Iron(III)–salen ion catalyzed s-oxidation of L-cysteine and s-alkyl-L-cysteines by H2O2: Spectral, kinetic and electrochemical study

Article abstract of DOI:10.1016/j.poly.2018.11.019

The H₂O₂ oxidation of L-cysteine and s-alkyl-L-cysteines (s-met-L-cys, s-et-L-cys & s-pro-L-cys) catalyzed by iron(III)–salen (salen = N,N′-bis(salicylidene)ethylenediaminato) complexes in aqueous CH₃CN proceeds through Michaelis–Menten kinetics. The rate constant (k) values correlate well with Hammett σ constants, which gives the positive reaction constant (ρ = 1.5–1.9) value. The CV of oxoiron(IV)-salen ion shows a clear oxidation peak at 1.28 V in 0.1 M phosphate buffer (PB) solution using 0.1 M tertiary butyl ammonium perchlorate (TBAP) as supporting electrolyte at 266 K. The rate of the reaction is highly sensitive to the length of the alkyl chains present in the L-cysteines, pH and solvent composition of the medium. The calculated binding constant values (K_f) in the range of 117–613 M^?1, indicate that iron(III)–salen complexes carrying electron donating substituents in the salen ligand have higher binding constant values compared to those carrying electron withdrawing substituents. Product analysis shows the conversion of L-cys to its disulfide and s-alkyl-L-cys to the corresponding sulfoxides. Based on the spectral and kinetic data the plausible mechanism has been proposed.

Full text of DOI:10.1016/j.poly.2018.11.019

Accepted Manuscript
Iron (III)-salen ion catalyzed s-oxidation of L-cysteine and s-alkyl-L-cysteines
by H O : Spectral, kinetic and electrochemical study
2
2
Periyakaruppan Karuppasamy, Dharmaraj Thiruppathi, Muniyandi Ganesan,
Thangamuthu Rajendran, Seenivasan Rajagopal, Veluchamy Kamaraj
Sivasubramanian
PII:
S0277-5387(18)30743-5
https://doi.org/10.1016/j.poly.2018.11.019
POLY 13566
DOI:
Reference:
To appear in:
Polyhedron
Received Date:
Revised Date:
Accepted Date:
5 May 2018
4 November 2018
7 November 2018
Please cite this article as: P. Karuppasamy, D. Thiruppathi, M. Ganesan, T. Rajendran, S. Rajagopal, V.K.
Sivasubramanian, Iron (III)-salen ion catalyzed s-oxidation of L-cysteine and s-alkyl-L-cysteines by H O : Spectral,
2
2
kinetic and electrochemical study, Polyhedron (2018), doi: https://doi.org/10.1016/j.poly.2018.11.019
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Iron (III)-salen ion catalyzed s-oxidation of L-cysteine and s-alkyl-L-cysteines by H O :
2
2
Spectral, kinetic and electrochemical study
a
a
a
Periyakaruppan Karuppasamy , Dharmaraj Thiruppathi , Muniyandi Ganesan ,
a,b
c,*
Thangamuthu Rajendran , Seenivasan Rajagopal ,
Veluchamy Kamaraj Sivasubramanian a,d,**
aPost Graduate and Research Department of Chemistry, Vivekananda College,
Tiruvedakam West- 625 234, Madurai District, Tamil Nadu, India.
bDepartment of Chemistry, PSNA College of Engineering & Technology, Dindigul,
Tamil Nadu, India.
c
School of Chemistry, Madurai Kamaraj University, Madurai-625 021, Tamil Nadu,
India.
d
K. L. N College of Information Technology, Pottapalayam, Sivagangai-630 611,
Tamil Nadu, India.
Synopsis:
The H O oxidation of biologically important amino acids such as L-
2
2
cysteine, s-met-L-cysteine, s-et-L-cysteine and s-prop-L-cysteine catalyzed by iron(III)-salen
complexes (1-6) in aqueous CH CN is an efficient and environmentally benign process. The
3
product analysis and the cyclic voltammetric and Difference Pulse Voltammetric studies
support the formation of active oxidant species and the products. The CV and DPV of
oxoiron(IV)-salen ion shows a clear oxidation peak at 1.28V V in 0.1M phosphate buffer
(PB) solution using 0.1M tertiary butyl ammonium perchlorate (TBAP) as supporting
electrolyte at 266K. The rate constant (k) values are correlate well with Hammett σ constants,
which gives the positive reaction constant (ρ=1.5-1.9) values. The rate of the reaction is
highly sensitive to the length of the alkyl chains present in the L-cysteines, pH and solvent
composition of the medium. Increases the temperature the rate of the reaction also increases.
The kinetic and spectral data reveals that the reaction follows Michaelis-Menton kinetics. The
-1
calculated binding constant values (K ) are in the range of 117-613 M , which indicates
f

electron donating substituents in the salen ligand has higher binding constant values than
electron withdrawing groups. The rate of reaction mainly depends on the substituents present
in the aryl moiety of the salen ligand and increase of chain length of the substrates. Based on
the spectral and kinetic data the possible mechanisms have been proposed.
Iron (III)-salen ion catalyzed s-oxidation of L-cysteine and s-alkyl-L-cysteines by H O :
2
2
Spectral, kinetic and electrochemical study
a
a
a
Periyakaruppan Karuppasamy , Dharmaraj Thiruppathi , Muniyandi Ganesan ,
a,b
c,*
Thangamuthu Rajendran , Seenivasan Rajagopal ,
Veluchamy Kamaraj Sivasubramanian a,d,**
aPost Graduate and Research Department of Chemistry, Vivekananda College,
Tiruvedakam West- 625 234, Madurai District, Tamil Nadu, India.
bDepartment of Chemistry, PSNA College of Engineering & Technology, Dindigul,
Tamil Nadu, India.
c
School of Chemistry, Madurai Kamaraj University, Madurai-625 021, Tamil Nadu,
India.
d
K. L. N College of Information Technology, Pottapalayam, Sivagangai-630 611,
Tamil Nadu, India.
Abstract
The H O oxidation of L-cysteine and s-alkyl-L-cysteines (s-met-L-cys, s-et-L-cys &
2
2
s-pro-L-cys) catalyzed by iron(III)-salen (salen=N,N-bis(salicylidene)ethylenediaminato)
complexes in aqueous CH CN proceeds through Michaelis–Menten kinetics. The rate
3
constant (k) values correlate well with Hammett σ constants, which gives the positive reaction
constant (ρ=1.5-1.9) value. The CV of oxoiron(IV)-salen ion shows a clear oxidation peak at
1
.28 V in 0.1M phosphate buffer (PB) solution using 0.1M tertiary butyl ammonium
perchlorate (TBAP) as supporting electrolyte at 266K. The rate of the reaction is highly

