Modeling Tryptophan/Indoleamine 2,3-Dioxygenase with Heme Superoxide Mimics: Is Ferryl the Key Intermediate?

Article abstract of DOI:10.1021/jacs.9b10498

Tryptophan oxidation in biology has been recently implicated in a vast array of paramount pathogenic conditions in humans, including multiple sclerosis, rheumatoid arthritis, type-I diabetes, and cancer. This 2,3-dioxygenative cleavage of the indole ring of tryptophan with dioxygen is mediated by two heme enzymes, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), during its conversion to N-formylkynurenine in the first and rate-limiting step of kynurenine pathway. Despite the pivotal significance of this enzymatic transformation, a vivid viewpoint of the precise mechanistic events is far from complete. A heme superoxide adduct is thought to be the active oxidant in both TDO and IDO, which, following O-O bond cleavage, presumably generates a key ferryl (Fe^IV=O) reaction intermediate. This study, for the first time in model chemistry, demonstrates the potential of synthetic heme superoxide adducts to mimic the bioinorganic chemistry of indole dioxygenation by TDO and IDO, challenging the widely accepted categorization of these metal adducts as weak oxidants. Herein, an electronically divergent series of ferric heme superoxo oxidants mediates the facile conversion of an array of indole substrates into their corresponding 2,3-dioxygenated products, while shedding light on an unequivocally occurring, putative ferryl intermediate. The oxygenated indole products have been isolated in a?31% yield, and characterized by LC-MS, ¹H and ¹³C NMR, and FT-IR methodologies, as well as by ¹⁸O_2(g) labeling experiments. Distinctly, the most electron-deficient superoxo adduct is observed to react the fastest, specifically with the most electron-rich indole substrate, underscoring the cruciality of electrophilicity of the heme superoxide moiety in facilitating the initial indole activation step. Comprehensive understanding of such mechanistic subtleties will benefit future attempts in the rational design of salient therapeutic agents, including next generation anticancer drug targets with amplified effectivity.

Full text of DOI:10.1021/jacs.9b10498

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Article
Modeling Tryptophan/Indoleamine 2,3-Dioxygenase with
Heme Superoxide Mimics: Is Ferryl the Key Intermediate?
Pritam Mondal, and Gayan B. Wijeratne
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b10498 • Publication Date (Web): 23 Dec 2019
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Modeling Tryptophan/Indoleamine 2,3-Dioxygenase with
Heme Superoxide Mimics: Is Ferryl the Key Intermediate?
Pritam Mondal† and Gayan B. Wijeratne*,†
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†Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35205, United States.
ABSTRACT: Tryptophan oxidation in biology has been recently implicated in a vast array of paramount pathogenic conditions
in humans, including multiple sclerosis, rheumatoid arthritis, type-I diabetes, and cancer. This 2,3-dioxygenative cleavage of
the indole ring of tryptophan with dioxygen is mediated by two heme enzymes, tryptophan 2,3-dioxygenase (TDO) and in-
doleamine 2,3-dioxygenase (IDO), during its conversion to N-formylkynurenine in the first and rate-limiting step of
kynurenine pathway. Despite the pivotal significance of this enzymatic transformation, a vivid viewpoint of the precise mech-
anistic events is far from complete. A heme superoxide adduct is thought to be the active oxidant in both TDO and IDO, which,
following O–O bond cleavage, presumably generates a key ferryl (FeIV=O) reaction intermediate. This study, for the first time
in model chemistry, demonstrates the potential of synthetic heme superoxide adducts to mimic the bioinorganic chemistry
of indole dioxygenation by TDO and IDO, challenging the widely accepted categorization of these metal adducts as weak oxi-
dants. Herein, an electronically divergent series of ferric heme superoxo oxidants mediates the facile conversion of an array
of indole substrates into their corresponding 2,3-dioxygenated products, while shedding light on an unequivocally occurring,
putative ferryl intermediate. The oxygenated indole products have been isolated in ~31% yield, and characterized by LC-MS,
1H and 13C NMR, and FT-IR methodologies, as well as by
O
labeling experiments. Distinctly, the most electron-deficient
18
2(g)
superoxo adduct is observed to react the fastest, specifically with the most electron-rich indole substrate, underscoring the
cruciality of electrophilicity of the heme superoxide moiety in facilitating the initial indole activation step. Comprehensive
understanding of such mechanistic subtleties will benefit future attempts in the rational design of salient therapeutic agents,
including next generation anticancer drug targets with amplified effectivity.
