The role of porphyrin peripheral substituents in determining the reactivities of ferrous nitrosyl species

Article abstract of DOI:10.1039/d0sc01625j

Ferrous nitrosyl {FeNO}7 species is an intermediate common to the catalytic cycles of Cd1NiR and CcNiR, two heme-based nitrite reductases (NiR), and its reactivity varies dramatically in these enzymes. The former reduces NO2- to NO in the denitrification pathway while the latter reduces NO2- to NH4+ in a dissimilatory nitrite reduction. With very similar electron transfer partners and heme based active sites, the origin of this difference in reactivity has remained unexplained. Differences in the structure of the heme d1 (Cd1NiR), which bears electron-withdrawing groups and has saturated pyrroles, relative to heme c (CcNiR) are often invoked to explain these reactivities. A series of iron porphyrinoids, designed to model the electron-withdrawing peripheral substitution as well as the saturation present in heme d1 in Cd1NiR, and their NO adducts were synthesized and their properties were investigated. The data clearly show that the presence of electron-withdrawing groups (EWGs) and saturated pyrroles together in a synthetic porphyrinoid (FeDEsC) weakens the Fe-NO bond in {FeNO}7 adducts along with decreasing the bond dissociation free energies (BDFENH) of the {FeHNO}8 species. The EWG raises the E° of the {FeNO}7/8 process, making the electron transfer (ET) facile, but decreases the pKa of {FeNO}8 species, making protonation (PT) difficult, while saturation has the opposite effect. The weakening of the Fe-NO bonding biases the {FeNO}7 species of FeDEsC for NO dissociation, as in Cd1NiR, which is otherwise set-up for a proton-coupled electron transfer (PCET) to form an {FeHNO}8 species eventually leading to its further reduction to NH4+.

Full text of DOI:10.1039/d0sc01625j

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The Role of Porphyrin Peripheral Substituents in Determining the Reactivities of
Ferrous Nitrosyl Species
Sk Amanullah, Abhishek Dey*
School of Chemical Sciences, IndianAssociation for the Cultivation of Science, 2A& 2B Raja SC Mullick
Road, Kolkata, India – 700032
Supporting Information Placeholder
ABSTRACT: Ferrous nitrosyl {FeNO}7 species are intermediates common to the catalytic cycles of
Cd1NiR and CcNiR, two heme-based nitrite reductases (NiR), and its reactivity vary dramatically in these
-
-
enzymes. The former reduces NO2 to NO in the denitrification pathway while the latter reduces NO2 to
+
NH4 in dissimilatory nitrite reduction. With very similar electron transfer partners and heme based active
sites, the origin of this difference in reactivity has remained unexplained. Differences in the structure of
the heme d1 (Cd1NiR), which bears electron-withdrawing groups and has saturated pyrroles, relative to
heme c (CcNiR) is often invoked to explain these reactivities. A series of iron porphyrinoids, designed to
model the electron-withdrawing peripheral substitution as well as the saturation present in heme d1 in
Cd1NiR, and their NO adducts were synthesized and their properties were investigated. The data clearly
show that the presence of electron-withdrawing groups (EWGs) and saturated pyrroles together in a
synthetic porphyrinoid (FeDEsC) weakens the Fe-NO bond in {FeNO}7 adducts along with decreasing
the bond dissociation free energies (BDFENH) of the {FeHNO}8 species. The EWG raises the Eº of
{FeNO}7/8 process, making the electron transfer (ET) facile, but decreases the pKa of {FeNO}8 species,
making protonation (PT) difficult, while saturation has the opposite effect. The weakening of the Fe-NO
bonding biases the {FeNO}7 species of FeDEsC for NO dissociation, as in Cd1NiR, which is otherwise
set-up for a proton-coupled electron transfer (PCET) to form a {FeHNO}8 species eventually leading to
+
its further reduction to NH4 .
1

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Introduction
Nitrite plays a vital role in the biochemical N-cycle.1, 2 Being generated from nitrate by the action of a
molybdenum-containing nitrate reductase3, nitrite is consumed via several pathways which involve
multiple heme and non-heme enzymes (Scheme 1A).2 Assimilatory ammonification, catalyzed by
siroheme containing nitrite reductase (CSNiR), and dissimilatory nitrite reduction, catalyzed by multi-c
+
heme-containing nitrite reductase (CcNiR, Figure 1A), leads to the formation of ammonium ion (NH4 )
directly, without releasing any intermediate nitrogenous species.4 Alternatively, denitrification involves
the reduction of nitrite to nitric oxide (NO), catalyzed by heme cd1 containing nitrite reductase (Cd1NiR,
Figure 1B).5 Further reduction of nitric oxide generates nitrous oxide,6 which eventually reduced further
+
to dinitrogen.7, 8 Thus, CcNiR reduces nitrite to NH4 without releasing any intermediate, and Cd1NiR
reduces nitrite to release NO. Both of these enzymes have heme cofactors in their active site with a very
similar distal environment and electron transfer partners (Figure 1).
A
AssimilatoryNH4+
ammonification
by CSNiR
N2
N2 fixation
NH2OH
Dissimilatory
Nitrite reduction
by CcNiR
Denitrification
NO2-
NO
N2O
B
O
-
O
O
H2O
FeII
H2O
+
N
+
N
H2O
2H
-
e-
NO2
FeIII
FeII
FeII
{FeNO}6
1H+/1e-
(in cd1NiR) (in CcNiR)
+
NH4
e-
NO
represents heme
O
-
NO2
H2O
H
O
N
in Cd1NiR
N
FeII
FeII
1H+/1e-
(in CcNiR)
{FeHNO}8
{FeNO}7

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Scheme 1. (A) Selected components of the biochemical cycle of “N” and (B) Proposed mechanistic
pathways of nitrite reduction catalyzed by CcNiR and Cd1NiR.2
The proposed mechanistic pathways of both Cd1NiR and CcNiR are quite similar (Scheme 1B).2 The
nitrite binds to the reduced ferrous iron center. With two protons from the distal residues, a molecule of
water is released, forming a {FeNO}6 intermediate (Enemark-Feltham notation).5, 9, 10 The CcNiR avoids
the formation of the dead-end intermediate, {FeNO}7 through two consecutive proton-coupled electron
transfer (PCET) to the {FeNO}6 species, generating a {FeHNO}8 intermediate,11 which, on further
+ 9
reduction, leads to the generation of NH4 . Alternatively, Cd1NiR forms {FeNO}7 through an electron
transfer (ET) from cytochrome c, and releases NO with the concomitant binding of nitrite to the ferrous
heme-d1 and the cycle continues.12-14 The different reactivity of {FeNO}7 species compels investigating
the difference in the active sites that control the competition between the PCET process and NO release.
A {FeNO}7 adduct generally possesses a very strong Fe-NO bond with a Kd ~10-9 and this displacement
of NO by nitrite is rather unexpected.15 Although the N-O stretch of the {FeNO}7 species of CcNiR is not
reported, the N-O stretch of Cd1NiR is higher than that of other known heme proteins like hemoglobin
and myoglobin.16-18 We find a strong positive correlation between the reported rate of NO dissociation
and the corresponding N-O frequency (Figure 2A). It suggests that the rate of NO dissociation is reflected
by the strength of Fe-NO bond, which is reflected in the N-O stretching vibration.16-21 Similar correlation
is also present between N-O stretching frequency and the rate of NO displacement by pyridine in different
synthetic meso-phenyl substituted Fe-porphyrins (Figure 2B).22 Iron-porphyrins bearing electron-
withdrawing groups (EWGs), having higher N-O stretching frequency, release NO easier. Previous work
from our group demonstrated that the iron-porphyrins bearing EWGs and/or saturated β-pyrrolic carbons
form weaker iron-nitrosyls due to the competitive back-bonding between macrocycle π* and NO π*-
orbitals from the filled Fe-dπ orbitals.23