sensitive to the length of the alkyl chains present in the L-cysteines, pH and solvent
composition of the medium. The calculated binding constant values (K ) in the range of 117-
f
-1
6
13 M , indicate that iron(III)-salen complexes carrying electron donating substituents in the
salen ligand have higher binding constant values compared to those carrying electron
withdrawing substituents. Product analysis shows the conversion of L-cys to its disulfide and
s-alkyl-L-cys to the corresponding sulfoxides. Based on the spectral and kinetic data the
plausible mechanism has been proposed.
Keywords: oxoiron(IV)-salen ion; biomimetic oxidation; substrate binding; mechanistic study
Corresponding
E-mail address: rajagopalseenivasan@yahoo.com (S. Rajagopal)
*Tel: +91-4543-258234; Fax: +91-4543-258358, E-mail address: sivavk1957@gmail.com
V.K.Sivasubramanian)
authors.
*Tel:
+91-452-2458246;
fax:
+91-452-2459139,
*
(
1
. Introduction
Model compounds play a significant role in establishing the structure of
intermediates formed during enzyme catalyzed biological redox reactions [1-14]. Horseradish
peroxidase (HRP), a hemoprotein peroxidase, serves as a catalyst in the oxidation reactions to
produce iron(III)-peroxo species (compound 0), which undergoes homo/heterolytic O-O
•+
IV
cleavage to give a ferryl-porphyrin radical cation [Por Fe =O] (compound I), a well
established active oxidant species. Spectroscopic techniques such as electronic absorption,
NMR, EPR, resonance Raman, XRD, EXAFS, ENDOR and Mössbauer have been
extensively used to understand the nature and structure of the active oxidant in biomimetic
studies [1-27]. Rajagopal and his coworkers [11-13] reported mechanistic details to establish
the role of iron(III)–salen complexes as model compounds for nonheme enzymes by using
them as catalysts for the oxygenation of organic sulfides, sulfoxides and amines utilizing

PhIO as well as H O as the oxygen source. They have successfully used the UV-vis, EPR,
2
2
resonance Raman(RR) and Mössbauer spectral methods to establish the nature of
intermediate Recently Nam and co-workers[25,26] have demonstrated that non-heme
iron(III)-hydroperoxo species is not capable of oxidizing organic sulfides at low temperature.
Interestingly when the reaction was carried out at room temperature the authors have
observed the disappearance of the intermediates and the formation of sulfoxide from organic
sulfide. They interpreted their results with the postulation that the O-O bond cleavage of
III
2+
[
(L)Fe -OOH] species occurred at room temperature to give high-valent oxo-iron
intermediates that are responsible for the oxidation of sulfides to sulfoxides. Ueda et al [27]
reported the reaction of
a
supramolecular complex 5,10,15,20-tetrakis(4-
III
sulfonatophenyl)porphyrinatoiron(III) (Fe TPPS) with hydrogen peroxide or cumene
hydroperoxide in an aqueous solution to form two types of hydroperoxo or alkylperoxo
intermediates, which underwent rapid homolysis to the corresponding ferryl oxo species.
Sulfur containing amino acids such as cysteine, methionine and glutathione undergo a wide
range of redox processes under physiological conditions [28-35]. S-methylcysteine and s-
ethylcysteine are two important sulfur containing amino acids present in garlic and onion
[36,]. The biological activity of garlic and other Allium plants is due to the presence of the
organosulfur compounds which have beneficial effect on human health [37-39]. The most
powerful medicinal properties of organosulfur compounds with rich garlic and onion
facilitate their role as anti-atherosclerosis [36], for sugar modulation [40] and as good
antioxidants. They have good antifungal, antimicrobial properties [41-44] and also they are
useful in the treatment of cataract formation, cancer and disorders in immune system [45-50].
The redox reaction of cysteine plays important role in the redox signaling pathway [51].
Cysteine plays a vital role in biochemical processes, cellular redox process, protecting the
cell from oxidative damage through its thiol functional group and gene expression [51,52].

Imbalance in cysteine metabolism has been identified in a range of other neurological
disorders such as Parkinson’s and Alzheimer’s diseases [53]. The thiols generally give more
complex kinetics and larger rate constants [54c]. The kinetics of the reactions of L-cysteine,
s-methyl-L-cysteine and L-cystine with potassium ferrate was investigated under pseudo and
non pseudo first order conditions [54]. Methyl cysteine is oxidized to sulfoxide and then
sulfone by permanganate and peroxide ions, but sulfone is formed incompletely under mild
conditions. In this present investigation we use hydrogen peroxide as oxygen source. It is
more advantageous to use the H O as the oxygen source, because it readily produces oxygen
2
2
for oxygenation, it is eco friendly, readily available reagent and it serves as peroxidase model
and leaves water as the only byproduct. The results obtained throws light on the mechanistic
details and the catalytic role on the H O oxidation of L-cysteines (I-IV) by iron(III)-salen
2
2
complexes (1-6) in aqueous CH CN with HEPES buffer.
3
2
. Experimental
2
.1. Materials
Salicylaldehyde, substituted salicylaldehyde (5-nitro, 5-methyl, 5-methoxy, 5-chloro & 5-
bromo) and substrates (L-cysteine, s-methyl-L-cysteine, s-ethyl-L-cysteine) were purchased
from Sigma Aldrich and used as such. s-Propyl-L-cysteine was purchased from Shanghai
Pengteng Fine Chemical Co.Ltd and used as such. Anhydrous ferric chloride (AnalaR grade),
ethylene diamine (AnalaR grade), HEPES buffer (AnalaR grade), HPLC grade acetonitrile
(
AnalaR grade) and 30% H O were purchased from Merck.
2 2
2
.2. Methods
De-ionized water was prepared by using EASY-STILL (Mark -2000DDQ-XL) instrument
and used to prepare the solutions for this study. HEPES buffer was used to maintain the pH
of the solvent medium. The UV-visible absorption spectral measurements have been carried