INTRODUCTION
Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-
dioxygenase (IDO) catalyze the rate-limiting first step of
Chart 1. Tryptophan 2,3-Dioxygenation to N-
formylkynurenine Mediated by TDO and IDO Enzymes Us-
ing Dioxygen.
kynurenine pathway, in which the indole ring of tryptophan
(Trp) is oxidatively cleaved at the 2,3-position to produce
N-formylkynurenine (NFK) using dioxygen (Chart 1).1-6
TDO and IDO are two evolutionarily related heme enzymes
(TDO is tryptophan-specific, while IDO can dioxygenate a
broader scope of substrates with indole moieties, such as
melatonin, serotonin, and tryptamine),7-9 however, unlike a
majority of such enzymes,10-12 their active oxidant is
thought to be a heme superoxo adduct (Figure 1), and
uniquely require only a single reduction event during com-
plete turnover.4, 13-14 These heme dioxygenases have been
the focal point of multiple human health related recent stud-
ies since; (1) accumulated intermediates of kynurenine
pathway are known to lead to various disease conditions in-
cluding multiple sclerosis,15 AIDS-related dementia,16 is-
chemic brain injury,17 cataract formation,18 depression,19-20
Huntington's disease,21-22 rheumatoid arthritis,23 and type-I
diabetes;24 (2) IDO serves in a critical immunoregulation
role by exerting antimicrobial/antiviral25 activity via Trp
degradation; (3) these enzymes have been recently impli-
cated in cell aging.26 Most importantly, TDO/IDO have been
found to engage in Trp catabolism in human T-cells, aiding
tumors to evade anticancer immunosurveillance of the
host.27-30 Thus, TDO/IDO inhibitors are rapidly emerging
anti-cancer and/or anti-aging therapeutic agents.31-37
The mechanism through which these enzymes operate
has been actively pursued for over half a century, however,
the key events/steps still remain elusive. The lack of such
mechanistic understanding can significantly impede the
ability to develop novel therapeutic agents with enhanced
activity. A base-catalyzed mechanistic proposal had been
widely accepted and reproduced in the literature,3, 38 how-
ever, recent studies including the structural characteriza-
tion of human IDO have led to its fall, since (1) the active site
of IDO lacked a distal histidine that was proposed to ab-
stract the indole ring proton of Trp (Figure 1B),1, 39 and (2)
N-methyltryptophan was found to be a slow, but active sub-
strate, whereas in a base-catalyzed scenario it would be in-
active.40 Accordingly, the mechanistic viewpoint has rapidly
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evolved, leading to proposals that differentiate the initial the indole to the electrophilic heme superoxide in a rate-
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heme superoxide attack on Trp between (1) an electro-
limiting initial substrate activation step. Isotopic labeling
experiments involving 18O2(g) reveal intriguing insights into
key mechanistic details of this reaction, suggesting the pos-
sibility of substrate dissociation upon the completion of the
initial monooxygenation step. The dioxygenated final or-
ganic products have also been isolated and unambiguously
identified. In addition to the significant advancement in
TDO/IDO modeling presented in this work, to the best of
our knowledge, this marks the first instance where unprec-
edented substrate oxidation capabilities of heme superox-
ide adducts are revealed, warranting reevaluation of their
conventional classification as sluggish oxidants.