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A
B
GLN 276
HIS 369
HIS 277
ARG 114
TYR 10
HIS 327
TYR 218
Heme c
Heme d1
LYS 134
HIS 182
Figure 1. The active site structure of the nitrite reductases at resting state; A) CcNiR (pdb: 1FS7)24 and B)
Cd1NiR (pdb: 1NIR)25; the figures are redrawn using software package Chimera 1.12rc.
A 1630
B 1710
FeTPFPP-NO
Cd1NiR-NO
1625
1700
FeTDCPP-NO
FeTDFPP-NO
1620
1615
1610
1690
1680
1670
Hemoglobin-NO
Myoglobin-NO
FeTTP-NO
-10
-5
0
5
-11
-8
-5
ln(koff)
-2
1
ln(koff)
Figure 2. Correlation between N-O stretching frequency16-18 and rate of NO dissociation19-21: A) in six-
coordinate heme nitrosyls in enzyme systems; B) in synthetic Fe-porphyrin nitrosyl complexes, TTP (p-
tolyl), TDFPP (2,6-difluorophenyl), TDCPP (2,6-dichlorophenyl), TPFPP (pentafluorophenyl).
CcNiR and Cd1NiR possess basic 2nd sphere distal residues and primarily σ-donor Histidine or lysine
-
axial ligands and same redox partner (cytochrome c). These residues assist in NO2 binding, proton
translocation and is likely to affect the dissociation constants of NOx ligands.9, 11, 26, 27 Another major
distinction is the difference in the nitrite binding sites: heme c in CcNiR, and heme d1 in Cd1NiR (Figure
1).24, 25 The major difference in heme d1, relative to heme c is the presence of two saturated β-pyrroles

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(i.e., sp3 hybridized peripheral carbons) along with two electron-withdrawing keto-groups (Figure 3).28, 29
Therefore, their divergent reactivity may stem from differences in the structure of iron-porphyrinoid
macrocycles. To evaluate this possibility, the electrochemistry of synthetic iron-porphyrin model
complexes (FeTPP, FeOEP, FeOEPone and Fe(2,4-OEPdione), Figure 3) and their nitrosyl adducts have
been investigated by several groups.30-39 Under the coulometric condition, nitrite could be reduced to
ammonium ion by the synthetic complexes mediated by a hydroxylamine bound species.40-43 The rate of
the reaction was strongly directed by the macrocycle i.e., FeOEP reacted faster than FeTPP and the
reaction was very slow in the Fe(2,4-OEPdione) complex. The basicity of {FeNO}8 species could
potentially explain the difference in reactivity.42 Alternatively, the greater Lewis acidity of {Fe(2,4-
OEPdione)-NO}7 (as suggested by facile pyridine binding to the Fe) was suggested to enhance the His-
coordination with heme d1 which might help the release of the trans NO.33 These results herald the intrinsic
nature of the macrocycle as a determinant of the different reactivity of the {FeNO}7 species i.e., NO
release vs PCET. A {FeHNO}8 species (proceed after PCET to {FeNO}7) is quite reactive and so far could
only be transiently observed in protected environments such as proteins,44, 45 or in bis-picket fence
porphyrin46 or in highly electron-rich FeOEP in the presence of weak acid such as substituted phenols.41
Alternately, under electrochemical conditions, {FeNO}8 yields the parent {FeNO}7 species and H2 .43 It
is important to understand the role of these peripheral modifications in the electronic structure and
reactivity of these iron nitrosyls, to understand the different reactivities of the {FeNO}7 species exhibited
by these enzymes.

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Heme c
FeOEP
e
FeTPP
Fe(2,4-OEPdione)
FeDEsC
FeTEsP
Figure 3. Structure of the naturally occurring heme and synthetic Fe-porphyrinoids. FeOEP, FeOEPone,
and Fe(2,4-OEPdione) were synthesized previously.32, 47, 48
In this manuscript, a series of synthetic iron-porphyrins were developed for systematically varying in their
peripheral substituents. By introducing EWGs and/or saturation at the β-pyrrolic positions, we were able
to decode the role of each substituent on the basic iron-porphyrin skeleton on the electronic structure and
reactivity of their corresponding {FeNO}7 species. The electrochemical and spectroscopic data of their
NO adducts and density functional theory (DFT) calculations help alienate the contribution of reduction
potential and pKa to the bond dissociation free energy (BDFENH) of the N-H bond in {FeHNO}8 species.
The results indicate a definitive role of EWG and saturation in tuning the Fe-NO bond strength, the

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reduction potential of {FeNO}7 and pKa of {FeNO}8 species, which can likely explain the origin of
differences in the reactivity of CcNiR and Cd1NiR.
Results
1. Synthesis
The heme involved in nitrite binding sites of CcNiR and Cd1NiR is heme c and heme d1, respectively. The
major difference between these being the presence of two electron-withdrawing-keto groups and two
saturated β-pyrrolic carbons in heme d1 (Figure 3). To rationalize the effect of EWG and/or saturation, a
series of iron-porphyrinoids were synthesized (Figure 3), namely, iron-tetraphenylporphyrin (FeTPP, fully
unsaturated); iron-diesterporphyrin (FeDEsP, having two electron-withdrawing ester groups); iron-
tetraesterporphyrin (FeTEsP, having four ester groups); iron-tetraphenylchlorin (FeTPC, having two
saturated β-pyrrolic carbons) and iron-diesterchlorin (FeDEsC, having two ester groups and two saturated
β-pyrrolic carbons). Those EWGs were designed to qualitatively emulate the –I (inductive) effect of the
keto-groups in heme d1. FeTPP, FeTPC, FeDEsP, and FeDEsC complexes were synthesized following
previously reported procedures.23, 49