out using JASCO V-530 spectrophotometer. The infra-red spectra have been recorded in a
JASCO FT-IR-410 spectrophotometer in a solid phase using KBr pellets. Mass Spectrum was
recorded by using Q-TOF micromass (YA-105) spectrometer to record high-resolution mass
spectrum (HRMS) both in the APCI (Atmospheric Pressure Chemical Ionization) and the ESI
1
(
Electrospray Ionization) modes. H NMR spectrum was recorded on a Jeol-400 MHz
Nuclear Magnetic resonance instrument. The CV and DPV were measured in aqueous
CH CN medium using electrochemical workstation (CH-Analyzer Model: CHI6087D), with
3
0
.1M TBAP as the supporting electrolyte and Ag/AgCl as the reference electrode.
.2.1. Synthesis of iron(III)-salen complexes (1-6)
2
In the present work, we synthesized six iron (III)-salen complexes (1-6) using
programmable solvothermal one pot method in a single step by the reaction of respective
salicylaldehydes with ethylenediamine and anhydrous ferric chloride in ethanol medium. The
one pot solvothermal technique provides the product in a high yield and relatively in the pure
form [56c] compared to the conventional synthesis. The preparation of iron(III)–salen
complexes by conventional method involves two stages. The first step involves the
preparation of salen ligand by the condensation of salicylaldehyde with ethylenediamine and
the second step involves the reaction of salen ligand with anhydrous ferric chloride to yield
iron(III)–salen complex. In every stage of conventional synthesis, the purification involves a
tedious procedure [11-13]. The scheme for the of synthesis of iron(III)-salen complexes (1-6)
is shown in Scheme.1.

N
O
N
O
HO
Fe
Cl
X
FeCl3
HN-CH -CH -NH
X
2
2
2
2
O
X
Y
Fe(III)Chloride Ethylene diamine
Salicylaldehyde
2.0 eq.
Y
Y
1
.0 eq.
1.0 eq.
iron(III)-salen chloride
1
2
3
4
5
6
. X = Y= H
1. X = Y= H
. X =NO , Y = H
2. X =NO , Y = H
2
2
. X = Me, Y = H
. X = OMe, Y = H
. X = Cl, Y = H
. X = Br, Y = H
3. X =CH , Y = H
3
4. X = OMe, Y=H
5. X = Cl, Y = H
6. X = Br, Y = H
Scheme.1. Solvothermal one pot synthesis of iron(III)-salen complexes (1-6)
.2.2. General procedure for the preparation of [Oxoiron(IV)-salen]-ion (1a-6a)
Oxoiron(IV)-salen]-ions were prepared by using eco-friendly greener oxidant,
hydrogen peroxide. Addition of H O to iron(III)-salen complex 1 in aqueous acetonitrile as
2
[
2
2
solvent, leads to decrease in intensity at 495 nm accompanying an increase in intensity at
about 390nm and formation of a broad band in the range 650 – 800 nm in the UV-visible
absorption spectrum. The spectral changes observed in the present work help us to conclude
that the possible active oxidant under the present experimental conditions is oxo-iron species
similar to the proposal of van Edlic and co-workers [12,13,55]. Conversion of iron(III)-salen
complexes (1-6) to [oxoiron(IV)-salen]-ions (1a-6a) is shown in Scheme 2.
Y
Y
2
4
3
1
3'
1'
Y
2
Y
2'
3' 4'
4
'
'
4
6
3
O
5
5'
2'
5
H O
2
5'
X
O O
Fe
N N
X
2
X
O
O
X
H O
2
III
IV
Fe
N N
6
6'
1
1'
7'
6
7
7'
7
Cl
Cl
1
. X=5,5'-H; Y=3,3'-H
. X=5,5'-NO ;Y=3,3'-H
. X=5,5'-Me;Y=3,3'-H
. X=5,5'-OMe; Y=3,3'-H
. X=5,5'-Cl; Y=3,3'-H
. X=5,5'-Br; Y=3,3'-H
1a. X=5,5'-H; Y=3,3'-H
2
3
4
5
6
2a. X=5,5'-NO2;Y=3,3'-H
3a. X=5,5'-Me;Y=3,3'-H
4a. X=5,5'-OMe; Y=3,3'-H
5a. X=5,5'-Cl; Y=3,3'-H
6a. X=5,5'-Br; Y=3,3'-H
2
Scheme 2. Preparation of [oxo(salen)iron(IV)] complexes by using eco-friendly greener
oxidant hydrogen peroxide.

