Biomimetic oxidation of acetaminophen prodrugs catalyzed by iron porphyrins: Effect of nitrogen and thiolate axial ligands on drug and metabolite formation

Article abstract of DOI:10.1016/j.apcata.2015.11.031

Metalloporphyrins (MP) are often employed in biomimetic catalysis due to their structural and functional similarity to the heme prosthetic group of oxygenating enzymes. In these enzymes, the iron porphyrin is primarily protein-bound at the active site by cysteine (cytochrome P450, CYP), histidine (peroxidase), and tyrosine (catalase), acting as axial ligands. The diverse functionality and variable oxidizing power exhibited by different oxygenating enzymes is [in part] attributed to the ligating amino acid, and the relationship between iron and the heteroatom. Attempts to mimic this relationship by developing MP systems that behave similarly to CYP enzymes have mainly focused on incorporating nitrogen (N)-heterocycles. Although it is widely known that different enzyme families favor distinctive products, sulfur is easily oxidized and prone to form disulfide bonds, and as such, thiolates for the most part remain unexplored. Herein, we report our findings from the biomimetic oxidation of phenacetin, methacetin, and acetanilide by meta-chloroperoxybenzoic acid (m-CPBA), catalyzed by hindered iron (III) porphyrins that are chelated by different axial ligands. Nine axial ligands that vary in pK_a (2.8-11.2), size, and heteroatomic identity were examined, to more accurately determine the factors that contribute to function and effect. The [three] thiolate ligands afforded the greatest yields of acetaminophen in the majority of cases. Furthermore, nitrogen- and sulfur-containing ligands were observed to promote different reaction pathways, regardless of pK_a or ligand size.

Full text of DOI:10.1016/j.apcata.2015.11.031

Accepted Manuscript
Title: Biomimetic oxidation of acetaminophen prodrugs
catalyzed by iron porphyrins: Effect of nitrogen and thiolate
axial ligands on drug and metabolite formation
Author: Chiara M. Chapman John M. Pruneau Chloe A.
Laverack Andrew S. Dutton Graham B. Jones
PII:
S0926-860X(15)30259-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.apcata.2015.11.031
APCATA 15658
To appear in:
Applied Catalysis A: General
Received date:
Revised date:
Accepted date:
10-9-2015
13-11-2015
16-11-2015
Please cite this article as: Chiara M.Chapman, John M.Pruneau, Chloe A.Laverack,
Andrew S.Dutton, Graham B.Jones, Biomimetic oxidation of acetaminophen
prodrugs catalyzed by iron porphyrins: Effect of nitrogen and thiolate axial
ligands on drug and metabolite formation, Applied Catalysis A, General
http://dx.doi.org/10.1016/j.apcata.2015.11.031
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Biomimetic oxidation of acetaminophen prodrugs catalyzed by iron porphyrins:
Effect of nitrogen and thiolate axial ligands on drug and metabolite formation
Chiara M. Chapman1, John M. Pruneau1, Chloe A. Laverack1, Andrew S. Dutton2, and Graham B. Jones1*
1Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA,
02115 *Fax: 617-373-8795 Email: gr.jones@neu.edu
2 Department of Chemistry and Biochemistry, Suffolk University, Boston, MA, 02108
Highlights:




Biomimetic studies on the metabolism of acetaminophen prodrugs were investigated
A number of key metabolic intermediates were observed, throwing light on potential alternative pathways
Nitrogen- and sulfur-containing axial ligands consistently afford different products
2-mercaptopyrimidine is employed as an axial ligand and does not suffer from degradation via dimerization or
oxidation
Abstract:Metalloporphyrins (MP) are often employed in biomimetic catalysis due to theirstructural and functional similarity to the
heme prosthetic group of oxygenating enzymes. In these enzymes, the iron porphyrin isprimarily protein-bound at the active site by
cysteine (cytochrome P450, CYP), histidine (peroxidase), and tyrosine (catalase), acting as axial ligands.The diverse functionality
and variable oxidizing power exhibited bydifferentoxygenating enzymes is [in part] attributed to the ligating amino acid, and the
relationship between iron and the heteroatom. Attempts to mimic this relationship bydevelopingMP systems thatbehavesimilarly to
CYP enzymeshave mainly focused on incorporating nitrogen (N)-heterocycles. Although it is widely known that different enzyme
families favor distinctive products,sulfur is easily oxidized and prone to form disulfide bonds, and as such,thiolates for the most
partremain unexplored. Herein, we report our findings from the biomimetic oxidation of phenacetin, methacetin, and acetanilide by
meta-chloroperoxybenzoic acid (m-CPBA), catalyzed by hindered iron(III) porphyrins that arechelated by different axial ligands.
Nine axial ligandsthat vary in pKa(2.8-11.2), size, and heteroatomic identitywere examined,to more accurately determinethe factors
that contribute to function and effect. The [three] thiolate ligands afforded the greatest yields of acetaminophen in the majority of
cases. Furthermore, nitrogen- and sulfur- containing ligands were observed to promote different reaction pathways, regardless of
pKaor ligand size.
1