41-44
philic-, or (2) a radical-addition (Figure 1C).4,
Intri-
guingly, both of these proposed mechanisms converge at a
common intermediate step: the formation of a ferryl inter-
mediate and the indole epoxide (Figure 1C). Lending cre-
dence, a ferryl intermediate has been spectroscopically ob-
served during human IDO turnover, where the formation
and decay of a ferryl-based Fe=O resonance Raman feature
has been evidenced at 799 cm-1.45-47 A handful of synthetic
TDO/IDO models exist,48-53 out of which, only a few has uti-
lized synthetic heme mimics.54-56 Nevertheless, insights into
the identity and properties of the active metal oxidant, key
reaction intermediates, and/or pivotal mechanistic events
are severely lacking.57-61
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Scheme 1. Generalized Reaction Scheme Depicting
Structural Variations of Heme Systems and Indole Sub-
strates Involved in this Study.
RESULTS AND DISCUSSION
Figure 1. Crystallographically characterized (A) ternary com-
plex of human TDO that includes the Trp substrate and the
heme-bound dioxygen moiety (PDB: 5TI9),2 and (B) active site
of human IDO (inhibitor bound; PDB: 2DOT) exhibiting the ab-
sence of a distal histidine residue.1 (C) Current mechanistic
proposals for the dioxygenation of tryptophan by IDO and TDO,
where the initial indole activation step is either an electrophilic
or a radical addition.
Formation and Dioxygenation Reactivity of Heme Su-
peroxide Adducts. We have thoughtfully selected three
porphyrinate systems for this study, which offer signifi-
cantly disparate electronic atmospheres for the heme iron
center: F20TPP (F20TPP = 5,10,15,20-tetrakis(pentafluoro-
phenyl)-porphyrin), TPP (TPP
phenylporphyrin), and TMPP (TMPP
=
5,10,15,20-tetra-
5,10,15,20-
=
tetrakis(4-methoxyphenyl)-porphyrin). The ferrous heme
complex, [(THF)2(TPP)FeII], exhibited significant elec-
tronic absorption perturbations from 426 (Soret;  = 16 ×
105 M-1cm-1), 536 ( = 6.2 × 104 M-1cm-1) and 558 ( = 6.5 ×
104 M-1cm-1) nm to 416 (Soret;  = 10 × 105 M-1cm-1) and 540
( = 9.0 × 104 M-1cm-1) nm in 9:1 DCM:THF solvent mixture
at –80 °C upon the bubbling of dry O2(g), indicating the for-
In sharp contrast to their biological,11, 62 and non-heme63-
64 counterparts, synthetic heme superoxo adducts have long
known to be weak oxidants.65-66 Accordingly, their organic
substrate oxidation reactivity is gravely understudied,67
and models of enzymes with a heme superoxide active spe-
cies are virtually non-existent. To this end, we herein de-
scribe the first series of synthetic heme superoxide models
that closely mimic the tryptophan oxidation (bio)chemistry
of TDO/IDO enzymes, cleaving the 2,3-double bond of an ar-
ray of indole substrates using dioxygen (Scheme 1). This
work, also for the first time in model chemistry, sheds light
on the ferryl species involved in this mechanism, and pre-
sents multiple spectroscopic and reactivity evidences that
support its occurrence as a reaction intermediate. Further,
the fastest dioxygenation reaction rate is observed between
the most electron-deficient superoxide adduct, and the
most electron-rich substrate, corroborating an addition of
mation of the EPR-silent, end-on ferric superoxo species,
68
[(THF)(TPP)FeIII(O2–•)] (Figure 2).12,
Isotope-sensitive
(Fe–O) and (O–O) resonance Raman frequencies for this
species were centered at 579 (18O2 = –26) and 1170 (18O2
= –60) cm–1, respectively (Figure 2). Subsequent addition of
2 equiv of 4 methylimidazole (Im) led to the formation of
–
the Im-coordinated superoxo adduct, [(Im)(TPP)FeIII(O2