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Scheme 2: Synthetic strategy of FeTEsP. In some cases, the –CO2Et group is abbreviated as “E” for clarity
in representation,
FeTEsP was synthesized to introduce four electron-withdrawing substitutions on the porphyrin ring. TEsP
(5 in Scheme 2) was synthesized from the propionic acid condensation of two dipyrromethanes (Scheme
2). One of which (TEsbpyr-dial, 3 in Scheme 2) contained four ester groups (at the 3 and 4 positions of
the respective pyrroles) as well as two aldehyde groups (at the 2 positions of the respective pyrroles) and
the other half was 5-phenyldipyrromethane (4 in Scheme 2). Base induced cyclization of diethyl fumarate
and p-toluenesulfonylmethylisocyanide (TosMIC) lead to the formation of pyrrole bearing two ester
groups (DEspyr, 1 in Scheme 2). Dipyrromethane of DEspyr (TEsbpyr, 2 in Scheme 2) was obtained with
the condensation with benzaldehyde under harsh acidic conditions. A Vilsmeier-Haack reaction was
performed upon TEsbpyr to obtain the corresponding dialdehyde, TEsbpyr-dial (3 in Scheme 2).

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The other half, i.e., the 5-phenyldipyrromethane (4 in Scheme 2), was prepared from acid-catalyzed
condensation of pyrrole and benzaldehyde following the Lindsey protocol.50 Zinc and iron metalation was
performed using established protocols.49 The zinc complex of TEsP was characterized by single-crystal
XRD. Needle-shaped purple crystals of ZnTEsP were grown from the diffusion of hexane into a DCM
solution of the complex (Figure 4C). It crystallized in a triclinic symmetry with a centrosymmetric P-1
space group. Structural analysis revealed that it was a dimer, formed by the coordination of a free carbonyl
“oxygen” atom with the zinc atom of another molecule. The structures of FeDEsP and ZnDEsC were
reported before and shown here for comparison (Figure 4A-B). Further investigations were performed
with the nitrosyl adducts of the iron-bound porphyrinoids.
A
B
C
Figure 4: Molecular structure of the crystals of A) a µ-oxo dimer of FeDEsP; B) ZnDEsC, and C)
ZnTEsP. Color code: C, black; Fe, brownish-red; Zn, green; N, blue; O, red. Hydrogen atoms are
omitted for clarity.
2. Iron-nitrosyl reduction potentials
The cyclic voltammograms of the nitrosyl complexes of FeTPP, FeDEsP, FeTEsP, FeTPC, and
FeDEsC showed an oxidation process at 0.20, 0.34, 0.38, -0.04 and 0.10 V, respectively, against
Fc+/Fc redox couple (Figure 5). For FeTPC and FeDEsC, the process was clearly observed only
under fast scan rates (Figure S23B). The process was irreversible at slow scan rates, indicating
dissociation of the NO during oxidation. Note that the CV of FeDEsC was performed under NO
saturated condition to prevent NO loss from the complex. Past research from Ryan and Kadish
group established the nature of these redox events: the oxidation wave of porphyrin {FeNO}7 leads
to the formation of FeIII-NO species, while in the case of chlorins, bacterio/iso-bacteriochlorins,

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and porphinone/porphinediones, the oxidation leads to the formation of FeII-NO species with the
macrocycle-cation radical.32, 33, 36, 51 The {FeNO}7/8 process was observed for FeTPP, FeDEsP,
FeTEsP, FeTPC, and FeDEsC at -1.41, -1.24, -1.14, -1.51 and -1.42 V, respectively, vs Fc+/Fc
redox couple (Figure 5). The values obtained for FeTPP were consistent with previous reports.38
The pre-wave observed in the case of FeTPP (-1.32 V), FeDEsP (-1.17 V) and FeTPC (-1.38 V),
might be due to ligand association, which disappeared at higher scan rates (Figure S23A) as
reported by Kadish group earlier.38, 52 In the case of FeDEsC, the irreversible pre-wave at ~ -1.22
V vs Fc+/Fc redox couple was likely due to the direct electrochemical NO reduction53 (NO
saturated solution), as observed by the Kadish group during the reduction of FeII-TPP-NO and FeII-
OEP-NO, in the presence of excess NO gas in the medium.38
FeTPP-NO
FeTEsP-NO
FeDEsC-NO
{FeNO}6/7
FeDEsP-NO
FeTPC-NO
{FeNO}7/8
0.65
0.15
-0.35
-0.85
-1.35
-1.85
Potential (V) vs Fc+/Fc
Figure 5: Cyclic voltammogram of the complexes in dichloromethane at room temperature. Working
electrode: glassy carbon; counter electrode: platinum; reference electrode: aqueous Ag/AgCl in 4M
KCl; Supporting electrolyte: tetrabutylammonium hexafluorophosphate (100 mM); scan rate: 50
mVps.

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A clear trend was observed in both the oxidation and reduction process of the {FeNO}7 species for the
series of iron porphyrinoids used here. With an increase in the number of EWGs attached to the β-pyrroles
relative to FeTPP-NO, both {FeNO}6/7 and {FeNO}7/8 couples shifted to higher potential (i.e., for
FeDEsP-NO and FeTEsP-NO in Figure 5). Alternatively, saturating one of the pyrroles of FeTPP-NO,
i.e., in the case of FeTPC-NO, both the reduction couples shifted to lower potentials. The FeDEsC-NO
complex, having both EWGs as well as saturated pyrrole centers, had both the reduction potentials almost
similar to those of FeTPP-NO. This implied that the EWG and saturation had opposite effects on the
electronic structure of the Fe-NO unit.
3. Fe-NO bond strength
The FTIR data of the five-coordinate {FeNO}7 complexes of FeTPP, FeDEsP, FeTEsP, FeTPC, and
FeDEsC showed the N-O stretch at 1676, 1686, 1688, 1680 and 1691 cm-1, respectively (Figure 6). The
data showed that when two EWGs were introduced (FeDEsP), the N-O vibration (str.) shifted to 1686 cm-
1
from 1676 cm-1 in FeTPP. Further addition of EWGs (FeTEsP), shifted the N-O vibration (str.) up to
1688 cm-1. Such high N-O stretching frequencies had only been reported for {FeNO}7 species of octa-
halogenated porphyrins and reflect poor back-bonding between the occupied iron and unoccupied NO π*-
orbitals.22, 54 The saturation of the pyrrole, by itself, exerted little effect on Fe-NO bonding as indicated
by the N-O stretching frequency of {FeTPC-NO}7 at 1680 cm-1 which was very similar to that of {FeTPP-
NO}7. But saturation along with EWGs caused a substantial weakening of the NO adduct, as indicated by
the N-O stretch of {FeDEsC-NO}7 at 1691 cm-1, relative to {FeTPP-NO}7 at 1676 cm-1. The Fe-N stretch
of the {FeNO}7 species of FeTPP-NO reproduced previously reported value. However, despite several
attempts with resonance Raman the Fe-N stretching frequencies could not be obtained for the other
compounds studied (Figure S24A).43 The strength of Fe-NO bond in the {FeNO}7 adducts was strongly
dictated by both σ-bonding and π-back-bonding.55-58 As reported recently, the presence of electron-
withdrawing substitutions on the porphyrin lowers the energy of the porphyrin π*-orbitals. This results in
competitive back-bonding from the filled dπ orbitals of Fe between porphyrin π* and NO π*-orbitals,
which eventually weakens the NO adducts.23 Saturation by itself had a minor effect on the back-bonding.
However, saturation along with two EWGs had an enhanced effect on the weakening of the NO-adduct.
Qualitatively, similar trend was observed for N-methylimidazole bound six-coordinate {FeNO}7 species
where the N-O stretching vibrations for FeTPP, FeDEsP, FeTEsP, FeTPC, and FeDEsC were observed at
1626 cm-1, 1641 cm-1, 1646 cm-1, 1635 cm-1and 1633 cm-1, respectively (Figure S25A). Therefore, axial
primarily σ donor nitrogeneous ligand has a very limited effect on the electronic structure of these
{FeNO}7 complexes. Note that the N-O stretch of the {FeNO}7 species in Cd1NiR was 1626 cm-1 with
heme d1, relative to 1612 cm-1 in myoglobin with heme b (which neither have EWG nor saturation).16-18