Keywords: Axial ligand, Biomimetic, Cytochrome P450, Iron porphyrin, Oxidation, Prodrug.
1. Introduction
Metalloporphyrins (MP) are often used as biomimetic catalysts for phase I cytochrome P450 (CYP) –mediated
metabolism [1-4], as they structurally mimic the active site analogous to all CYP isozymes, featuring an iron
protoporphyrin IX known as heme B. The super-family of CYP oxygenating enzymes is estimated to be responsible for
over 90% of pharmaceutical biotransformations [5-7]. Although many oxygenating enzymes contain a heme-active site,
none possess the catalytic efficiency of CYP enzymes, the major differentiating factor being the amino acid the active
site is bound by.
In CYP enzymes, heme B is protein-bound by cysteine (Cys), whereas other enzymes are histidine (His)- or
tyrosine (Tyr)-bound, i.e. peroxidase and catalase, respectively. This relationship between iron(heme B) and
sulfur(Cys) creates the “push-pull” effect that is attributed for the oxidizing power of CYP [8-10]. Moreover, other
oxygenating enzymes have different mechanisms, create a dissimilar “push” onto iron, and have significantly slower
auto-oxidation ratesthan CYP enzymes [11,12]. Attempts to more closely mimic CYP oxidation has primarily
involvedthe incorporation of imidazole, or other nitrogen(N)-heterocycles, that ligate to theMP in situ. As sulfur is
easily oxidized and prone to dimerization, thiols largely remain unexamined[12].
H
(Promoiety)
N
Fe
=
+ m-CPBA
N
SH
R
R
O
X
X
N
H
APAP prodrug
X
N
X
X
R
R
R
R
Fe
S
Cl
N
N
N
O
HO
FeIII
N
APAP (1)
N
N
+
Oxidized
moiety
X
other product(s)
X
X
R
R
R = F, Cl, or Br.
X = H or Br.
Scheme 1. Promoiety approach to an acetaminophen (APAP, 1) prodrug, generally shown using the heme-enzyme mimic employed
here; featuring a hindered iron (III) porphyrin catalyst, mercapto-N-heterocycle to ligatein situ, and m-CPBA as an oxygen donor.
In recent years, MP catalysts have primarily been used to increase reaction stereo- or regio- selectivity, as well as
produce and/or scale-up metabolites. However, targeting prodrugs offers an even greater range of applicability.
Biomimetic systems provide a simpler platform to assess a compound’s efficiency as a prodrug, and streamline the
process of evaluating prodrug libraries, as opposed to employing time-consuming in vitro or in vivo models.In order to
tangentially explore these areas, the effectsof different sulfur(S)- and/or (N)-containing axial ligands were assessed in
the biomimetic oxidation of acetaminophen (paracetamol, APAP) prodrugs, using iron (III) porphyrin catalysts, a
general example for which is illustrated in Scheme 1.
APAP is one of the most widely used analgesic and antipyretic agents, particularly with
children where sudden adverse reactions from alternatives have been reported [13,14].
Unfortunately, APAP is associated with dose-dependent toxicity, stemming from the
electrophilic metabolite, N-acetyl-p-benzoquinone imine (NAPQI, Fig. 1), generated during
first-pass metabolism by CYP2E1, 1A2, and 3A4. At therapeutic doses, NAPQI accounts for
less than 10% of the metabolic products, a small amount that then undergoes glutathione
(GSH)- conjugation and is excreted [15]. Conversely, at high concentrations, GSH-stores
N
O
O
Fig. 1. NAPQI.
deplete and excess NAPQI becomes covalently bound to cellular proteins via Cys[16]. NAPQI-protein adducts have
made APAP the leading cause of acute liver failure [17].To circumvent NAPQI-induced liver toxicity, many have
taken the promoiety approach to prodrug design, capping the APAP phenol-group using ether- [18,19], amino acid-
[20], and/or other similar derivatizations [21]. Moreover, this approach has proven to be beneficial for improving the
poor solubility and bioavailability [22] that is commonly exhibited by drugs containing phenolic functional groups [23].
H
H
H
H
N
N
N
N
O
O
O
O
O
H3CO
HO
(2)
(3)
(4)
(1)
2

Fig. 2. Acetaminophen (APAP, 1) and prodrugs
analyzed in this study: phenacetin (2), methacetin (3),
and acetanilide (4).
Table 1
Axial ligand structure, pKa, and molecular weight (MW).
MW
(g/mol)
Entrya
Axial Ligand
Piperidine
pKa
11.2
9.7
Structure
N
Here, we report our findings from iron(III)
A
85.2
86.1
H
porphyrin
(FeP)-mediated
biomimetic
[29, 30]
oxidations of APAP (1) prodrugs, with
particular emphasis on the effect of axial ligands.
Phenacetin (2), methacetin (3), and acetanilide
(4) are examined (Fig. 2), 2-4 being chosen as
representative compounds that reportedly
undergo different biotransformations to (1). The
activity of three hindered FeP complexes (Fig.
3) are compared in this work, asrecent literature
has demonstrated variable catalytic efficiency
from β-pyrrole substituted MPs [24-28]. To
examine the role of sterics and electronics,
Fe(TDCPPβBr8)Cl and Fe(TDCPP)Cl are used
N
B
H
Piperazine
H
[29, 30]
N
SH
C
[31]
Benzylmercaptan
9.4
8.7
124.2
112.2
N
SH
2-
Db
Mercaptopyrimidine
N
to
assess
the
effect
of
increased
electronegativity at the β-positions, compared to
potential steric hindrance from the bulky β-
pyrrole bromines surrounding the iron active site. Employing Fe(TDFPP)Cl then allows a more direct comparison of
the effects of electronegativity. In one aforementioned study assessing the effect of β-pyrrole bromination[25], meta-
chloroperoxybenzoic acid (m-CPBA)was found to afford the largest conversion of carbamazepine, regardless of the
degree of bromination (0-8). Based on these findings,m-CPBA decidedly provided the most equal starting point for the
FeP catalysts.
While a number of other variables are discussed, axial ligands are the primary focus (Table 1), as our aim is to
identify chelating agents that promote behavior more similar to what is observed of Fe–S(Cys) in CYP enzymes. We
show that optimal FeP/axial ligand combinations are different for each respective transformation, providing a starting
point for potential prodrugs of (1) derived from similar starting materials. Furthermore, reactions in which S-containing
reagents are employed afford greater yields of (1), and generate different products than reactions with N-containing
reagents.
2. Experimental
2.1. Materials and methods
R
R
X
X
X
X
X
X
X
R
R
R
R
Cl
N
N
N
FeIII
N
X
R
R
(I) Fe(TDFPP)Cl: R = F, X = H
(II) Fe(TDCPP)Cl: R = Cl, X = H
(III) Fe(TDCPPβBr8)Cl: R = Cl, X = Br
Fig. 3. Structures ofiron(III) porphyrin catalysts (FeP)
used in the biomimetic oxidation of APAP-prodrugs.
3