•)] (418 (Soret;  = 7.5 × 105 M-1cm-1) and 543 ( = 6.5 × 104
M-1cm-1) nm);69-72 (Fe–O): 577 (18O2 = –23) and (O–O):
1170 (18O2 = –56) cm–1 (Figure 2).72-74 Similarly, the rest of
the ferric superoxo compound series was prepared and
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[(THF)(F20TPP)FeIII(O2–•)],
(red) (*due to subtraction of intense heme bands). Solvent: 9:1
DCM:THF.75
characterized:
[(Im)(F20TPP)FeIII(O2–•)], [(THF)(TMPP)FeIII(O2–•)] and
[(Im)(TMPP)FeIII(O2–•)] (Figures S1 and S2).12
1
2
To probe the reactivity of these ferric superoxide adducts,
100 equiv of the indole substrates were added in at –80 °C;
however, no spectral changes were evident. Markedly, upon
warming this mixture up to –40 °C, the superoxide adducts
initiated reactivity with added indole substrates, with the
intermediacy of a distinct heme species. For example, for
[(THF)(TPP)FeIII(O2–•)], a subtle, but prominent red shift
(from 416 to 417 nm) followed by a blue shift (417 to 414
nm) of the Soret band was observed along with overall de-
crease in the absorption intensity (Figure 3A) in the pres-
ence of 3-methylindole (Scheme 1; 1a). Such sequential bi-
phasic spectral changes were even more prominent for the
reaction between [(Im)(TPP)FeIII(O2–•)] and 1a (Figure
3B). Similar changes were also observed for
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[(THF)(F20TPP)FeIII(O2–•)]
[(Im)(F20TPP)FeIII(O2–•)]
[(THF)(TMPP)FeIII(O2–•)]
(Figure
(Figure
(Figure
S4),
S4),
and
S3)
[(Im)(TMPP)FeIII(O2–•)] (Figure S3) at –40 °C (or –55 °C
(vide infra)). As well, other substituted indoles shown in
Scheme 1 (i.e., 1b and 1c) displayed similar reactivity (Fig-
ures S5 and S6). Noticeably, no reaction was observed with
indole, 2-methylindole, or 1,3-dimethylindole. This lack of
reactivity is well precedented by previous studies, where a
3-position substituent was found to be essential for dioxy-
genation, while N-substituted indoles exhibited either very
slow or no reactivity.40, 54, 76 In fact, the latter observation
was thought to be “evidence” for the previously accepted,
now obsolete base-catalyzed mechanism of TDO/IDO.76 We
note here that the decay rates of the heme superoxide com-
plexes in the absence of substrate were at least an order of
magnitude slower than the indole oxidation rates, and that
self-decay does not possess any distinct reaction intermedi-
ates (Figure S8).
Figure 2. (A) UV-vis spectra for 10 µM solutions of
[(THF)2(TPP)FeII] (red), [(THF)(TPP)FeIII(O2–•)] (green),
and [(Im)(TPP)FeIII(O2–•)] (blue) at –80 oC; (B) EPR spectra
for frozen 2 mM solutions of [(THF)(TPP)FeIII(O2–•)] (green),
and [(Im)(TPP)FeIII(O2–•)] (blue) compared with that of a
[(TPP)FeIII(Cl)] (red) at 7 K; resonance Raman spectra (ex
=
406.7 nm) of 2 mM (C) [(THF)(TPP)FeIII(O2–•)] and (D)
[(Im)(TPP)FeIII(O2–•)] prepared with 16O2(g) (black) and 18O2(g)
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Characterization of the Putative FeIV=O (Ferryl) Inter-
mediate and Mechanistic Details. Inspired by the unam-
biguous spectroscopic observation of a heme-based reac-
tion intermediate, and the proposed ferryl intermediate in
the TDO/IDO mechanistic proposals (vide supra), we set to
probe the plausibility of the putative formation of a ferryl
intermediate. In this, we have generated authentic ferryl ad-
ducts of all three heme systems, [(Im)(TPP)FeIV(O)],
[(Im)(F20TPP)FeIV(O)], and [(Im)(TMPP)FeIV(O)], by re-
acting the corresponding FeII complexes with m-CPBA, and
the subsequent addition of Im at –40 °C (Scheme 2; Figures
3 and S9).77-79 Remarkably, the Soret band positions of the