2
.2.3. Electrochemical study
The redox potentials (E ) were measured using electrochemical Workstation (CH-
1
/2
Analyzer Model: CHI6087D). The three electrodes used for CV and DPV studies are the
glassy carbon (GC) electrode as the working electrode, Pt wire as counter electrode and
Ag/AgCl/KCl (saturated) as reference electrode. 0.1M Tertiary butyl ammonium perchlorate
(
TBAP) was used as supporting electrolyte. The phosphate buffer (NaH PO +Na HPO4) was
2 4 2
prepared by mixing 0.1 M solution of sodium dihydrogen phosphate with disodium hydrogen
phosphate to maintain the pH= 7.2.
2
.2.4. Kinetic study
The kinetic studies were carried out using spectrophotometer JASCO V-530 at the
-4
desired wavelength. The concentrations of iron(III)-salen complex and H O taken are 2x10
2
2
-3
M and 5x10 M respectively. The strength of H O is carefully monitored in all the
2
2
measurements. The kinetic study on the H O oxidation of L-cysteine and s-alkyl-L-cysteines
2
2
catalyzed by iron(III)-salen ion in CH CN/H O (70:30 v/v ratio) was conducted under pseudo
3
2
first order conditions. The progress of the reaction was followed by maintaining the
concentration of substrates at least 10 times higher than that of oxidant concentration. The
rate of the reaction was measured by following the decay of absorbance of the oxidant with
time at appropriate wavelength and collected in Table 1.
2
.3. Product analysis
A solution containing 5 mM substrate, 0.2mM iron(III)-salen complex and 5mM
H O in 5 ml of CH CN/H O (70:30 v/v ratio) was stirred at 298 K for 0.5-1h depending on
2
2
3
2
the nature of the substrate and complex. The reaction mixture after completion of the reaction
was extracted with diethyl ether to recover the organic products. It was dried over sodium

sulfate. The solvent was evaporated by rota evaporator and the product was analyzed using
1
FT-IR, H NMR, ESI-MS and GC-MS spectral techniques. The detailed product analyses are
elaborated in the discussion part.
3
. Results and discussion
3
.1. Electochemical study
Cyclic voltammetric (CV) response of iron(III)-salen complex at GC electrode
in 0.1M TBAP at 50mV scan rate is shown supporting information (Fig.S1). The potential at
0.37 V corresponds to the reversible reduction of Fe(III) to Fe(II) and another peak at
0.96V to irreversible oxidation of Fe(II) to Fe(III). These results are similar to the report on
-
+
the redox properties of iron(III)-salen complex.[56a-c] In the present study we have also
recorded the CV of iron(III)-salen-H O system at 266K in CH CN-H O(90%-10%(v/v))
2
2
3
2
mixture using 0.1 M TBAP as the supporting electrolyte in pH= 7.2 and the observed cyclic
voltammagram is shown in Fig.1. The CV exhibits quasi-reversible redox wave at E =1.28
1
/2
V versus Ag/AgCl as and the obtained CV is similar to the CV reported for oxoiron species
•+
IV
IV
◦
(
TDCP )Fe (ClO ) [57a] and (TMP)Fe O [57b] in acetonitrile at -35 C. The DPV of
4
oxoiron(IV)-salen ion shows oxidation peak potential at 1.28V which infers that the
formation of oxoiron(IV)-salen ion from iron(III)-salen with the addition of hydrogen
peroxide as shown in Fig.2.
3
.2. UV-visible absorption spectral study
The absorption maxima (λmax) of the iron(III)-salen complexes were reported in pure
acetonitrile medium[11]. Here we recorded the absorption spectra of iron(III)-salen
complexes (1-6) in aqueous acetonitrile. Interestingly, addition of water leads to substantial
red shift in the absorption maximum of LMCT of iron(III)-salen complexes. The λmax value
of parent iron(III)-salen complex 1 is shifted from 470 nm to 495 nm when the medium is

changed from CH CN to 70% acetonitrile -30% water (v/v) (Fig.3). The same trend of
3
shifting of λmax to higher wavelength (red shift) is observed for all iron(III)-salen complexes
(1-6) when the medium is changed from acetonitrile to aqueous acetonitrile. The λmax value of
iron(III)-salen complexes leads to more red shift in the presence of electron donating
substituents in the ligand but to the small extend in the presence of electron withdrawing
substituents. The λmax values of a) iron(III)-salen complex b) iron(III)-salen complex on the
addition of H O and c) iron(III)-salen complex-H O mixture on the addition of substrates
2
2
2
2
(I-IV) are given in Table 1 (supporting information Figs S2-S5).
Table 1
UV-vis absorption spectral data of iron(III)-salen complexes (1-6), [oxoiron(IV)-salen] ions
(
1a-6a) and on the addition of substrates (I-IV) to 1a-6a in CH CN/H O (70% :30% (v/v))
3 2
mixture at 298 K, pH=6.5. Oxidation potential values of substrates (I-IV) are given at the
bottom.
Complex
UV-vis spectral data
λmax in
λmax values
of oxo
iron(IV)-
salen
λmax values of oxo iron(IV)-salen after the addition of
CH CN/H O
substrate
2
3
I
II
III
IV
(7:3 v/v
ratio)
(1a-6a )
1
2
3
4
5
6
351, 495
471
321, 488
320, 494
399,468
325,512
319,548
358,494
351,494
310,495
385,468
328,514
314,547
356,493
350,494
308,495
380,469
325,514
314,549
357,495
350,495
306,494
376,470
327,516
313,549
356,496
351,496
467
350,516
354, 541
361,496
352,494
---
342,502
349, 534
352,485
348,489
---
*
Oxidation
potential
1
.46±0.0V 1.63±0.02V
1.72±0.02V 1.80±0.02V
(
E )
ox
Addition of H O to iron(III)-salen complex 1 in CH CN/H O (7:3(v/v) ratio), leads to a
2
2
3
2
decrease in intensity at 495 nm accompanying an increase in intensity at about 390nm and