2.1.1. Reagents
H3C
N
2,4-
E
[32]
CH3
8.3
7.7
96.1
82.1
The iron porphyrin complexes, iron (III)
Dimethylimidazole
N
H
meso-
tetrakis(2,6-difluorophenylporphyrin)
chloride [Fe(TDFPP)Cl], iron (III) meso-
tetrakis(2,6-dichlorophenylporphyrin) chloride
[Fe(TDCPP)Cl], iron (III) meso- tetrakis(2,6-
dichlorophenylporphyrin-β-octabromo)chloride
[Fe(TDCPPβBr8)Cl], were purchased from
Frontier Scientific (Logan, UT, U.S.A.).
Catalyst purity was assessed by UV-Visand
compared to literature [35,36]. All analytical
standards, axial ligands, oxidants, substrates,
and solvents were purchased from Fisher
Scientific, Sigma-Aldrich, or T.C.I. America,
and used without further purification.Preparative
thin-layer chromatography (TLC) plates were
purchased from Silicycle (Québec, Canada).
N
F
[33]
CH3
2-Methylimidazole
N
H
N
G
1H-Imidazole
Benzenethiol
6.9
6.6
68.1
[29, 33]
N
H
SH
N
H
[34]
110.2
F3C
4-(Trifluoromethyl)
Imidazole
Ib
2.8
n/a
136.1
n/a
N
H
2.1.2. Equipment
Qualitative analysis was performed by gas
chromatography-mass spectrometry (GC-MS)
using Agilent Technologies (Wilmington, DE,
U.S.A.) HP 6890 Series GC system G1530A
Control: No ligand
n/a
J
a Letters correspond to following charts. ; b Predicted pKa ±0.3.
with an Agilent HP 7683 autosampler coupled to Agilent HP 5973 quadrupole mass-selective detector (transfer line
275°C, source 230°C, ionization potential 70 eV, scan range 30-400 amu). Helium was used for carrier gas flow at a
rate of 1.4 mL/min. Separations were performed on an Agilent 19091Z-002 HP-1 methyl siloxane capillary column
(25.0 m, 200 μm internal diameter, 0.11 μm film thickness). GC-MS data acquisition was controlled by MSD
ChemStation G1701DA Software.
The samples were quantitated by HPLC-ultraviolet (UV) (Agilent 1100 Series, G1322A Degasser, serial:
JP73021014), using a prepared calibration curve of each standard (λ = 254 nm). Separation of acetaminophen and its
precursors was carried out on a Phenomenex Aqua C18 Column, particle size 5μm (150 mm x 4.6 mm). HPLC-UV
data acquisition was controlled by ChemStation for LC 3D Software Rev.A.10.01(1635), Agilent Technologies (1990-
2003). Proton nuclear magnetic resonance (1H NMR) spectra were recorded at 400 MHz on a Varian NMR instrument;
spectra were prepared using ACD/Labs (Toronto, Canada).
The iron porphyrin complexes, Fe(TDFPP)Cl, Fe(TDCPP)Cl, Fe(TDCPPβBr8)Cl, were examined by UV-Vis
Spectrophotometry (UV-Vis), using ThermoScientific (Madison, WI, U.S.A.) BioMate 3S (v4.000I, 2K2NO20001).
2.1.3. pKa predictive model
Computational calculations to predict pKa-valuesfor 2-mercaptopyrimidine (D) and 4-(trifluoromethyl)imidazole (I)
consisted of gas phase structure optimization using Gaussian 09, andapplication of the density functional method BPBE
basis set 6-31+G(d) (T= 298.15 K)[37].Free energy of the SMD aqueous solvationfor the gas phase structures was
modeled using single point calculations[38]. Predictederror margin values were determined to be± 0.3pKa-units.
2.2. General procedure for catalytic oxidations
The conditions for a typical reaction were as follows: Fe(TDCPPβBr8)Cl(2.7 mg, 1.67 x10-3 mmol) and imidazole
(1.2 mg, 0.01674 mmol) were added to 500 μL of 3:2 (v/v) acetonitrile/ 2-propanol in a 2-dram(7.39 mL) vial
containing a screw cap. The vial was covered in aluminum foil, to protect from exposure to light, and the reaction was
stirred at room temperature. After30-minutes(to allow for axial ligation, if necessary),phenacetin (30 mg, 0.1674
mmol) was added to the reaction, followed by the slow addition ofm-CPBA (77 wt%) (37.5 mg, 0.1674 mmol) over a
1-hour period. Following the addition of oxidant, the reaction was stirred for 24 hours at room temperature, in the
absence of light. Typical catalyst/ axial ligand/ oxidant/ substrate molar ratio was 1:5-10:100:100 (axial ligand: thiol or
pKa> 9.0, 5mol%; N-heterocycle, 10mol%).
4

Gas and liquid chromatographic analyses (GC, LC) were performed on aliquots withdrawn directly from the
reaction vessel. Compounds were identified by comparison of their retention times to those of authentic
standards.Yield (substrate)(2-4) and yield (product)(1) were determined using prepared calibration curves (λ = 254 nm).
Conversionand selectivity were calculated viathe following Eq. (1-3):
Control reactions were carried out in the absence of the porphyrin catalysts, both with and without axial ligands, as
well as in the absence of oxidant (FeP only), to assess the contribution of atmospheric oxygen.
2.2.1. 5-acetamido-2-ethoxyphenyl acetate (6)
Following the catalytic oxidation of (2), where Fe(TDCPP)Cl and 4-(trifluoromethyl)imidazole (I) were employed
under the previously described conditions, the crude reaction mixture was concentrated in vacuo and reconstituted in
CH2Cl2. Chromatography and isolation was performed on a preparative TLC plate (SiO2, 60Å, 1000μm thickness)
using two mobile phases (40% EtOAc in hexane, 2% MeOH in CH2Cl2), and extracted from the stationary phase using
1
10% MeOH in CH2Cl2. H NMR (400 MHz, CD2Cl2): δ 7.349 (s, 1H), 7.174 (m, 1H), 6.908 (d, 1H), 4.01 (q, 2H),
2.265 (s, 3H), 2.098 (s, 3H), 1.349 (t, 3H). Attempts to further analyze (6) resulted in rearrangement of the acetate
moiety (photo-Fries[39]).
O
3. Results and discussion
FeIV
"Field effect"
Bond length dictates
3.1. Axial Ligands
"QM effect"
σ- and π- donor
capabilities of ligand.
charge-charge repulsion.
L
The axial ligand push-pull effect in oxygenating
enzymes is partially attributed to both quantum
mechanical (QM) and field effects (Fig. 4), resulting
from orbital mixing and the charge from the axial
ligand (L) [40-42], respectively. Generally guided by
the amino acid ligates of these enzymes (Cys, His, and
(Cpd I)
Fig. 4. Quantum mechanical (QM) and field effects from axial
ligands (L) in Compound I (Cpd I).
Tyr), all nine of the axial ligands employed feature nitrogen, sulfur, or both (Table 1, A-I), and were chosen for one or
more of the following characteristics: pKa, size, and reactivity toward oxidizing agents [43,44].
IV
+

The formation of the high-valent oxygenating species, Compound I (Cpd I) in FeP, Fe (=O) , has been shown
and/or proposed with the majority of the imidazole [45] and thiol [46,47] reagents being examined as axial ligands.
Yamaguchi et al. found the axial coordination of imidazole to FeP increased the rate of O−O bond cleavage in m-
CPBA, and that the rate could be directly correlated to the donor effect of the axial ligand [48]. Furthermore, Takahashi
et al. not only showed imidazole and phenolate axial ligands accelerated the rate of formation of Cpd I, the chelation
also increased the rate of oxygen transfer to the substrate (in electron-rich FeP) [45].
3.2. Phenacetin (2)
Phenacetin (2) was originally prescribed as an antipyretic/analgesic, butwas removed from market in the 1980s after
it was linked to nephritis and possible carcinogenicity[49]. Although it is well-established (2) is metabolized by CYP
enzymes, the toxicity is still not fully understood;this has ledto investigations into its metabolic pathway years after
being banned. For these reasons we chose to include (2) in this study.
3.2.1. FeP-catalyzed (2) oxidation by m-CPBA
5