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The higher N-O stretching frequency in the Cd1NiR (by 14 cm-1) was indicative of a weaker Fe-NO
bonding. And it was associated with a ~106 fold enhancement in NO dissociation rate from the {FeNO}7
intermediate (Figure 2).13, 19-21, 59 The 15 cm-1 upshifting of the N-O vibration observed here between
FeDEsC and FeTPP mirrored the 14 cm-1 shift observed between Cd1NiR and myoglobin suggesting a
-
weakening of the Fe-NO bond, raising the possibility of ligands like NO2 displacing the bound NO.
{FeDEsP-14NO}7
{FeDEsP-15NO}7
{FeTPP-14NO}7
{FeTPP-15NO}7
difference
1643
1656
difference
1676
1686
1700
1900
1800
1700
1600
1500 1900
1800
1600
1500
wave number (cm-1)
wave number (cm-1)
{FeTEsP-14NO}7
{FeTEsP-15NO}7
{FeTPC-14NO}7
{FeTPC-15NO}7
1646
difference
1658
difference
1680
1688
1900
1800
1700
1600
1500 1900
1800
1700
1600
1500
wave number (cm-1)
wave number (cm-1)
{FeDEsC-14NO}7
{FeDEsC-15NO}7
1660
difference
1691
1900
1800
1700
1600
1500
wave number (cm-1)
Figure 6. FTIR data of the {FeNO}7 adducts in dichloromethane at room temperature.

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The displacement of the bound NO from the N-methylimidazole bound six-coordinated {FeNO}7 species
-
by NO2 was investigated using absorption spectroscopy (See SI, section 8). The Kd for the process
(ꢀꢁꢂꢂ − ꢃꢄ + ꢃꢄ2− ⇄ ꢀꢁꢂꢂ − ꢃꢄ2− + ꢃꢄ) was determined to be 0.09 for FeTPP and 0.46 for FeDEsC
(Table 1). The higher Kd for FeDEsC, relative to FeTPP, translated to a ∆G difference of ~1 Kcal/mol and
correlated very well with its stronger N-O stretching frequency and demonstrated clearly how the EWG
-
and saturation of the porphyrin ring aid the displacement of NO by NO2 , as proposed in Cd1NiR. Asimilar
effect was observed on the displacement rate of NO by pyridine in {FeNO}7 complexes in a series of iron
porphyrins, where the octa-halogenated derivative of TPP was ~106 times faster than that of FeTPP.22
Spectroelectrochemistry was used to access the N-O vibrations of the {FeNO}6 and {FeNO}8 species
(Table 1, Figure S26-S28). The N-O vibrations for the six-coordinate {FeNO}6 for FeTPP, FeDEsP, and
FeTEsP were obtained at 1914 cm-1, 1923 cm-1, and 1927 cm-1, respectively (Figure S27). The higher N-
O vibration for the porphyrins containing EWGs relative to FeTPP mirrored the trend observed for the
corresponding {FeNO}7 species. The N-O vibrations for both five and six-coordinate {FeNO}6 species
of FeTPC and FeDEsC could not be obtained, which was consistent with the irreversible CV observed for
these species indicating that these {FeNO}6 species dissociate within the time scale of the experiment
(Figure S23B and S27). The inability to identify five-coordinate {FeNO}6 species of FeDEsP and FeTEsP
again suggested the formation of a labile {FeNO}6 adduct. The N-O vibrations for the {FeNO}8 species
could be observed for FeTEsP and FeDEsC at 1550 cm-1 and 1537 cm-1, respectively (Figure S28, Table
S2). The values obtained were consistent with the previously reported values for the five-coordinate
{FeNO}8 porphyrins.54, 60, 61 Note that the frequencies were much higher than the value reported for FeTPP
at 1496 cm-1.43 Here, too, the inclusion of the EWG and saturation together lead to a substantial increase
in the N-O vibration indicating a weakening of the Fe-NO bonding. Thus, the FTIR data for the {FeNO}6,
{FeNO}7, and {FeNO}8 species all showed that the inclusion of EWG and saturation at the periphery of
the porphyrin macrocycle substantially weakened the Fe-NO bonding.

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Table 1. Properties of {FeNO}6//7/8 species for the synthetic porphyrins
e
{FeNO}6
{FeNO}7
{FeNO}8
Kd


a
νN-O
νN-O
5C
νN-O
5C
E0,b
E0
5C
(6C)
(6C)
1676
(1626)
(6C)
149643
FeTPP-NO
1844
(1914)
0.09±0.05
0.20±0.04
0.20
0.34
-1.41
-1.24
-1.14
-1.51
-1.42
FeDEsP-NO
c
1686
c
(1923)
(1641)
FeTEsP-NO
FeTPC-NO
c
1688
(1646)
1680
1550
0.23±0.02
0.13±0.04
0.38
(1927)
d
d
-0.04d
0.10d
c
(1635)
FeDEsC-NO
1691
1537
0.46±0.04
(1633)
a. stretching frequency in cm-1, b. Potentials are reported vs Fc+/Fc in
dichloromethane, c. Not observed and d. irreversible cathodic waves, e. ꢅꢆꢇꢇ −
ꢈꢉ + ꢈꢉ−ꢊ ⇄ ꢅꢆꢇꢇ − ꢈꢉ−ꢊ + ꢈꢉ
A plot of the experimentally observed N-O vibrations (Table 1) with ln(Kd) for the series of complexes
investigated here showed a reasonably linear correlation (Figure 7) in line with the linear correlation
observed between ν(N-O) and ln(koff) (Figure 2). Thus, the electronic structure responsible for the
correlation in the enzyme active site was captured in the series of porphyrins used here – primarily the
competitive back-bonding between porphyrin π* and NO π*-orbitals adding credence to the use of
electron-withdrawing –COOEt group to mimic the keto-group in heme d1. The Kd was larger for {Fe-
NO}7 species with higher ν(N-O) frequency. The stronger back-bonding with the porphyrin π* will reduce
back-bonding to the NO π* thus tuning its pKa. It is conceivable that this will affect the thermodynamics
of the PCET to {FeNO}7 to form {FeHNO}8.