independently generated authentic ferryl intermediates
were strikingly similar to those of the corresponding reac-
tion intermediates (Figure 3; Figure S9), albeit the Q-band
regions exhibited either a mixture of features and/or the
peak positions were not clearly distinguishable. This reac-
tion intermediate was also observed to be EPR-silent, as
would be expected for a ferryl adduct (Figure S7).77, 80 To
further interrogate this observation in detail, we carried out
low-temperature 2H NMR studies utilizing the pyrrole-posi-
tion deuterated F20TPP-d8 (see experimental section for de-
tails). [(THF)2(F20TPP-d8)FeII] and [(THF)(F20TPP-
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d8)FeIII(O2–•)] exhibited a single 2H NMR feature at δpyrrole
=
89.2 and δpyrrole = 8.8 ppm, respectively, as would be ex-
pected according to literature precedence (Figure 4).81 The
–
addition of 3-methylindole into [(THF)(F20TPP-d8)FeIII(O2
•)], and subsequent increment in temperature from –80 °C
to –55 °C led to the immediate formation of a new diamag-
netic 2H NMR feature at δpyrrole = 3.2 ppm (Figure 4), which
is identical to that of the authentic ferryl adduct, while being
in close agreement with previous reports.77, 81 Finally, when
the indole oxidation reaction was carried out in the pres-
ence of excess triphenylphosphine (PPh3) at –40 °C, tri-
phenylphosphine oxide (O=PPh3) formation was observed
in the expense of indole oxidation product formation, as
confirmed by 31P NMR (Figure S11).82 Separate control ex-
periments were carried out in the absence of heme complex,
dioxygen, or indole substrate, none of which demonstrated
PPh3 oxidation (Figures S12 – S14). Thus, the (1) excellent
agreement of Soret, and (2) low-temperature 2H NMR spec-
troscopic features, while (3) being EPR-silent, and (4) ex-
hibiting triphenylphosphine oxidation impart strong cor-
roboration that the observed reaction intermediate is most
likely the anticipated ferryl species, resembling similar ob-
servations in the IDO mechanism.45 To the best of our
knowledge, this is the first instance where any evidence in
support of the anticipated, biologically-relevant ferryl inter-
mediate has been presented for indole oxidation with any
model system. In fact, high-valent heme intermediates have
never been observed for any reactivity landscape initiated
by synthetic heme superoxides.
Figure 3. Electronic absorption spectral changes observed dur-
ing the reaction of a 10 µM 9:1 DCM:THF solution of (A)
[(THF)(TPP)FeIII(O2–•)] and (B) [(Im)(TPP)FeIII(O2–•)] with
100 equiv of 3-methylindole at –40 °C (Red = initial ferric su-
peroxo complex; green = intermediate species; blue = final fer-
ric product). (C) Electronic absorption spectral features of 10
µM [(THF)2(TPP)FeII] (red), independently prepared
[(TPP)FeIV(O)] (green) and [(Im)(TPP)FeIV(O)] (blue) in 9:1
DCM:THF at –40 °C. Insets show the expanded Soret and Q-
band regions, and arrows indicate the direction of peak transi-
tion.
Scheme 2. Schematic Representation of the Independ-
ent Generation of Six-coordinate FeIV=O (ferryl) Com-
plexes.
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Figure 4. 2H NMR spectral data in 9:1 DCM:THF for (A)
[(THF)2(F20TPP-d8)FeII] at –80° C, (B) [(THF)(F20TPP-
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d8)FeII(O2–•)] at –80 °C, (C) authentic ferryl intermediate,
[(THF)(F20TPP-d8)FeIV(O)], and (D) reaction intermediate be-
tween [(THF)(F20TPP-d8)FeIII(O2–•)] and 3-methylindole at –
55 °C, and (E) the final heme product at room temperature.