The results of the FeP-mediated oxidation of (2) with m-CPBA are illustrated in Fig. 5A-C, in which the total
oxidative conversion of (2) and the selectivity for (1) are highlighted. With no axial ligand (J), oxidative conversion of
(2) reached 70, 78, and 55% with Fe(TDFPP)Cl, Fe(TDCPP)Cl, and Fe(TDCPPβBr8)Cl, respectively. With one
exception (Fig. 5A, J), little selectivity for (1) was observed in the absence of an axial ligand (4-12%). Overall,
reactions employing (C), (D), and (H) afforded the most favorable combinations of conversion and selectivity for (1),
subsequently generating the highest yields, with only one outlier (Fig. 5A, C vs. J).
(A)
(B)
(C)
Fe(TDFPP)Cl
Fe(TDCPP)Cl
Fe(TDCPPβBr8)Cl
50 100
50 100
45 90
40 80
35 70
30 60
25 50
20 40
15 30
10 20
100
90
80
70
60
50
40
30
20
10
0
50
45
40
35
30
25
20
15
10
5
45
40
35
30
25
20
15
10
5
90
80
70
60
50
40
30
20
10
0
5
0
10
0
0
0
A. B. C. D. E. F. G. H. I. J.
A. B. C. D. E. F. G. H. I. J.
Axial Ligand
A. B. C. D. E. F. G. H. I. J.
Fig. 5. Results of the oxidation of (2), under the following conditions: (2) (1 equiv), Fe(TDFPP)Cl (A)/ Fe(TDCPP)Cl (B)/
Fe(TDCPPβBr8)Cl (C) (1mol%), axial ligand A-I (5-10mol%), m-CPBA (1 equiv), in MeCN/IPA (3:2, v/v), rt, 24 h.
Made apparent in Fig. 5A, Fe(TDFPP)Cl was least effected by the nature of the axial ligands, leading to
inconsistent trends relative to Fe(TDCPP)Cl and Fe(TDCPPβBr8)Cl. The greatest yields of (1) were observed using
axial ligands (H) and (D), and with no axial ligand (J), affording 16.5, 15.8, and 14.4%, respectively (Table 2). Notably,
imidazole (G), arguably the most commonly used axial ligand that is examined here, afforded both the lowest
selectivity (13%) and yield (9%) of (1). When Fe(TDCPP)Cl was employed (Fig. 5B), the greatest selectivity for (1)
was observed with (E), where (1) was produced almost exclusively, affording 13% yield. The greatest yields of (1)
overall using Fe(TDCPP)Cl were found with axial ligands (C), (D), and (H), generating 19.2 (27.1), 17.9 (24.5), and
16.6 (25.8)% yield (and selectivity), respectively. The effect of the axial ligands was most evident with
Fe(TDCPPβBr8)Cl (Fig. 5C), where little to no selectivity for (1) was observed from any of the axial ligands except
those containing thiols (C, D, H). Yield (and selectivity) of (1) reached 19.2 (33.9), 16.5 (36.6), and 9.8 (14.4)% with
axial ligands (D), (C), and (H), respectively. It is plausible the bulky bromine substituents at the β-pyrrole positions of
Fe(TDCPPβBr8)Cl are creating steric hindrance and inhibiting active-site access.A number of studies have observed
similar findings[3,24,25], some showing a less-substituted counterpart to be more active, i.e. 4 vs. 8
bromines,highlighting the possible role of sterics.
Comparing the pKa values of (A-I), no overall correlation to the results could be ascertained. However, reactions
with thiols (C) and (D) generally afforded higher yields of (1) than (H), possibly related to pKa. Reactions employing
imidazole ligands (E, F, G, I) also followed a general trend, where (E) afforded the greatest yields of (1) in the
majority of cases. Interestingly, this was followed by (F) or (I), two compounds with very different pKa’s.
Table 2
Phenacetin (2) oxidation resultsa: acetaminophen (1) yield and product trendsb.
Entry Axial Ligand
Fe(TDFPP)Cl
Fe(TDCPP)Cl
Yield (1, %) (5) (6)
12.0
Fe(TDCPPβBr8)Cl
Yield (1, %) (5) (6)
1.0
Yield (1, %) (5) (6)
X
X
1
2
3
4
Piperidine (A.)
Piperazine (B.)
Benzylmercaptan (C.)
2-Mercaptopyrimidine (D.)
13.0
10.5
13.4
15.8
X
X
9.0
4.8
X
X
X
X
X
19.2
17.9
16.5
19.2
6