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1695
1690
1685
1680
1675
FeDEsC-NO
FeTEsP-NO
FeDEsP-NO
FeTPC-NO
FeTPP-NO
-2.5
-2
-1.5
ln(Kd)
-1
Figure 7: Correlation between experimentally measured N-O stretching frequency and NO dissociation
constant
4. PCET to {FeNO}7
The {FeNO}7 species of CcNiR, with heme c, accepts 1H+/1e- to form {FeHNO}8 on its way to form
+
NH4 without releasing any intermediate species and does not dissociate NO.9 The lower N-O stretch in
{FeNO}7 of heme c (1651-1671 cm-1)62, in general, was suggestive of a weak NO dissociation in CcNiR.
But, for a facile PCET, the BDFE of the N-H bond in {FeNHO}8 should be high as well.63 The BDFE of
the N-H bond in {FeHNO}8 species can be calculated using the following equation:64
BDFENH = 1.37 pKa + 23.06 Eº + C
Where, the pKa was that of {FeNO}8 species, which was calculated from the change in Gibb’s free energy,
∆Gº of the protonation equilibrium between {FeNO}8 and {FeHNO}8. The Eº represents the one-electron
reduction potential of {FeNO}7/8 redox process, which was directly obtained from the cyclic
voltammogram. C is a constant which depends on the solvent.64 The pKa of the {FeNO}7 species was
difficult to determine as the protonation leads to an irreversible reaction.42 Thus, the ∆Gº of protonation
was computed using DFT calculations. The BDFENH values estimated using these were normalized
relative to FeDEsC-NO which was set at 0 Kcal/mol (Table 2). These calculations indicated that the
protonation of {FeNO}8 species gradually became less favorable from FeOEP to FeOEPone to Fe(2,4-
OEPdione). It was consistent with the previously reported trend for nitrite reduction to ammonia, using
moderately strong acids like phenols, under controlled potential electrolysis i.e., the rate of the reaction:
FeOEP> FeOEPone > Fe(2,4-OEPdione), where the protonation of {FeNO}8 species was proposed to be
the rate-limiting step.42

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Table 2. Values of pKa, Eº, and BDFENH relative to FeDEsC-NO
∆Eº
(mV)
10
∆BDFENH
(Kcal/mol)
2.38
Complexes
∆pKa
FeTPP-NO
FeDEsP-NO
1.57
-0.32
-3.12
1.92
-
180
280
-90
-
3.71
FeTEsP-NO
2.18
FeTPC-NO
0.56
FeDEsC-NO
-
FeOEP-NO
5.36
0.91
-1.76
-70
140
260
5.73
FeOEPone-NO
Fe(2,4-OEPdione)-NO
4.47
3.58
B
A
2224
2154
1684
1657
Figure 8. Formation of N2O during electrolysis of {FeDEsP-NO}7 at -1.31 V (vs Fc+/Fc). On applying
potential, N2O is generated (νN-O (14/15): 2224/2154 cm-1), with the expanse of {FeDEsP-NO}7 peaks. A)
in the presence of 14NO and B) in the presence of 15NO.
Comparing the ΔGº for PCET to the {FeNO}7 species of FeDEsC-NO and FeDEsP-NO (without saturated
pyrroles), the major contribution to the difference was derived from the of the {FeNO}7/8 redox process.
The Eº was increased by ~180 mV in FeDEsP-NO, making the reduction more facile. As a result, the
BDFENH of {FeHNO}8 species of FeDEsP-NO was increased by ~3.71 Kcal/mol, relative to FeDEsC-
NO, suggesting that the {FeNO}7 species of FeDEsP-NO should be more prone to undergo PCET reaction
than FeDEsC-NO. Gratifyingly, during the electrochemical reduction of FeDEsP-NO, it generated N2O

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in the presence of methanol as the proton source. This was evidenced by the growth of a vibrational band
at 2224 cm-1 as cathodic potential was applied (Figure 8A), which shifted to 2154 cm-1 when 15NO was
used (Figure 8B). The formation of N2O from NO can be mediated by {FeHNO}8 which is difficult to
characterize under electrochemical conditions.65 Alternatively, when {FeDEsP-NO}7 was reduced
chemically by cobaltocene in presence of methanol and PPh3, it readily generated Ph3P=O and Ph3P=NH
(see SI, section 10, Figure S33-35), suggesting the generation of HNO in the solution. This can only
happen if the reaction proceeds through the PCET process forming a {FeHNO}8 species.66 Note that these
results do not imply that FeDEsP mirrored the reactivity of CcNiR because CcNiR does not release any
+
HNO, it generates NH4 via {FeHNO}8 without releasing HNO or N2O. The fact that FeDEsP-NO could
-
produce HNO from a weak proton donor while the NO could be displaced by NO2 in FeDEsC-NO,
suggested that the electronic structure of the porphyrin ring resulting from EWG and saturation can
discriminate NO release vs PCET to a {FeNO}7 species under physiological conditions.
Discussion
It has been proposed that CcNiR undergoes two consecutive PCET reactions on the {FeNO}6
intermediate. The first PCET forms a {FeNO}7 species (with protonation of Arg114 residue) which
subsequently generates {FeHNO}8 species through another PCET process, releasing only a trace amount
of the {FeNO}7 intermediate.11, 67 In contrast, Cd1NiR undergoes an electron transfer (ET) from
cytochrome c, forming the {FeNO}7 intermediate, which releases NO rapidly (koff ~200 s-1).13, 19 There is
an extensive debate on whether the NO is released from the ferric state or the ferrous state of the NO
adduct.1, 68 Recent data suggest that it is likely that NO is released from the ferrous, {FeNO}7 state.2, 13, 59
The fast release of NO from {FeNO}7 intermediate may be attributed to a weak Fe-NO bonding in the
ferrous nitrosyl adduct formed. The higher N-O stretching frequencies in the {FeNO}n (n=6,7, and 8)
species of FeTEsP and FeDEsC suggested weak Fe-NO bonding, i.e., four electron-withdrawing ester
groups or two ester group with two saturated carbon centers had an almost similar effect. Thus, heme d1,
which had two EW-keto groups and two saturated pyrrolic carbons, was likely to have a weaker Fe-NO
bond relative to heme c, which has neither. A weaker Fe-NO in FeDEsC is associated with a large Kd for
-
NO2 replacement relative to FeTPP consistent with the strong linear correlation between νN-O and Kd
observed for both enzymatic and synthetic systems. Similarly, a weaker Fe-NO in heme d1 active site of
-
Cd1NiR, evident from a higher νN-O, should result in higher NO (product) Kd, relative to NO2 (substrate).
It might be envisaged that due to the ruffled nature of isobacteriochlorin ring in heme d1, they form weak
{FeNO}7 species. But calculations of the ruffling parameters69 suggested maximum ruffling was present
in {FeTPC-NO}7 species, which possess a chlorin ring (see SI, section 9), and had a relatively strong Fe-
NO bond.