(*solvent peaks (black) and an instrumental glitch (red)).
Patent differences in reaction rates among
[(THF)(TPP)FeIII(O2–•)], [(THF)(F20TPP)FeIII(O2–•)], and
[(THF)(TMPP)FeIII(O2–•)] were also observed, in that,
[(THF)(F20TPP)FeIII(O2–•)] reacted the fastest, while
[(THF)(TMPP)FeIII(O2–•)] was the slowest (Figure 5).
Indeed, [(THF)(F20TPP)FeIII(O2–•)] initiated reactivity with
indole substrates even at –55 °C, while others required the
increment in reaction temperature up to –40 °C. As seen in
Figure 5, the kinetic traces collected at Soret wavelengths
are clearly biphasic, indicating the fomation and decay of a
Figure 5. UV-vis spectral changes in 9:1 DCM:THF at –40 °C for
the reaction of 10 µM (A) [(THF)(TPP)FeIII(O2–•)] (B)
[(THF)(TMPP)FeIII(O2–•)] and (C) [(THF)(F20TPP)FeIII(O2–•)]
with 100 equiv of 3-methylindole (Red = initial ferric superoxo
complex; blue = final ferric product; insets show kinetic time
traces for Soret regions).
perceptible
intermediate.
Intruigingly,
for
[(THF)(F20TPP)FeIII(O2–•)] both of these phases are much
more rapid compared to [(THF)(TPP)FeIII(O2–•)] or
[(THF)(TMPP)FeIII(O2–•)], revealing superior reactivity of
both the superoxo adduct, and the (ferryl) intermediate.
Moreover, the most electron-rich indole substrate, 2,3-di-
methyl indole reacts the fastest with all three superoxides.
These findings in concert suggest that an attack from the in-
dole substrate on the electrophilic iron superoxo adduct is
most likely the initial step of the mechanism. The feasibility
of such an attack is in line with the significant “ferrous-oxy”
character possessed by these synthetic superoxide ad-
ducts.83 Similar trends have also been observed for heme-
modified TDO enzyme models, where electron-withdraw-
ing heme substituents increased the rate of tryptophan oxi-
dation.84
The rate-limiting regions for TDO/IDO have been dis-
cussed in detail,44 and are both proposed to be pre-ferryl.
For TDO, the initial attack on the heme superoxide is the
slow-most step, making the ferryl intermediate virtually un-
observable. In contrast, IDO encompasses the ferryl species
in its slowest step, allowing its detailed characterization.44-
46
With this background, the rate-limiting events for these
synthetic heme superoxide adducts presumably incorpo-
rate both the initial superoxide attack and ferryl formation,
permitting the determination of a clear interdependency
between the reaction rates and the heme electronic struc-
ture, while allowing the characterization of the (ferryl) in-
termediate. Accordingly, under pseudo-first-order reaction
conditions (50 – 300 equiv of 3-methylindole), the rate of
indole dioxygenation by [(THF)(TPP)FeIII(O2–•)] displayed
linear dependence on the indole substrate concentration,
leading to a second order rate constant of 0.47 M-1 s-1 (Fig-
ure 6). Noteworthily, no reactivity was observed if the su-
peroxide adducts were warmed up to –40 °C prior to the ad-
dition of substrates, reasonings for which, are currently un-
clear.
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Scheme 3. Plausible Pathways for the Overall Dioxygen-
ation Reaction.
6
7
8
9
O
THF
Ar
Ar
O
N
H
N
N
N
O2
N
N
FeII
N
FeIII
Ar
Ar
Ar
Ar
No reaction
N
N
9:1 DCM:THF
-80 ºC
9:1 DCM:THF
-80 ºC
THF Ar
B
Ar
(THF)2(Por)FeII
UV, 2H NMR, EPR
[(B)(Por)FeIII(O2 )]
UV, 2H NMR, Resonance Raman, EPR
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9:1 DCM:THF
-40 ºC
Figure 6. The dependence of pseudo-first-order dioxygenation
rate constants (initial rate fits) on the 3-methylindole substrate
concentration for 10 µM [(THF)(TPP)FeIII(O2–•)] in 9:1
DCM:THF at –40 °C.