X
X
X
X
X
X
X
5
6
7
8
9
10
2,4-Dimethylimidazole (E.)
2-Methylimidazole (F.)
Imidazole (G.)
Benzenethiol (H.)
4-(Trifluoromethyl) Imidazole (I.)
None (J.)
14.2
11.1
8.5
16.5
13.9
14.4
13.0
10.6
3.2
16.6
8.9
2.0
2.8
trace
9.8
4.6
2.1
X
X
X
X
X
X
X
X
9.4
a Conditions: Catalyst/oxidant/substrate molar ratio of 1:100:100, 5-10mol% ligand (A-I), 500μL MeCN/IPA (3:2, v/v), rt, 24 h.
b Observed product denoted by ‘X’.
3.2.2. (2) Mechanism
In addition to (1), one of two other major products was observed in each reaction, depending on the FeP/axial
ligand combination. Generally speaking, the thiolate ligands (C, D, H) favored the formation of hydroquinone (5), and
the remaining favored (6), identified as 5-acetamido-2-ethoxyphenyl acetate. These results are shown in Table 2, where
the alternative major product is also denoted (‘X’). Of note, products (5-6) were never observed in the same reaction
mixture, indicating they are formed via different mechanisms.
OH
H
H
N
N
N
N
A.
B.
Ac
OH
Ac
H
(2)
C
Et
O
Et
Et
O
O
O
O
O
C.
(- CH3CHO)
(- H2O)
N
H
N
NH2
OH
+
O
O
O
O
O
HO
p- phenetidine
AcOH
OEt- NAPQI
(1)
O
H
O
O
N
Et
O
OH
N
H
N
N
OH
+ H2O
(6)
(- H2O)
O
O
O
O
HO
N-hydroxy-(1)
HO
HO
(5)
NAPQI
(1)
Scheme 2. Proposed pathway(s) to major products (1), (5), and (6), in the oxidation of (2).
Significant mechanistic insight was obtained from the analysis of (6).Mass spectrometric analysis of the isolated
compound showed a parent peak at m/z 237 [(2) + 58], secondly abundant peak at m/z 195 [M+ - 42], base peak at m/z
124 [M+ - 113], and fragmentation pattern similar to (2), indicating the addition of oxygen directly to the aromatic ring
[50]. The full structure of (6) could then be determined using NMR.Following structure elucidation, the origin of the
acetate moiety prompted investigation. The oxidation of (3) under analogous conditions eliminated the possibility of
the acetate originating from the acetaldehyde by-product, as the same relative product was observed in these reactions;
the parent, secondly abundant, and base peaks were observed at m/z 223 [(3) + 58], m/z 181 [M+ - 42], and m/z 124 [M+
- 99], respectively. Additionally, substituting methanol (MeOH)for 2-propanol (IPA) excluded any solvent interaction
hypotheses. Thus, the formation of (6) is likely a result of an interaction between the highly reactive imine of (2),
ethoxy-N-acetyl-p-benzoquinone imine (OEt-NAPQI), and acetic acid (AcOH), the by-product of p-phenetidine,
though the latter compound was not detected.
These findings support the mechanistic pathway(s) proposed in Scheme 2, adapted in part from metabolic pathways
of (2) [51-53]. In vivo, (2) is reported to primarily undergo O-dealkylation by CYP1A2, resulting in(1) and
acetaldehyde. Alternatively, minor metabolites are formed through N-hydroxylation and N-deacetylation, by CYP1A2
and amidase enzyme hydrolysis, respectively. Following its formation in the body, (1) is further oxidized by CYP2E1
via N-hydroxylation to the electrophilic intermediate, NAPQI, and is finally conjugated by transferase enzymes. Based
on this mechanism, we propose reactions in which thiolate ligands (C), (D), and (H) were employed, (2) is
7

hydroxylated to the hemiketal intermediate, followed by O-dealkylation to afford (1), subsequently generating (5) from
NAPQI1in the absence of these biological conjugates.
This mechanism suggests a competing reaction with (1), explaining the lower yields in reactions where (6) is
observed. The formation of OEt-NAPQI is supported by the relatively weak nucleophilicityof AcOH (N= 1/ω),
predicted to be 1.62 eV [54]. OEt-NAPQI is estimated to have an electrophilicity index (ω) of 19.429 eV [51], giving it
the electrophilic strength required for the weak nucleophile to accomplish the addition. Control reactions were
performed under the following conditions: in the absence of m-CPBA (FeP only), in the absence of FeP catalysts (m-
CPBA only), and in the absence of FeP catalysts with each axial ligand (m-CPBA and ligand), none of which afforded
any products.
3.2.3. Solvent effect
Based on the success of the prior reaction, the oxidation of (2) using m-CPBA, Fe(TDCPPβBr8)Cl, and axial ligand
(D), under analogous conditions to those reported previously(Fig. 5, Table 2), was carried out in 100% acetonitrile
(MeCN). In this reaction, none of the previously observed products (1, 5-6) were detected, and in general little
conversion of (2) was seen. Focusing on the low overall conversion, the reaction was repeated using axial ligand (I),
albeit with the same result. Although solubility left limited options for the reaction solvent, varying mixtures of
MeCN/PhMe, MeCN/trifluorotoluene (PhCF3), and MeCN/THF were examined, none of which were successful, and
appeared to hinder catalytic activity in almost all cases.
Fig. 6. Effect of MeOH on the oxidation of (2), under the following conditions: (2) (1 equiv), Fe(TDFPP)Cl (A)/ Fe(TDCPP)Cl (B)/
Fe(TDCPPβBr8)Cl(C) (1mol%), ligands (C-G, I) (5-10mol%), m-CPBA (1 equiv), MeCN/MeOH (1:1, v/v), rt, 24 h.
It is well-established solvent properties can dramatically effect the outcome of FeP oxidations, i.e. polarity,
viscosity, and proticity.The latter two are proposed to play a role in thesolvent-cage effect, believed to increase reaction
selectivity by containing the substrate radical andiron oxygenating species following hydrogen-abstraction.No trends in
solvent polarity could be deduced, in agreement with cage effect literature[55].While IPA is the only protic solvent, it
is also significantly more viscous(2.1 cP[56]) than MeCN, PhMe, or THF (0.37 − 0.57 cP[56]), prompting further
investigation.
The oxidation of (2) was repeated in MeCN/MeOH (1:1, v/v), using m-CPBA, Fe(TDFPP)Cl, Fe(TDCPP)Cl, or
Fe(TDCPPβBr8)Cl, and select axial ligands. The findings from these studies (Fig. 6A-C) indicate protic solvents are
(A)
(B)
(C)
Fe(TDFPP)Cl
Fe(TDCPP)Cl
Fe(TDCPPβBr8)Cl
50 100
50 100
45 90
40 80
35 70
30 60
25 50
20 40
15 30
10 20
100
90
80
70
60
50
40
30
20
10
0
50
45
40
35
30
25
20
15
10
5
45
40
35
30
25
20
15
10
5
90
80
70
60
50
40
30
20
10
0
5
0
10
0
0
0
C.
D.
E.
F.
G.
I.
J.
C.
D.
E.
F.
G.
I.
J.
C. D. E.
F. G. H.
I.
J.
Axial Ligand
necessary for the reaction to be successful, as the viscosity of MeOH is only 0.55 cP[56].
3.2.4. Solvent interaction
1 Preliminary analyses (GC-MS) indicate NAPQI is observed at initial reaction time-points (< 3 h), in crude reaction aliquots. Studies to trap the
unstable intermediate are currently underway.
8