formation of a broad band in the range 650 – 1000 nm (Fig. 4). The absorption spectral
changes shown in the Fig.4 with clear isosbestic point at 640 nm and absorption in the region
6
50-1000 show the conversion of iron(III) to oxoiron(IV) species and the process involves no
complex formation[11-13]. We have also studied the same system at 268K and the spectral
changes are shown in Fig. 4a. Fig. 4a shows rapid decay at 495 nm and increase in intensity
at 390nm and broad absorption in the region 650-1000 nm with a peak at 740 nm (Fig. 4a).
The formation of this broad weak band in the near-IR region is characteristic of [oxoiron(IV)-
salen] species[13b-13f]. The new species with λmax = 740 nm is stable at 268K but decays in
the presence of L-cysteines. The absorption spectral changes are also observed for all other
iron(III)-salen complexes on the addition of H O . The similar trend of spectral changes are
2
2
observed for complex 2 in CH CN/H O (7:3v/v ratio), with addition of H O shown in
3
2
2
2
supporting information Fig. S4.The absorption spectral changes with clear isosbestic point
III
shows that the conversion of Fe to oxoiron(IV) species involves no complex formation
(
Scheme 1) at 298K. [12,13] We followed the spectral changes of iron(III)-salen-H O
2 2
mixture in the presence of L-cysteine and s-alkyl-L-cysteines (I-IV)(Chart 1)
spectrophotometrically. On the addition of I to complex 1, we observe an initial increase of
OD followed by regular decrease at 495 nm and continuous decrease in OD at 390 nm and in
the region 500-800nm (Fig. 5). The appearance of broad absorption in the region 600-800 nm
for the iron(III)-salen–H O and a decrease in its intensity in the presence of substrate,
2
2
clearly indicates the formation of the active oxidant [oxoiron(IV)-salen)] radical cation and
its decay (supporting information Fig. S6, S7 & S7a). This observation indicates that the
present system leads to initial oxidant-substrate complex formation followed by the oxidation
of substrate to its oxidized form by the active oxidant. The plot of log OD vs time gives linear
fit as shown in supporting information Fig. S8. Almost in all the cases with the addition of
substrates to the iron(III)-salen complex-H O mixture the λ values obtained are very close
2
2
max

to the λmax values of iron(III)-salen complexes. This particular observation helps us to
conclude that after effecting oxygenation the [oxoiron(IV)-salen] species forms back the
starting material, the iron(III)-salen complex. Here the iron(III)-salen complexes behave as
good catalysts.
NH2
OH
SH
L-cysteine
S
OH
H N
2
O
s-methyl-L-cysteine
II
I
NH2
H N
2
O
S
OH
S OH
s-propyl-L-cysteine
O
s-ethyl-L-cysteine
III
IV
Chart 1. Structures of L-cysteine and s-alkyl-L-cysteines (I-IV) used in the present study
.3. Kinetics of oxidation reactions
3
The rate constants for the iron(III)-salen catalyzed H O oxygenation of L-cysteine
2
2
and s-alkyl-L-cysteines (I-IV) are obtained by measuring the rate of reaction at different
substrate concentration. The values of pseudo first order rate constant (k ) for the iron(III)-
1
salen complexes (1-6) catalyzed H O oxidation of cysteine as a function of concentration of
2
2
I are shown in Fig 6.
From Fig.6 it is obvious that the k value increases with increase in substrate
1
concentration but attains saturation at high substrate concentration. These results indicate that
the reaction proceeds through Michaelis-Menten type catalysis. The inference from the
saturation kinetics is that the substrate binds to the oxidant before the rate controlling step.
Thus the kinetics of iron(III)-salen catalyzed H O oxidation of L-cysteine and s-alkyl-L-
2
2
cysteines proceeds through Michaelis-Menten kinetics (eqs (1) and (2)). The observed

kinetics is analyzed in terms eqs (3) and (4) and the rate constant, k, for the formation of
products and the Michaelis-Menten constants are obtained from the plot of 1/k vs
1
1
/[substrate] (supporting information Fig.S9) and the k and K data are collected Tables 2
M
and 3 respectively. The low K values (Table 3), indicate the strong binding of substrates
M
with oxidant [45, 49] and also high catalytic efficiency of iron(III)-salen complex.
Oxidant + Substrate
Oxidant-substrate complex
Products
(
1)
Oxidant-substrate complex
(2)
k [substrate]
k1 = kobs
=
(3)
(4)
K  [substrate]
M
The rearrangement of eq. (3) leads to eq. (4)
1
1
KM
1
k



k1 kobs k [substrate]
Table 2
Rate Constants k obtained from Michaelis-Menten kinetics for H O oxidation of cysteines
2
2
(
I-IV) catalyzed by iron(III)-salen complexes (1-6) in CH CN:H O (70:30, v/v) at 298 K,
3 2
pH= 6.5.
Substrate
Rate constant k values of oxo iron(IV) salen complexes oxidation of cysteines
2
-1
k x10 s
1
2
3
4
5
6
Ρ
r
I
5.3±0.2 105±5
3.0±0.2 69.9±3
1.6±0.05 1.1±0.06 8.1±0.4 7.5±0.4 1.9±0.1 0.99
1.0±0.05 0.6±0.03 4.5±0.2 3.1±0.2 1.9±0.1 0.98
II

III
IV
1.4±0.1 35.3±2
0.7±0.1 16.3±1
0.8±0.04 0.5±0.03 2.7±0.1 1.9±0.1 1.7±0.1 0.98
0.6±0.03 0.3±0.02 1.1±0.1 1.5±0.1 1.5±0.1 0.97
Table 3
Michaelis-Menten constant values, K obtained from Michaelis-Menten kinetics for H O
2
M,
2
oxidation of cysteines (I-IV) catalyzed by iron(III)-salen complexes (1-6) in CH CN:H O
3
2
(70:30, v/v) at 298 K pH= 6.5.
Michaelis-Menten constant (KM X 105 M ) values of oxo iron(IV) salen complex
-1
Substrate
oxidation of cysteines
1
2
3
4
5
6
I
17±0.9
8±0.4
4±0.2
2±0.1
99±5
33±2
15±1
10±1
9±0.5
5±0.3
3±0.2
0.8±0.0
8±0.4
5±0.3
3±0.2
0.5±0.0
7±0.4
3±0.2
1±0.1
0.4±0.0
6±0.3
4±0.2
1±0.1
0.3±0.0
II
III
IV
3
.4. Substituent effect
The rate of iron(III)-salen ion catalyzed H O oxidation of L-cysteine and s-alkyl-L-
2
2
cysteines are highly sensitive to the nature of the substituents present in the phenolic part of
the salen ligand of iron(III)-salen complexes (1-6). The electron donating groups like methyl,
methoxy present in the salen ligand decrease the rate of the oxidation while electron
withdrawing groups such as chloro, bromo, nitro increase the rate of oxidation. It is found
that the correlation of log k values of iron(III)-salen complexes catalyzed reaction with
Hammett’s sigma constant is excellent (r = 0.99, n = 6) shown in Fig. 7 and the ρ value is in
the range1.5-1.9 (Table 2).