N
H
Ar
NH
N
N
N
FeIII
Ar
Ar
+
Ar
N
N
HN
N
N
N
FeIII
Ar
Ar
O
Ar
Ar
N
O
N
N
N
Ar
N
O
N
Bulk-scale Indole Oxygenation Reactions and Overall
Reaction Landscape. To further rationalize the hypothe-
sized indole 2,3-dioxygenation chemistry, we designed
scaled-up reactions using [(THF)(TPP)FeIII(O2–•)], permit-
ting rigorous characterization of the desired organic prod-
ucts by 1H and 13C NMR, FT-IR, and LC-MS methods (Figures
S15 – S20). For example, when 3-methylindole (1a) was
subjected to heme superoxide mediated oxidation, ortho-
formamidoacetophenone (2a) was found to be the major
product. Likewise, the predominant organic product follow-
ing the oxidation of 1b and 1c (Scheme 1) were revealed to
be 2b and 2c, respectively.85 However, the maximum yield of
product isolated capped at ~31%; the balance of the added
substrate was leftover, i.e., unreacted. For imidazole-coordi-
nated superoxide adducts, as also previously reported,54
both the reaction rates and the yield of the final dioxygen-
ated product were diminished (yield = ~20%), presumably
due to competitive interactions exerted by imidazole (Fig-
ure S21). We note that the present study, therefore, offers
the first fully characterized series of organic products for a
FeIII
Ar
Ar
N
Ar
B= THF
O2
B= Im
O2
N
FeIV
Ar
Ar
N
N
HN
Ar
[{(Por)FeIII}2(µ-O)]
[(Im)2(Por)FeIII
]
+
B
Ar
+
O
+
O
[(B)(Por)FeIV(O)]
UV, NMR (2H and 31P), EPR
CH3
CH3
NH
NH
CHO
CHO
NMR (1H and 13C),
FT-IR, LC-MS
NMR (1H and 13C),
FT-IR, LC-MS
CONCLUSIONS
TDO/IDO-dependent tryptophan 2,3-dioxygenation in bi-
ology is fundamental for a battery of human health related
concerns, including some of the most supreme challenges in
human pathogenesis.15-24, 31-37 Both TDO and IDO are heme
enzymes, and their active oxidant is thought to be a heme
superoxide adduct.4, 10-13 However, the current literature on
heme superoxide intermediates, irrespective of protein or
synthetic model-based, acutely lack a direct linkage to in-
dole dioxygenation. To this end, this study communicates
the first report where a medley of synthetic heme superox-
ide and indole substrate models have been utilized to mimic
the aforementioned tryptophan oxidation chemistry of TDO
and IDO heme enzymes. The thermal instability of such di-
oxygen-derived synthetic heme superoxide models has
warranted the use of cryogenic conditions, and upon warm-
ing up the superoxide/indole mixtures from –80 to –40 °C
(or –55 °C), the indole substrates were observed to undergo
2,3-bond cleaving dioxygenation, yielding ~31% product.
Interestingly, we report the detection of a low-temperature
reaction intermediate (UV-vis, 2H NMR, EPR, and PPh3 oxi-
dation), which we propose to be the biologically-relevant
ferryl species (Figure 1C).