With respect to the effect of the axial ligands, when MeOH was used in place of IPA the same relative trends in
conversion of (2) and selectivity for (1) were observed, in thatthiolate ligands afforded the greatest yields of (1).
Overall however, the conversion of (2) increased and a number of new compounds were observed, leading to
loweryields of (1). While (5) was not detected in any of the MeCN/MeOH reactions, (6) was again observed in
reactions withN-containing axial ligands, and generally absent in reactions with the thiolatecounterparts.
In addition to (1) and (6), five other oxidative products of (2) were detected in relatively significant quantities. Two
of these compounds were identified as methacetin (3), and benzoquinone (7), by comparison to authentic standards.
Two of the three remaining unknown compounds ([M+] = m/z 195 and m/z 154), were detected in all reactions, whereas
the third ([M+] = m/z 356) was only detected with ligands (C), (E), (G), and (I).
3.2.5. Deuterated-d3 methanol studies
The formation of (3) suggested the possibility of a solvent interaction, as it was deemed unlikely the C−C bond of
the ethoxy-moiety is being cleaved. For the dual-purpose of identifying the unknown products and elucidating the
mechanism of formation, MeOH was replaced with (1,1,1)-trideuteromethanol (MeOH-d3) to repeat
reactionspreviously established to promote different pathways or products. Ligands (D) and (E), and a control (J) were
examined with Fe(TDCPPβBr8)Cl.
By comparison of GC-MS retention time (rt) and ion fragmentation pattern to the original reactions, two of the
three remaining unknown compounds were identified as N-(3,4-dimethoxyphenyl) acetamide (8, [M+] = m/z 195) and
3,4-dimethoxyphenol (9, [M+] = m/z 154). Fig. 7-8 exemplify the well-aligned mass spectra of MeOH (top) and
MeOH-d3(bottom) reactions for (8) and (9). Scheme 3 illustrates how structures were elucidated using the MS ion
fragmentation mechanisms from MeOH/MeOH-d3 reaction data, demonstrated with the multiple-fragmentation
pathways from (9). The final unknown (Unk-1, [M+] = m/z 356) has been preliminarily identified as a dimer of (2); the
MS fragmentation corresponds well to the monomer, and the MeOH-d3 studies had no effect on the outcome. However,
as no mechanistic correlation to the axial ligands was ascertained, no attempts to further identify (Unk-1) were made.
Calculating the relative product distribution for each ligand at timepoints 1.5 and 18 hours (h) provides a general
outline to the mechanism of the methoxy-addition, viz. Table 3. For example, (3) was observed in the majority of
reactions at 1.5 h (Table 3, entries 3-8),but was not detected in any reaction at 18 h, indicating (3) is further oxidized
and therefore can be viewed as an intermediate, to both (1) and (8). Assuming (3) follows the trend consistent with (1)
and (2), it will form a reactive imine-intermediate, methoxy-N-acetyl-p-benzoquinone imine (OMe−NAPQI),
facilitating the addition of a second methoxy-moiety to the next available electrophilic site to produce (8). Over the
timespan of 1.5 to 18 h, the
concentration of (8) decreased in all
reactions, while the concentration of
(9) increased (Table 3), supporting our
hypothesis that (9) is a degradation
product of (8). The proposed
mechanism(s) for the oxidation of (2)
in MeOH is illustrated in Scheme 4
CH3
O
OH
(8)
H3C
O
[M+] = m/z 154
(d6)-[M+] = m/z 160
(9)
(- CH4)
(- CH3OH)
OH
O
O
O
H3C
H3C
O
Fig. 8 (left).
Mass spectra of GC-MS
peaks at rt = 3.98 min, from
the oxidation of (2) in
MeOH (top) and MeOH-d3
(bottom) solvent mixtures;
identified as (9).
m/z 139
m/z 123
d6-(8)
(d3)- m/z 142
(d3)- m/z 126
(- CO)
(- CO)
Fig. 7. Mass spectra of GC-MS peaks at rt = 9.32 min, from the oxidation of (2)
in MeOH (top) and MeOH-d3 (bottom) solvent mixtures; identified as (8).
OH
H3C
H3C
Scheme 3 (right).
Proposed GC-MS ion frag-
mentation mechanism of (9).
O
O
9
m/z 95
m/z 111
(d3)- m/z 114
(d3)- m/z 98

A. O-dealkylation:
(abbreviated).
Similar to AcOH, MeOH has
a
[hemiketal-(2)]
(2)
nucleophilicity index (N) of 1.92 eV [54]; on
this basis, it is more probable OEt−NAPQI,
rather than NAPQI, undergoes nucleophilic
substitution to (3), when considering the
relative electrophilicity indices of the imine-
intermediates are 19.429 and 4.230 eV [51],
respectively. Moreover, (7) is reported to form
by hydrolysis of NAPQI [57] suggesting even a
weak nucleophile is more likely to attack the
NAPQI ring at the carbon of the imine, rather
than at the para- carbonyl.
(7)
(1)
NAPQI
B. N-hydroxylation:
[N-hydroxy-(2)]
(2)
(3)
OEt-NAPQI
(8)
(1)
(9)
C. Hydrolysis:
(2)
(6)
Combining results from the aforementioned
studies highlight several salient points. Given
the substitution byMeOH at the position para
to the acetamide, it is more plausible the AcOH
was unable to complete the addition at the
carbonyl (when comparing the leaving group
+
p-phenetidine
AcOH
Scheme 4. Proposed pathway(s) for the biomimetic oxidation of (2) in
MeCN / MeOH.
strength of the two functional groups), and consequently is solely observed at the position meta to the acetamide. Based
on these findings we conclude that the absence of (6) in reactions where thiolates were employed, both with
MeCN/IPA and MeCN/MeOH, is a result of the hydrolysis not occurring, and therefore no AcOH is available to react
(Scheme 4C). Furthermore, control reactions in the absence ofFePafforded no products, implying the porphyrin catalyst
plays a role in the hydrolysis of (2).
As previously stated, it is reported that in vivothe hydrolysis of (2) is catalyzed by amidase enzymes; the only one
of the three major metabolic routes not mediated by CYP. Thus, the thiolate-ligands increased catalyst selectivity
whilepromoting pathways analogous to CYP enzymes.
3.3. Methacetin (3)
Methacetin (3) was chosen for this work based on its use as a liver function test (i.e.LiMAxtest[58]), where 13C-
labelled (3) is utilized for patients undergoing liver surgery to establish function perioperatively.13C-(3) is metabolized
exclusively by CYP1A2, a CYP isozyme with ubiquitous activity throughout the liver, to (1) and 13C-formaldehyde
Table 3
Phenacetin (2) oxidation in MeCN/MeOHa: relative product distributionsb (%) at 1.5 and 18 hours.
Relative Product Yield (1.5 and 18 h, %)
O
O
O
O
O
OH
HN
HN
HN
HN
Entry Axial Ligand
(Unk-1)
O
OMe
OAc
OMe
OMe
OH
OEt
OMe
OMe
(7)
(9)
(1)
(3)
(6)
(8)
1.5h 18h 1.5h 18h 1.5h 18h 1.5h 18h 1.5h 18h 1.5h 18h 1.5h 18h
1
2
3
4
5
6
7
8
Benzylmercaptan (C.)
39 54
47 58
−
−
24
31
t
13
28
9
−
−
−
−
−
−
−
−
t c
−
58
60
44
−
−
−
29
33
26
t
7
8
10
10
−
−
−
−
−
−
34
37
6
10
14
7
8
8
15
10
9
15
9
−
−
−
4
16
18
10
14
9
16
13
16
10
−
11 19
t
−
2-Mercaptopyrimidine (D.)
2,4-Dimethylimidazole (E.)
2-Methylimidazole (F.)
Imidazole (G.)
Benzenethiol (H.)
4-(Trifluoromethyl) Imidazole (I.) 11 48
−
−
−
36
45
46
−
−
−
−
−
−
9
−
−
41
63
33
4
14 12
20 66
−
t
−
−
9
−
23
51
20
22
t
7
None (J.)
29 53
aConditions: Fe(TDCPPβBr8)Cl/oxidant/substrate molar ratio of 1:100:100, 5-10mol% ligand (A-I), 500μL MeCN/MeOH (1:1, v/v), rt, 24 h.
bRelative product distribution calculated from GC-MS corrected peak area values; percentage based on total area of products ; c trace ≤ 3%.
10