The positive ρ value points out that negative charge is developed on the oxidant in the
transition state. It is interesting that the positive ρ values in the range 1.5 to 1.9 obtained in
the present work (supporting information Figs. S10-12) are similar to the positive ρ value 1.4-
2
.3 as obtained by Premsingh et al [58]for the [oxo(salen)chromium(V)] oxidation of
aromatic amines.
The electron withdrawing groups in the salen part of the [oxo(salen)iron(IV)]
complexes will have the inherent character to destabilize the oxo-species making them as
highly reactive. However the [oxo(salen)iron(IV)] species, the radical cations, are ultimately
stabilized by the electron donating groups in the salen ligand. [11-13] This difference in the
.
-
IV
stability of the oxidant [(salen )Fe =O)] is attributed to the difference in the reactivity of
iron(IV)-complexes having electron withdrawing and electron donating substitutions. The log
k values are plotted against the reduction potentials of iron(III)-salen complexes (1-6) and
this plot is linear and the slope value is 8.87 as shown in Fig. 8. The linear relationship with
high slope value supports the postulation of the electron transfer (ET) in the rate controlling
step for the L-cysteine oxidation reaction. The dimerization of sulfur radicals to form
disulfide (cystine) for the L-cysteine oxidation reaction can formulated in Scheme 2. The
plots of log k vs oxidation potentials of cysteines (I -IV) are also linear and the slope value is
in the range of -1.5- -1.9 as shown in supporting information Fig S13 for complex 1. From
Fig S13, the slope values obtained from the linear plots are also comparable with the values
obtained by Goto et al [59]. This linear relationship with low ρ values support the postulation
of oxygen atom transfer (OAT) in the rate controlling step for the oxidation of s-alkyl-L-
cysteines. However the oxidation of L-cysteine follows the electron transfer (ET) mechanism.
The mechanistic details to the formation of selective products are presented in Schemes 2 and
3
.

3
.4. Substrate binding with the catalyst
In order to understand the nature of interaction between the substrate and
catalyst used in the present study, we have recorded the optical density (OD) of
iron(III)-salen complexes in the presence of different concentration of L-cysteine as
shown in supporting information Fig. S13. As the L-cysteine concentration increases,
there is a substantial increase in the absorbance. It shows that the addition of substrate
results an increase in the absorbance of iron(III)-salen complex at 500 nm indicating
the binding of substrate with the catalyst. The binding constant values have been
calculated using Benesi-Hildebrand method and a sample plot is shown in Fig. 9 and
the calculated K values are given in the Table 4. The calculated binding constant
f
-1
values (K ) are in the range of 117-612 M . It is interesting to note that iron(III)-salen
f
complex with electron releasing substituents in the salen ligand has higher binding
constant values than the parent iron(III)-salen complex but the complex with electron
withdrawing groups in the salen ligand has lesser value than the parent iron(III)-salen
complex.
Table 4. Values of K for the binding of I with iron(III)-salen complexes (1-6) calculated by
f
Benesi-Hildebrand method.
-1
Complex
K M
f
1
2
3
4
5
6
242
613
135
460
117
157
3
.4.1. Radical scavenger effect

In this present study, we have carried out radical scavenger activity of L-cysteines
against iron(III)-salen complex and H O in aqueous CH CN in the presence of HEPES
2
2
3
-4
-3
buffer. 0.3mL of iron(III)-salen complex [2x10 M] was added to 0.3 mL of H O [5x10
2
2
-3
M] and 0.3mL of L-cysteine [2x10 M] solution and remaining 2.1 mL of solvent with 0.1M
of HEPES buffer was taken in a 3 mL UV-visible spectrophotometer cuvette. The absorbance
of the sample was recorded at wavelength 230 nm for H O and 495nm for iron(III)-salen
2
2
complex. The scavenging effect was calculated using the following equation
Scavenging effect (%) = {1- (Abssample / Abscontrol)} x 100
The iron(III)-salen complex and H O radical scavenging activity of L-cysteine was
2
2
linearly increased with increasing the concentrations of L-cysteine (0.02mM -2.0mM) as
shown in supporting information Fig.S24. H O continuously provides to form reactive
2
2
oxidative species (ROS) through iron(III)-salen complex to L-cysteine. Scavenging effect of
H O with L-cysteine was also studied but during scavenging of H O , cysteamine was
2
2
2
2
generated by the degradation of L-cysteine with the loss of thiol group. The H O scavenging
2
2
of L-cysteine shows only less than >10.0% compared with the reported values [60a-c]. This
result was indicated that the H O scavenging activity of L-cysteine has adversely associated
2
2
in the presence of thiol functional group
3
.5. Effect of changing pH
The kinetic study of the reaction has been carried out at different pH values. HEPES
buffer was mainly used to maintain the pH of the reaction medium. The rate constant data
measured at different pH values are collected and given in the supporting information Table
S2 and shown in Fig S14. These data show a sharp increase in the rate of the reaction with an
increase of pH upto 6.5 followed by a decrease at pH>6.5 (supporting information Fig. S14).
Cysteines are zwitter ions at neutral pH. At low pH both amino and carboxyl groups are