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The ΔGº for PCET to the {FeHNO}8, the competing reaction to NO release, was affected by both Eº and
pKa of the {FeNO}7 species. Between FeDEsC-NO and FeTPC-NO, where the latter was devoid of
EWGs, the pKa of {FeNO}8 species increased by 1.92 unit (Table 2), but it lowered the Eº of {FeNO}7/8
redox process by ~90 mV (Table 2), making the PCET to the {FeNO}7 species of FeTPC-NO slightly
favorable, relative to FeDEsC-NO. By omitting both saturation and EWGs (FeTPP-NO), the Eº decreased
while the pKa of {FeNO}8 species increased which resulted in higher BDFENH of the {FeHNO}8 species
but not as high as of FeDEsP-NO. In the case of FeTEsP-NO, where the saturation was absent but two
more EWGs were present (relative to FeDEsC-NO), the Eº increased by ~280 mV but the pKa of {FeNO}8
species becomes too low to be protonated, resulting BDFENH lower than FeTPP-NO. Therefore, for a
facile PCET, there needs to be a balance between Eº and pKa, which was attained here in FeDEsP-NO.
This model was equally applicable to FeOEP system, where the high BDFENH was due to the greater pKa
of the {FeNO}8 species. Introducing EW-keto groups increased the Eº but at the expense of the pKa of
{FeNO}8 species (Table 2), resulting in a gradual decrease in BDFENH. However, it should also be noted
that {FeHNO}8 species is not very stable due to the disproportionation of Fe-HNO unit. The {FeHNO}8
species could only been stabililized either with steric protection (bis-picket fence porphyrin46 or globin
chain in hemoglobin44, 45) or in a highly electron-rich porphyrin like FeOEP (νNO/{FeNO}7 = 1665 cm-1). 70,
41, 71, 72
Hence, it is not surprising, that {FeHNO}8 species in electron-deficient
7
octabromo[tetrakis(pentafluorophenyl)]porphyrin, Fe(TFPPBr8)(NO) (νNO/{FeNO} = 1726 cm-1)54 could
not be isolated. The weak BDFENH and weaker {FeNO}7 adduct likely biases the FeDEsC (which have
two EWGs along with two saturated pyrrolic carbons, like heme d1 in the active site of Cd1NiR) for NO
dissociation. On the contrary, stronger BDFENH driven by a favourable balance between pKa (due to better
back-bonding to the NO π*-orbitals) and Eº is responsible for facile PCET to {FeNO}7 to produce
+
{FeHNO}8 which is necessary to eventually release NH4 in CcNiR.
Conclusion
In summary, the results on structural variants of iron-porphyrins suggested that Cd1NiR does not proceed
with the PCET process to form {FeHNO}8 intermediate, due to its lower BDFENH arising from the weaker
back-donation from heme d1 where the EWGs and sp3 peripheral carbons enhance competitive back-
bonding from the iron to the porphyrinoid π* and NO π*-orbitals. The weaker back-bonding to the bound
NO results in weaker Fe-NO bond and hence, it releases NO. In CcNiR, on the other hand, heme c has
greater back-bonding to the NO from iron, which strengthens the Fe-NO bond and tunes the pKa allowing
+
to undergo PCET to form {FeHNO}8 species, which is crucial for the further reactions to release NH4 .

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Experimental Details
Materials: All reagents were of the highest grade commercially available. Iodine, trifluoroacetic acid
(TFA), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), ethanol, aqueous ammonia solution, ceric
ammonium nitrate (CAN), sarcosine, potassium tert-butoxide, benzaldehyde, magnesium sulphate, p-
toluenesulphonic acid (PTSA), p-toluenesulfonylmethyl isocyanide (TosMIC), phosphorus oxychloride
(POCl3), dichloroethane (DCE), propionic acid were purchased from Spectrochem Ltd. Diethyl ether,
Tetrahydrofuran (THF), Acetonitrile, Dichloromethane, and Toluene were purchased from RANKEM
Ltd., paraformaldehyde, anhydrous Ferrous bromide (FeBr2), 2,4,6-Collidine, Tetrabutylammonium
hexafluophosphate (TBAPF6) were purchased from Sigma-Aldrich chemical company. Na2SO4, Zinc
acetate were purchased from MERCK and used without any further purification. Unless otherwise
mentioned all reactions were performed at room temperature. The column chromatography was performed
with silica gel (mesh size: 60-100, 100-200 and 230-400) and neutral Alumina, preparative TLC was
performed with Silica gel GF-254 (~13% CaSO4, 0.5 H2O binders with fluorescent indicator). These were
purchased from SRL Pvt. Ltd. THF was dried using K-metal in the presence of benzophenone until the
colour of benzophenone turned intense bluish-green. Toluene was dried using Na-metal in the presence
of benzophenone until the colour of benzophenone turned intense blue. MeOH was first dried like toluene
using sodium after that it was distilled from Mg-cake. DCM and chloroform were distilled with both
anhydrous CaCl2 followed by CaH2.
Instrumentation: All electrochemical experiments were performed using CH Instruments (model
CHI700E and CHI710D Electrochemical Analyzer). Biopotentiostat, reference electrode (standard single-
junction silver/silver chloride filled with 4M KCl with AgCl solution) were purchased from CH
Instruments. The absorption spectra were measured in the SHIMADZU spectrograph (UV-2100). The
aerobic and anaerobic cuvettes were purchased from Starna Scientific. The FT-IR data were measured on
the Shimadzu FTIR 8400S instrument. The CaF2 windows for IR spectroscopy were purchased from
Sigma Aldrich. The anaerobic setup for IR spectroscopy was purchased from PerkinElmer. The optically
transparent thin-layer electrochemical cell (OTTLE) was purchased from the University of Reading for
spectroelectrochemistry. All the NMR spectra were recorded on the Bruker DPX-300, Bruker DPX-400
or DPX-500 spectrometer at room temperature. The mass spectra were recorded by the QTOF Micro
YA263 instrument. Resonance Raman data were collected using a Trivista 555 spectrograph (Princeton
Instruments) and using 413.1 nm excitation from a Kr+ laser (Coherent, Sabre Innova SBRC-DBW-K).
The X-band EPR spectra were recorded on a JEOL instrument. X-ray single-crystal data were collected
at 120 K using radiation on a SMARTAPEX diffractometer equipped with CCD detector. Data collection,
data reduction, structure solution refinements were carried out using the software package of APEXIII.