(Scheme 5), the latter of which is further oxidized to 13CO2 that is then measured for the test[58].
H
N
N
A.
B.
OH
(1) +
H2C O
(3)
Me
O
O
O
O
Liver
function test,
when (3)
OMe-NAPQI
C.
13C-labelled
(
13CO2).
O
NH2
+
AcOH
(6)
CO2
H
O
N
H3CO
p- anisidine
O
H3CO
(10)
Scheme 5. Proposed pathway(s) to major products (1) and (10) in the oxidation of (3).
3.3.1. FeP-catalyzed (3) oxidation by m-CPBA
Results of the oxidation of (3) by m-CPBA catalyzed by Fe(TDFPP)Cl, Fe(TDCPP)Cl, and Fe(TDCPPβBr8)Cl are
summarized in Fig.9A-C, respectively. Thiolate ligands afforded the greatest selectivity for (1); where the order, (C) >
(H) > (D), was consistent with each FeP. Reactions catalyzed by Fe(TDFPP)Cl and Fe(TDCPP)Cl in the absence of
axial ligands (Fig. 9A-B, J) gave the greatest conversions of (3) at approximately 90%, albeit with 10% selectivity for
(1), demonstrating the loss of selectivity when no axial ligand was employed.
Fig. 9. Results of the oxidation of (3), under the following conditions: (3) (1 equiv), Fe(TDFPP)Cl (A)/ Fe(TDCPP)Cl (B)/
Fe(TDCPPβBr8)Cl(C) (1mol%), axial ligand A-I (5-10mol%), m-CPBA (1 equiv), in MeCN/IPA (3:2, v/v), rt, 24 h.
Consistent with results from the oxidation of (2), an unknown with [+ 58 amu] greater mass than (3) (m/z 223) was
observed in the majority of reactions with N-heterocycle ligands. Based on identical relative GC-MS ion
fragmentations (parent, secondly abundant, base peak: m/z 223 [(3) + 58], m/z 181 [M+ - 42], m/z 124 [M+ - 99]), this
compound could be identified as the 4-methoxy analogue of (8), 5-acetamido-2-methoxyphenyl acetate (10). Deviating
from the trend with (2), (10) was observed in trace amounts in reactionswith ligands (D) and (H)when Fe(TDFPP)Cl
was employed. However, relative to quantities in reactions with N-containing ligands (corrected peak area), it is clear
(10) is still not a favored product when employing these ligands. Furthermore, (5) was detected as a major product with
ligands (C) and (H) in reactions catalyzed by Fe(TDFPP)Cl and Fe(TDCPPβBr8)Cl, and with ligands (C), (D), and (H)
with Fe(TDCPP)Cl.
11

The results from the oxidation of (3), with respect to major products and yield of (1), are detailed in Table 4.
Overall, reactions with thiolate ligands afforded the greatest yield of (1), with one outlier (Table 4, entry 3 vs. 9,
(A)
(B)
(C)
Fe(TDFPP)Cl
Fe(TDCPP)Cl
Fe(TDCPPβBr8)Cl
50 100
50 100
45 90
40 80
35 70
30 60
25 50
20 40
15 30
10 20
100
90
80
70
60
50
40
30
20
10
0
50
45
40
35
30
25
20
15
10
5
45
40
35
30
25
20
15
10
5
90
80
70
60
50
40
30
20
10
0
5
0
10
0
0
0
A. B. C. D. E. F. G. H. I. J.
A. B. C. D. E. F. G. H. I. J.
Axial Ligand
A. B. C. D. E. F. G. H. I. J.
Fe(TDCPP)Cl). In many cases, the use of N-containing ligands afforded lower yields of (1) than reactions where no
axial ligand was employed (J), whereas thiolate ligands afforded yields greater than (J) without exception. Of note,
selectivity for (1) was remarkably consistent in the oxidation of (3), which can be easily ascertained from Fig. 9A-C.
Although literature coverage on the metabolism of (3) is much less prevalent than (1) and (2), it is highly plausible,
based on structural similarities, that (3) follows analogous metabolic trends. Moreover, (3) is primarily metabolized by
the same isozyme (CYP1A2) as (2). As such, (3) would undergo N-hydroxylation and hydrolysis (phase II), aligning
with the results of this work (Scheme 5). The axial ligand trends observed with (3) are in agreement with those from (2),
and possibly even more clear when considering the selectivity for (1) in the remaining reactions.
Fig. 10. Results of the oxidation of (4), under the following conditions: (4) (1 equiv), Fe(TDFPP)Cl (A)/ Fe(TDCPP)Cl (B)/
Fe(TDCPPβBr8)Cl(C) (1mol%), axial ligand A-I (5-10mol%), m-CPBA (1 equiv), in MeCN/IPA (3:2, v/v), rt, 24 h.
3.4. Acetanilide (4)
Late in the 1800s, CahnandHepp unintentionally discovered the antipyretic effect of acetanilide (4)[49]. However,
since the toxicity viamethemoglobinaemia (disruption to the oxygen transport by hemoglobin)was established shortly
thereafter[49], derivatives were sought almost immediately, leading to (1) and (2). As the biotransformation of (4) to
(1) requires aromatic-ring hydroxylation, it offers a valuable platform for development of a biomimetic system.
Table 4
Methacetin (3) oxidation results a: acetaminophen (1) yield and product trends b.
Entry Axial Ligand
Fe(TDFPP)Cl
Yield (1, %) (5) (10)
5.6
Fe(TDCPP)Cl
Yield (1, %) (5) (10)
4.8
Fe(TDCPPβBr8)Cl
Yield (1, %) (5) (10)
0
1
2
3
4
5
6
7
8
9
10
Piperidine (A.)
Piperazine (B.)
t
X
4.8
4.9
9.8
13.2
7.1
7.8
X
6.8
8.8
11.1
3.7
3.2
4.0
12.4
5.3
5.0
X
Benzylmercaptan (C.)
2-Mercaptopyrimidine (D.)
2,4-Dimethylimidazole (E.)
2-Methylimidazole (F.)
Imidazole (G.)
17.7
13.5
7.8
X
X
X
X
X
t
X
X
X
t
X
X
X
X
X
X
X
6.0
5.6
12.2
2.2
Benzenethiol (H.)
4-(Trifluoromethyl) Imidazole (I.) 7.8
X
15.5
10.3
9.3
X
X
X
X
X
None (J.)
8.6
a Conditions: Catalyst/oxidant/substrate molar ratio of 1:100:100, 5-10mol% axial ligand (A-I), 500μL MeCN/IPA (3:2, v/v), rt, 24 h.
b Observed product denoted ‘X’, or t= trace.
12