protonated and at high pH carboxyl group is deprotonated and amino group is in the
unprotonated form. Thus at low and neutral pH the site of attack of the oxidant on the
substrate is the sulphur (thioether) centre but at the pH > 7 both the sulfur centre and amino
group are the likely sites of attack of the oxidant. These kinetic studies establish that
cysteines are highly sensitive to the change of pH of the medium [60d].
3
.6. Influence of solvent
The effect of changing the solvent composition on the rate of the reaction has also
been investigated and the data presented in the supporting information Table S3. The data
show that the value of rate constant decreases as the water content in the reaction medium
increases from 10% to 50% [61-63a]. We are not able to carry out the reaction at high water
content due to solubility problem of iron(III)-salen complexes. Water being a solvent with
high polarity will have greater affinity with oxidants of the present reaction system having the
positive charge. As a result with increase of water content there is more solvation and the
solvation sheath is having a negative impact for the formation of oxidant- substrate complex
which is a pre-requite for the oxygenation process. Also the reactive oxygen of the active
oxidant [oxo(salen)iron(IV)] species is surrounded by water molecules. These twin effects
may be the cause for the observed reaction rates of the reactions [62].
3
.7. Effect of temperature
The rate constant data for the oxidation of L-cysteine with [oxoiron(IV)-salen]
complexes (1a-6a) at five different temperatures are collected in Table 5. For all the
‡
‡
‡‡
substrates the increase in temperature increases the rate constant. The ∆∆ HH and ∆∆ SS vv aa ll uu ee ss
evaluated from the kinetic data are presented in Table 5. Eyring’s plots log k/T versus 1/T for
all complexes (1a-6a) with L-cystine is shown in supporting information Fig S16. The
‡
‡
‡‡
thermodynamic data show that both ∆∆ HH and ∆∆ SS values vary substantially with the change in
‡
‡
‡‡
nature of the oxidant. It is interesting to observe that the values of ∆∆ HH and ∆∆ SS are

complementary to each other, rather to say there is a compensatory effect with respect to ∆∆ HH ‡
‡
‡
‡
‡‡
and ∆∆ SS .. The negative ∆∆ SS vv aa ll uu ee ss indicate that the transition state is more orderly compared
‡
‡
‡‡
to the reactants. The plot of ∆∆ HH vv ss .. ∆∆ SS shown in Fig. 10 is linear with good correlation
coefficient (r=0.98, n=6) and the isokinetic temperature is 433 K. The linear isokinetic
relationship indicates that the oxidation of L-cysteine follows the similar mechanism. The
fact that the isokinetic temperature is well above the experimental temperature indicates that
the application of Hammett equation to the title reaction is valid.
Table 5
2
-1
Rate constant kx10 s values at five different temperatures and thermodynamics parameters
for complexes 1-6 catalyzed H O oxidation of L-cysteine in CH CN-H O (7:3 v/v ratio) at
2
2
3
2
2
98K,pH= 6.5
2
-1
Complexes
Thermodynamic
parameters
k x10 s
‡
H
‡
2
83K 288K 293K 298K 303K
∆
-∆S
kJ/mol
J/K/mol
1
2
3
4
5
6
0.8
2.0
3.2
5.3
7.4
134
172
92.0 96.4 100.5 104.9 110.0
2
182
122
87
0.4
0.7
1.2
0.7
6.0
5.4
1.6
1.1
8.1
7.5
2.2
1.6
10.2
9.6
33
44
24
31
0.07 0.3
1.9
1.0
4.1
3.2
147
124
3
.9. Product analysis
1
We have used FT-IR, H-NMR, ESI-MS and GC-MS techniques for the analysis of
products obtained during the course of the reaction under pseudo first order experimental
condition. Generally amino acids are pH dependent and on dissolving in a polar solvent they

easily ionize due to ionic nature. Basic groups such as -NH get ionized in positive mode
2
while acidic groups -COOH group get ionized easily in the negative mode of ionization. This
is because in positive mode of ionization proton gets added while in negative mode of
ionization proton gets removed. So it may be difficult to run GC-MS spectra for these zwitter
ionic products due to easy ionization. Even though it may be difficult we tried to get the clear
GC-MS spectra for our reaction product. Due to ionization of our product it gets fragmented
easily during ionization with loss of small molecules like CO or H O and not able to provide
2
2
an exact m/z value for s-oxidation of L-cysteines. The IR spectrum shows the stretching
-1
-1
frequency of –S-S- (cystine), a disulfide, at 518 cm and a band at 692 cm to the stretching
frequency of unreacted cysteine carrying C-S function (supporting information Fig. S17).
This study shows that L-cystine is the product during the H O oxidation of I catalyzed by
2
2
1
iron(III)-salen complexes. H NMR spectral data of I and its disulfide (L-cystine) are
comparable with the authentic values supporting information Fig. S18. The ESI-MS data
show distinct peaks at m/z values 121, 239 formed during the course of the title reaction. It
strongly conforms the conversion of I (m/z=121) to L-cystine (m/z=239.07) during iron(III)-
salen ion catalyzed H O oxidation of I supporting information Fig. S19. s-Alkyl-L-cysteines
2
2
1
give the corresponding sulfoxide as the product. H NMR spectral data of the product δ =
.1–2.24 (2H, q), 2.73–3.03 (2H, m), 3.71–3.79 (1H, m), 2.62 and (s, SOCH ) values are in
2
3
good agreement with reported values [38,60,61] and also supports the formation of sulfoxide,
supporting information Fig.S20. The ESI-MS data also support the formation of sulfoxide as
the major product. It strongly confirms the conversion of II (m/z=135.0) to its corresponding
sulfoxide (m/z=157.03) during the H O oxidation of II catalyzed by iron(III)-salen
2
2
complexes as shown in supporting information Fig.S21. The GC-MS spectrum H O
2
2
oxidation of I catalyzed by iron(III)-salen complex is shown in the supporting information