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The structure was solved by the direct method and refined in a routine manner. The non-hydrogen atoms
were treated anisotropically. All the hydrogen atoms were located on a difference Fourier map and refined.
Electrochemical Measurements: Since {FeNO}8 readily react with a trace amount of water present in
the solvents,43 every solvent used in electrochemical measurements were first dried following the
aforementioned protocol. After that, they had been super-dried with activated 4Å molecular sieves and
kept inside the glove box for 1 week. Nitrosyl adduct of the five complexes (FeTPP, FeDEsP, FeTEsP,
FeTPC, and FeDEsC) were considered for electrochemical analysis. All CV data were collected under
anaerobic conditions in a custom made electrochemical cell. 6 ml of NO-complex (concentration: 1 mM)
was taken in presence of 100 mM TBAPF6 as supporting electrolyte. The glassy carbon electrode was
taken as a working electrode, standard single-junction silver/silver chloride filled with 4M KCl with AgCl
solution as the reference electrode and a Pt electrode was taken as the counter electrode. Ferrocene (Fc)
was used as an internal reference and the potential scale is normalized with respect to the potential of the
Fc+/Fc couple. The potential was swept starting from 0V to a positive potential (oxidation) followed by
negative potential (reduction), except for FeDEsC-NO, here sweeping positive potentials, lead to
irreversible CV. Hence, for this, the potential was swept from 0V to negative potential (reduction)
followed by positive potential (oxidation).
NO complex preparation: Dry degassed NO gas was generated upon the dropwise addition of a
deaerated saturated solution of sodium nitrite to the deaerated 6M H2SO4. The gas was passed through
two 4N KOH solution bubbler followed by one concentrated H2SO4 solution. The solution of each
complex was reduced by 0.5 equivalent Na2S solution (in methanol) inside a glove box, sealed properly
and kept out of the box. NO gas was purged through the samples (kept in an ice bath to reduce solution
evaporation) for 5 mins. The vials were tightly sealed and used for further investigations. NO complexes
were also prepared using Ph3CSNO (as well as Ph3CS15NO), which were prepared through reported
procedures.73, 74 To the reduced samples, 1 equivalent of Ph3CSNO in THF/DCM (whichever required)
was added. The vials were perfectly sealed and used for further investigations. The detailed
characterization of the five and six-coordinated N-methylimidazole bound {FeNO}7 adducts are given in
the main text and the supporting informations (Figure S24A-B, S24A-C, Table S1).
FTIR data collection: DCM/THF solution of the complexes were injected in the anaerobic FTIR setup
or OTTLE cell and tightly sealed inside a glove box. The cell was removed from the box and data
collected. Spectroelectrochemistry was performed using the OTTLE cell connecting with the
electrochemical analyzer. The sample solution contained 100 mM TBAPF6 as a supporting electrolyte.
The FTIR spectra were taken at different time intervals under electrolysis conditions.

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UV-Vis absorption data collection: For all anaerobic data were collected taking the samples from the
glove box in a tightly sealed anaerobic cuvette. The cuvette was removed from the box and data collected.
The background was corrected before the experiments, by an identical amount of solvent mixture.
Computational Details: All calculations were performed at the IACS computer cluster using Gaussian
03 software.75 BP86 functional reproduced better agreement with the experimental frequencies and hence
further calculations were performed with that functional. A mixed basis set with 6-311g* on Fe and 6-
31g* on C, O, N and H atoms were used for optimization.76, 77 For the final energy and ground-state
calculations, a 6-311+g* basis set was used on all atoms. The solvent effect was corrected using the
Polarizability Continuum Model (PCM).78 For all complexes spin-unrestricted schemes have been
adopted which distinguishes between α and β-spin orbitals. Frequency calculations were performed using
the basis set used for optimization, and no negative frequencies were found for the structures reported.
Synthetic details
The FeTPP, FeTPC, FeDEsP and FeDEsC complexes were synthesized following the reported
procedures.23 The synthetic procedure of FeTEsP is described below:
Diethyl 1H-pyrrole-3,4-dicarboxylate (DEspyr): 110 ml dry THF was added to Potassium tertiary
butoxide (15.57 g, 138.7 mmol) in a flask attached to a Schlenk line under N2 atmosphere. A solution of
tosyl methyl isocyanide (TosMIC) (13.5 g, 69.4 mmol) and diethyl fumarate (10 ml, 69.4 mmol) in 60 ml
dry THF was prepared and added dropwise to the flask keeping in the ice water bath. Stirring was
continued for 6 to 7 hours at room temperature. Then THF was evaporated and the reaction was quenched
using saturated NH4Cl solution. The mixture was worked up with ethyl acetate and dried over Na2SO4
and evaporated through a rotary evaporator. Purification was done by recrystallization from methanol.
Crystals obtained were washed with cold ethyl acetate. 1H NMR (CDCl3): δ (ppm) 1.30 (t, 6H), 4.25 (q,
4H), 7.38 (d, 2H), 10.57 (bs, 1H). 13C {1H} NMR (CDCl3): δ (ppm) 14.33, 60.26, 115.39, 126.59, 166.42.
ESI-MS (Positive ion mode, CH3CN): m/z 234.05 (100%; [M+Na]+), 212.09 (45%; [M+H]+), 250.05
(30%;[M+K]+)
Tetraethyl 2,2'-(phenylmethylene)bis(1H-pyrrole-3,4-dicarboxylate) (TEsbpyr): p-tolunesulphonic
acid (0.89 g, 4.7 mmol) and MgSO4 (0.23 g, 1.9 mmol) were taken in a flask. Benzaldehyde (194 µl, 1.9
mmol) was added to it under N2 atmosphere. The solid mixture was heated under vacuum until yellow
colouration. Then THF (2 ml) was added and it was heated for 5 minutes. Then a solution of DEsPyr (1
g, 4.7 mmol) in dry CHCl3 was added to it. The solution was refluxed for 5 hours. The reaction was
quenched using concentrated NaOH solution and was worked up with dichloromethane. The organic layer
was dried over Na2SO4, evaporated through a rotary evaporator and was dissolved in a minimum amount