3.4.1. FeP-catalyzed (4) oxidation by m-CPBA
Overall, the oxidation of (4) afforded low yields of (1), and in many cases low overall conversion as well (Fig.
(A)
(B)
(C)
Fe(TDCPP)Cl
Fe(TDCPPβBr8)Cl
Fe(TDFPP)Cl
50 50
45 45
40 40
35 35
30 30
25 25
20 20
15 15
10 10
50 50
45 45
40 40
35 35
30 30
25 25
20 20
15 15
10 10
50
45
40
35
30
25
20
15
10
5
50
45
40
35
30
25
20
15
10
5
5
0
5
0
5
0
5
0
0
0
A. B. C. D. E. F. G. H. I. J.
A. B. C. D. E. F. G. H. I. J.
Axial Ligand
A. B. C. D. E. F. G. H. I. J.
10A-C), an observation consistent with literature reports. The biomimetic oxidation of unactivated alkanes and
aromatic rings remains a challenge for researchers, turning many to immobilized catalysts in an attempt to address
common issues. Nevertheless, as our objective is to establish trends of porphyrin catalyst/ axial ligand combinations,
more emphasis was placed on relative reactivity rather than overall yields.
In the oxidation of (4), Fe(TDCPP)Cl and Fe(TDCPPβBr8)Cl were significantly more effected by the nature of the
axial ligands than Fe(TDFPP)Cl, illustrated in Fig. 10A, where the selectivity for (1) is fairly consistent (A-J).
Regarding the trends with (2) and (3), the effect of the axial ligands observed in the oxidation of (4) was inconsistent.
The greatest yields of (1) in reactions catalyzed by Fe(TDFPP)Cl and Fe(TDCPP)Cl were with axial ligands (D) and (I),
and with no ligand (J). Only varying slightly, ligands (D), (H), and (I) afforded the greatest yields of (1) in reactions
catalyzed by Fe(TDCPPβBr8)Cl (see S13, Supplementary Material).
5. Conclusion
2-Mercaptopyrimidine (D) was chosen for these studies based on the premise that pyrimidine is less susceptible to
oxidation (relative to benzene) and the lower reactivity may hinder disulfide bond and/ordimer formation. Considering
the findings from the oxidations of (2-4), the thiolate axial ligands show clear superiority above the others examined
herein. Moreover, while it was not the objective of this work, it is possible yields could be further improved following
reaction optimization.
Although benzylmercaptan (C) and benzenethiol (H) remained effective, both were observed to form dimers and
undergo oxidation (SOx), even at catalytic amounts (5mol%). In contrast, no dimer or oxidized forms of (D) were
observed in any reaction. Additionally, (D) was the only ligand that was effective with all three substrates and
porphyrin catalysts employed here, notably in the oxidation of (4), where the remaining thiolate ligands did not follow
suit.After thoroughly examining relevant literature, we believe this is the first instance where a nitrogen-heterocycle
mercaptanhas been successfully employed as an axial ligand in biomimetic metalloporphyrin catalysis.The findings of
this study indicate that this class of compounds may be a promising alternative to other, more easily oxidized, sulfur-
based reagents.
Biomimetic catalysis has made great strides in recent years, progress that has been significantly aided by new
mechanistic insight from both natural enzymes, and computational studies. Studies focusing on the effect of axial
ligands[12,59], or long-chain substituents that interact with the metal-center[60], have identified alternative
mechanisms and pathways, and on the basis of the current findings we are motivated to explore numerous applications.
Abbreviations
13

AcOH
APAP
CAT
CH2Cl2
Cpd I
CYP
Cys
EA
EtOAc
FeP
GC-MS
h
His
HPLC-UV
HRP
IPA
m-CPBA
MeCN
MeOH
MeOH-d3
NAPQI
PhMe
QM
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
Acetic acid
Acetaminophen
Catalase enzyme
Dichloromethane
Compound I
Cytochrome P450
Cysteine
Electron affinity
Ethyl acetate
Iron(III) porphyrin
Gas chromatography-mass spectrometry
Hours
Histidine
High performance liquid chromatograph- ultraviolet
Horseradish peroxidase enzyme
2-Propanol
meta-Chloroperoxybenzoic acid
Acetonitrile
Methanol
(1,1,1)-Trideuteromethanol
N-acetyl-p-benzoquinone imine (OMe: Methoxy ; OEt: Ethoxy)
Toluene
Quantum mechanical
(retention) time
meso-Tetrakis(2,6-dichlorophenylporphyrin)
meso-Tetrakis(2,6-dichlorophenylporphyrin-β-octabromo)
meso-Tetrakis(2,6-difluorophenylporphyrin)
Tetrahydrofuran
Thin-layer chromatography
Tyrosine
(r)t
TDCPP
TDCPPβBr8
TDFPP
THF
TLC
Tyr
Acknowledgments
We thank Empiriko Inc for financial support of this work, Pam Randhawa for useful discussions, Roger Kautz for
performing NMR studies, and Marek Domin and James Glick for mass spectrometric analysis.
Appendix A: Supplementary material
Tables S1-S13.
References
[1]
[2]
J.T. Groves, T.E. Nemo, R.S. Myers, J. Am. Chem. Soc. 101 (1979) 1032-1033.
D. Mansuy, C. R. Chimie. 10 (2007) 392-413.
[3]
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