Fig.S22. The formation of product during the iron(III)-salen complexes catalyzed H O
2
2
oxidation of L-cysteines are shown in Scheme.3 in the supporting information Fig.S23.
4
. Mechanism
The Michaelis- Menton kinetics of the present reaction helps us to postulate that the
reaction proceeds through the complex formation between the oxidant and the substrate. The
observed low KM values indicate the strong binding of substrate with the oxidant.
Interestingly the plots of log k values against oxidation potentials of substrates (E ) are linear
ox
and a sample plot is shown in supporting information Fig S15 (slope = -1.94, r = 0.98, n=6)
for complex 1. The plots of other complexes are presented in supporting information
Fig.S15a-13f. The slopes obtained from the linear plots are also compared with the values
obtained by Goto et al [59]. Attempt has been made to find nature of intermediate formed
under the present experimental conditions. The UV-vis absorption spectral study clearly
III
shows the formation of Fe -OOH which undergoes heterolytic cleavage resulting in the
IV
•+
formation of reactive species [O=Fe -(salen)] as the oxidant. Based on the experimental
observations that (i) the reaction is fractional-order in the substrate and attains saturation
kinetics at high [substrate] and (ii) the shift in the max and the initial increase in the
absorbance on the addition of the substrate to the active oxidant indicate the formation of a
complex between the oxidant and substrate. Thus with the addition of substrate, initial
complex formation takes place via either oxygen atom of oxidant or the metal ion. After the
formation of oxidant-substrate complex the formation of disulfide or sulfoxide as the final
product may be formulated by the mechanisms shown in Schemes 2 and 3. In most of the
other oxidation reactions of sulfides [64] and cysteines [65-67] the kobs depends linearly on
the substrate concentration and no saturation kinetics is observed. This linear dependence on
substrate was taken as evidence for large K values (loose enzyme–substrate complexes).
M

The saturation kinetics observed here and low K values (Table 3) point out the strong
M
binding of substrate with the oxidant. We also got the slopes of –1.3 to –2.4 in the plots of
log k vs E (supporting information Fig. S15-S20) as observed by Goto et al [59] for a
ox
reaction proceeding via direct oxygen transfer. Also the oxidation potentials (1.46, 1.63,
1
1
.72, 1.80V) of the substrates (I-IV) are comparable with the oxidation potentials (1,.13,
.24, 1.34, 1.37, 1.41, 1.61, 1.70) of sulfides used by the above research group. They have
proved that these sulfides prefer the path of oxygen atom transfer rather than electron
transfer. They also indicated that the substrate1,5-dithiacyclooctane (DTCO) having low
oxidation potentials (0.70V) proceeds through electron transfer pathway with high ρ
values(≈8) in the plot of log k vs. Eox. All these facts support the oxygen atom transfer
mechanism in the present study. The active oxidant in the present system facilitates electron
transfer (ET) for the oxidation of L-cysteine to L-cystine (disulfide) (Scheme 4) and the
oxygen atom transfer (OAT) for the oxidation of s-alkyl-L-cysteines to its corresponding
sulfoxides (Scheme 5).

O
O
OH
III
III
IH2V
Fe
H O
Fe
Fe
2
2
Compound 0
Compound I
Active oxidant
Iron(III)-salen
R
R'
S
O
O
R
R'
Fe(IV)
S
IV
Fe
L-cysteine
Active oxidant
oxidant-substrate complex
R'
R
S
NH2
O
ET
R=
R'=H
OH
R
S
H O
2
S
R
L-cystine
III
Fe
Iron(III)-salen
Scheme 4. Electron transfer mechanism for the iron(III)-salen catalyzed H O oxidation
2
2
I to L-cystine (disulfide) in CH CN/H O (7:3 v/v ratio) at pH= 6.5
3
2

O
O
OH
III
III
IV
Fe
H O
Fe
Fe
2
2
Compound 0
Compound I
Active oxidant
Iron(III)-salen
R
R'
S
O
FeIV
O
R
R'
IV
S
Fe
L-cysteine
Active oxidant
oxidant-substrate complex
R
R'
NH2
S
O
R'=-CH ,-C H ,-C H
R=
3
2
5
3
7
O
OH
III
Fe
OAT
O
S
III
Fe
R
R'
Iron(III)-salen
S-Alkyl-L-cysteine sulfoxide
Scheme 5. Oxygen atom transfer (OAT) mechanism for the iron(III)-salen catalyzed H O
2
2
oxidation II-IV to its corresponding sulfoxides in CH CN/H O (7:3 v/v ratio) at pH= 6.5
3
2
5
. Conclusions
The H O oxidation of biologically important amino acids L-
2
2
cysteine, s-met-L-cysteine, s-et-L-cysteine and s-prop-L-cysteine catalyzed by iron(III)-salen
complexes (1-6) in aqueous CH CN is an efficient and environmentally benign process. The
3
product analysis and the cyclic voltammetric and Difference Pulse Voltammetric studies
support the formation of active oxidant species and the products. The kinetic data reveal that
the reaction follows Michaelis-Menton kinetics. The rate of reaction mainly depends on the
substituents present in the aryl moiety of the salen ligand and increase of chain length of the

substrates. Based on the spectral and kinetic data the possible mechanisms have been
proposed.
6
. Abbreviations
Salen- N.N’-bis(salicylidene)ethylenediaminato
Tpps-5, 10, 15, 20-tetrakis(4-sulfonatophenyl)porphyrinato
HRP- harseradishperoxidase
HEPES- (4-(2-hydroxyethyl)piperazine-1-ehtanesulfonicacid
TBAP- Tetrabutylammoniumperchlorate
GCE- Glassy carbon electrode
DPV-Differential pulse voltammogram
CV-Cyclic voltammogram
ESI-MS- Electron spray ionization mass spectrometry
FT-IR- Fourier transfer infrared spectroscopy
NMR- nuclear magnetic resonance
OD – Optical density
OAT- Oxygen atom transfer
ET-electron transfer
DTCO- 1, 5-dithiacyclooctane
Acknowledgement
The authors thank University Grants Commission (UGC) for financial support and the
Management of Vivekananda College, Tiruvedakam West, Madurai- 625 234 for providing
research facilities.
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