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of DCM and charged on GF-254 silica gel preparative TLC plate and eluted with 30% ethyl acetate-
hexane mixture. The product band was scratched off and extracted with ethyl acetate. 1H NMR (CDCl3):
δ (ppm) 1.14 (t, 6H), 1.33 (t, 6H), 4.03 (q, 4H), 4.26 (q, 4H), 6.27 (s, 1H), 6.88 (d, 2H), 7.22 (m, 5H),
10.64 (bs, 2H), 1.26, 2.04, 4.12 (for EtOAc). 13C {1H} NMR (CDCl3): δ (ppm) 13.75, 14.26, 40.51, 60.11,
61.08, 113.21-139.15, 164.01, 167.00. ESI-MS (Positive ion mode, CH3CN): m/z 533.35 (100%;
[M+Na]+), 549.34 (45%;[M+K]+), 511.38 (35%; [M+H]+).
Tetraethyl 5,5'-(phenylmethylene)bis(2-formyl-1H-pyrrole-3,4-dicarboxylate) (TEsbpyr-dial):
POCl3 (450 µL, 4.8 mmol) was added slowly to DMF (380 µL, 4.8 mmol) taken in a flask, kept in an ice
bath to form the Vilsmeier-Haack reagent. The reagent was dissolved in DCE, degassed and then added
dropwise to another flask containing TEsbpyr (250 mg, 0.48 mmol) in DCE keeping the flask on ice bath
under N2 atmosphere. The reaction mixture was degassed for 15 minutes and then refluxed for 5 hours.
The reaction was monitored by TLC after charring the TLC plate using 2,4-DNP solution. The reaction
was then quenched by adding a saturated solution of sodium acetate and worked up with dichloromethane.
The organic layer was dried over Na2SO4, evaporated through a rotary-evaporator and purified by column
chromatography on silica gel (100-200 mesh) with EtOAc: toluene (1:10). 1H NMR(CDCl3): δ (ppm) 1.21
(t, 6H), 1.39 (t, 6H), 4.15 (q, 4H), 4.40 (q, 4H), 6.72 (s, 1H), 7.01 (d, 2H), 7.26 (m, 5H), 9.92 (s, 2H),
13
11.35 (bs, 2H). C {1H} NMR (CDCl3): δ (ppm) 13.97, 14.23, 29.63, 40.43, 114.19-139.67, 163.12,
164.87, 180.80. ESI-MS (Positive ion mode, CH3CN): 589.66 (75%, [M+Na]+) , 605.65 (25%, [M+K]+),
567.65 (10%,[M+H]+).
5-phenyldipyrromethane: 5-phenyldipyrromethane was synthesized following the reported protocol.79
Tetraethylesterdiphenylporphyrin (TEsP): TEsbpyr-dial (200 mg, 0.36 mmol) and 5-
phenyldipyrromethane (79.5 mg, 0.36 mmol) was taken in a round bottom flask. Propionic acid (70 ml)
was added and the system was refluxed for 30 minutes. The acid was distilled out from the reaction
mixture. The solid product obtained was washed with warm water to remove the remained acid. It was
then dissolved in dichloromethane and dried over Na2SO4, evaporated through a rotary-evaporator.
Polypyrrole formed during the reaction was removed through column chromatography on silica gel (100-
200 mesh) with EtOAc-hexane (1:4). The product was purified by second column chromatography using
0.1% dichloromethane-methanol (99:1) mixture. 1H NMR(CDCl3): δ (ppm) -2.31 (br s, 1H), -2.05 (br s,
1H), 1.38 (t, 6H), 1.61 (t, 6H), 4.05 (t, 4H), 4.76 (q, 4H), 7.64-8.20 (m, 10H), 8.97 (d, 2H), 9. 36 (d, 2H),
11.23 (s, 2H). 13C {1H} NMR (CDCl3): δ (ppm) 13.92, 14.56, 22.83, 29.50, 29.84, 61.96, 62.10, 106.44,
119.45-141.19, 146.99, 151.09, 164.70, 166.85. ESI-MS (Positive ion mode, CH3CN): m/z 751.11 (50 %,
[M+H]+).

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FeTEsP: TEsP (30 mg, 0.04 mmol) was dissolved in dry THF (15 ml), 2,4,6-collidine (20.8 µL, 0.16
mmol) was added under N2 atmosphere to generate the porphyrin base. Then FeBr2 (34.5 mg, 0.16 mmol)
was added to it. The reaction mixture was stirred for 10 hrs at room temperature and the progress of the
reaction was monitored by TLC. On full conversion, THF was evaporated using a rotary evaporator and
-
workup was done using dichloromethane and HCl (to remove excess FeBr2 as FeCl4 ). The organic layer
was dried over Na2SO4, evaporated through a rotary-evaporator and purified by column chromatography
1
on silica gel (100-200mesh) with 3.5% dichloromethane-methanol (96.5:3.5). H NMR-Paramagnetic
(CDCl3): δ (ppm) 75.99, 79.94, 81.83. ESI-MS (Positive ion mode, CH3CN): m/z 804.86 (100 %, [M]+).
ZnTEsP: TEsP (30 mg, 0.04 mmol) was dissolved in dry THF (15 ml), 2,4,6-collidine (20.8 µL, 0.16
mmol) was added to generate the porphyrin base. Then Zn(OAc)2 (29.35mg, 0.16 mmol) was added to it.
The reaction mixture was stirred for 10 hrs and the progress of the reaction was monitored by TLC. On
full conversion, THF was evaporated and it was purified by column chromatography on silica gel (100-
200 mesh) with 0.3% dichloromethane-methanol (99.7:0.3). 1H NMR (CDCl3): δ (ppm) -2.31.07 (t, 3H),
1.38 (t, 3H), 3.28 (q, 2H), 4.34 (q, 2H), 6.94-8.21 (m, 10H), 8.95 (d, 2H), 9.25 (d, 2H), 10.66 (s, 2H). 13C
{1H} NMR (CDCl3): δ (ppm) 13.54, 14.42, 61.42, 61.70, 106.79, 119.79-144.89, 151.35, 153.86, 164.47,
167.10. ESI-MS (Positive ion mode, CH3CN): m/z 835.04 (100 %, [M+Na]+), 812.09 (80%, [M]).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at pubs.acs.org.
Additional NMR, mass, UV−vis, and FTIR data and coordination of the DFT-optimized structures (PDF).
Accession Codes
CCDC 1854376 (FeDEsP) , 1854377 (ZnDEsC) and 1959224 (ZnTEsP) contain the supplementary
crystallographic data for this paper. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting
The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223
336033.
AUTHOR INFORMATION

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DOI: 10.1039/D0SC01625J
Corresponding Author
Abhishek Dey, Email: icad@iacs.res.in.
ORCID
Abhishek Dey: 0000-0002-9166-3349
Sk Amanullah: 0000-0003-3288-5942
Present Addresses
School of Chemical Sciences, IndianAssociation for the Cultivation of Science, 2A& 2B Raja SC Mullick
Road, Kolkata, India – 700032
Author Contributions
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This research is funded by the Council of Scientific and Industrial Research (CSIR) Grant
01(2874)/17/EMR-II and Department of Science and Technology grant SERB/EMR/008063. S.A.
acknowledges CSIR-SRF. Ms. Paramita Saha is greatly acknowledged for her help in synthesis.
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