻(anion of 6)+[Fe(TPP)Cl]→[Fe(TPP)

NO]+[Fe(LN₄^R)Cl]⁻(anion of 2). Further support for this chemistry

comes from IR analysis. The FTIR spectrum of the bulk reaction mixture

of [Fe(TPP)Cl]/complex 6 (1:1) conﬁrmed this postulate and the only

ν_NObands observed are from [Fe(TPP)NO] (ν_NO: 1701 cm⁻¹in KBr),

which shifted when 6–¹⁵NO was used (ν_NO: 1669 cm⁻¹in KBr) (see

the Supporting information). Furthermore, the UV–vis spectrum of

independently-prepared complex 2 and [Fe(TPP)NO] (mixed in a 1:1

ratio) in THF at 298 K nearly resembles the ﬁnal spectrum of the reac-

tion described above (see Supporting information). The control reaction

of [Fe(TPP)Cl] with the {FeNO}⁷complexes 3 and 4 also appears to gen-

erate [Fe(TPP)NO]; however, the reaction time is long (>12 h) and

incomplete according to IR and UV–vis spectroscopies. Additionally,

Darensbourg observes a similar reaction of [Fe(TPP)Cl] or [Fe(OEP)Cl]

with a 5C {FeNO}⁷–N₂S₂complex that also takes place over a similar

(24–72 h) timescale [82]. Thus, while {FeNO}⁷can ultimately result in

the same product as with {FeNO}⁸(5 and 6) and dinitrosyl iron

complexes (DNICs, the reactions studied by Darensbourg), the reaction

path appears very different.

(v) These air-sensitive {FeNO}⁸materials appear to be indeﬁnitely

stable in the solid-state when stored under anaerobic/anhydrous

conditions in the dark. Their solution-state solubility is different

resulting in slow re-oxidation to the {FeNO}⁷derivatives 3 and 4

over the course of 6–9 h in both polar and apolar organic solvents.

The ultimate explanation behind this reactivity is unknown at

present, but it is consistent with the label for this class of FeNO

systems as “elusive”. However, it is important to point out that

these are slow processes with respect to the potential for such

molecules to serve as HNO sources.

(vi) Finally, complexes 5 and 6 exhibit a nitroxyl-like reactivity with

the ferric porphyrin complex, [Fe(TPP)Cl], to afford [Fe(TPP)NO]

in stoichiometric yield. UV–vis monitoring of this reaction sug-

gests that a potential 6C {FeNO}⁷is an intermediate traversed in

the reaction path which may occur through a “transnitroxylation”

process.

In sum, unraveling the chemistry of iron–nitroxyl/{FeNO}⁸deriva-

tives is challenging as they represent synthetic targets that are difﬁcult

to achieve and maintain. Once stabilized, at least to some extent, they

offer new avenues of MNO reactivity that have never been explored.

To the best of our knowledge a “transnitroxylation” process has not

been proposed before (in stark contrast to transnitrosation), presum-

ably due to a lack of reasonably well-characterized and stable

Fe-nitroxyl systems. We expect that systems like these and others will

ultimately be used as synthetic analogues of biological Fe-nitroxyls

and HNO-donors with therapeutic application.

4. Conclusions

The following are the summary of the main ﬁndings of this work:

(i) We have described the synthesis and characterization of several

non-heme FeNO complexes supported by the planar LN₄

diimine-dipyrrolide ligand framework, which represent new

examples of the {FeNO}⁷(3 and 4) and {FeNO}⁸(5 and 6) classes

of metal nitrosyls in hybrid heme architectures. The synthesis

of the rare {FeNO}⁸-type complexes 5 and 6 could be achieved

by stoichiometric chemical reduction with [Co(Cp*)₂] or KC₈.

These {FeNO}⁸species add to the small list of this type of

FeNO complex that can be isolated under standard lab condi-

tions.

Acknowledgments

T.C.H. acknowledges the NSF, the University of Georgia Research

Foundation (UGARF), and the University of Georgia Chemistry De-

partment for funding this research. We also wish to thank Prof. Mi-

chael K. Johnson for his assistance with EPR spectroscopy, and Prof.

I. Jonathan Amster and Mrs. Melissa Stoudemayer for their help

with the high-resolution mass spectral studies.

Appendix A. Supplementary data

(ii) The {FeNO}⁷complexes display spectroscopic and structural

properties that are mostly consistent with other 5C FeNO species

in this class. One particular outlying feature was demonstrated in

the X-ray crystal structure of 3, which afforded an atypical Fe–

N–O angle of ~155° for this EF-notation. This unusual metric

parameter has been explained with respect to the relative

amount of Fe p_zcharacter in the largely d_z²-based HOMO of

this and similarly disposed 5C {FeNO}⁷complexes. Indepen-

dent calculations by another group have assigned the oxida-

tion states in such systems as LS-Fe(I)–NO⁺(S=1/2) with a

majority spin-density on Fe.

Supplementary data to this article can be found online at http://

dx.doi.org/10.1016/j.jinorgbio.2012.08.026.

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