heme dioxygenase enzyme mimic. Interestingly, when iso-
18
topically-labeled
O
was utilized, the mass of (major)
2(g)
product 2c shifted from 293.11 to 297.12 m/z, indicating
the incorporation of two 18O atoms (Figure S18). When a 1:1
16
O
:18O2(g) mixture was used to oxidize the indole
2(g)
substrate 1c, a 1:1:1 ratio among the 2 헑 16O:16O18O:2 헑 18O
incorporated 2c products was observed (Figure S19),
shedding light on the stepwise oxygen insertion mechanism
at play (similar to TDO/IDO; see Figure 1C). This crucial,
unprecedented result also indicates the likelihood of indole
epoxide dissociation following the initial oxygenation step
(Figure 1C), which presumably dictates the modest reaction
yields. This observation underscores the importance of the
extended protein structure that likely restricts a similar
substrate dissociation in TDO/IDO, allowing the generation
of stoichiometric dioxygenated products. In line with the
UV-vis experiments (vide supra), none of the substrates that
lacked 3-position indole ring substituents or the proton on
the indole N-atom produced the desired product in bulk re-
actions. The final heme product in these dioxygenation re-
actions was characterized to be either the oxo-bridged (µ-
oxo) diferric compound, [{(TPP)FeIII}2(µ-O)], or the bis-Im
ferric complex, [(Im)2(TPP)FeIII]+, in the absence and pres-
ence of Im, respectively (Scheme 3; Figures S23 and S24).
The electronic properties of both the heme oxidants and
the indole substrates employed in this interrogation offer
clear trends in rates of indole oxidation, strongly suggesting
that an indole addition to an electrophilic heme superoxide
moiety is the initial step of the reaction. Our findings here
are therefore in favor of one of the proposed mechanisms
for TDO/IDO (Figure 1C), paralleling previous studies on
heme-modified TDOs.84 This initial indole addition step is
also rate-limiting, and in support, the rate of indole oxida-
tion is linearly dependent on the indole concentration. Our
scaled-up approach has allowed the detailed
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1
characterization of oxidized organic products by H NMR,
NMR Spectrometer. 31P NMR and low-temperature 2H NMR
18
13C NMR, FT-IR, and LC-MS analyses, as well as
O
la-
spectroscopic studies were carried out on a Bruker DRX 400
MHz NMR Spectrometer. All NMR spectra were recorded in
5-mm (outer diameter) tubes. The chemical shifts were re-
ported as δ (ppm) values calibrated to natural abundance
deuterium or proton solvent peaks. Infrared (IR) vibra-
tional spectra were collected on a Bruker FT-IR spectrome-
ter (Vertex 70) at room temperature. A SCIEX 5600 Triple-
Tof mass spectrometer (SCIEX, Toronto, Canada) was used
to analyze the mass profiles of the organic products. The
IonSpray voltages for positive modes were +/- 5000 V, and
the declustering potential was 80 V. Ionspray GS1/GS2 and
curtain gases were set at 40 psi and 25 psi, respectively. The
interface heater temperature was maintained at 400 oC.
Electron paramagnetic resonance (EPR) spectra were col-
lected in 4 mm (outer diameter) quartz tubes using an X-
band Bruker EMX-plus spectrometer coupled to a Bruker
ER 041 XG microwave bridge, and a continuous-flow liquid
helium cryostat (ESR900) controlled by an Oxford Instru-
ments TC503 temperature controller (experimental condi-
tions: microwave frequency = 9.41 GHz; microwave power
= 0.2 mW; modulation frequency = 100 kHz; modulation
amplitude = 10 G; temperature = 7 K). 5,10,15,20-tetra-
phenylporphyrin iron(III) chloride, [(TPP)FeIIICl], and
5,10,15,20-tetrakis(4-methoxyphenyl)-porphyrin,
1
2
3
4
5
6
7
8
2(g)
beled studies that uncover strong evidence supporting a
stepwise/sequential dioxygen insertion mechanism, as
would be expected for TDO/IDO. In contrast to TDO/IDO,
however, the indole epoxide intermediate in these model
systems presumably escape following the first oxygen in-
sertion step, likely due to the absence of sterically encum-
bering protein mass that limit/prevent substrate dissocia-
tion in nature. The final heme product is Fe(III) rather than
the mechanistically expected Fe(II) species. This anomaly is
possibly due to the rapid oxidation of the resultant Fe(II)
(from indole oxidation) by dissolved dioxygen in solution;
indeed the oxo-bridged (µ-oxo) diferric compound,
[{(TPP)FeIII}2(µ-O)], is known to be the main product of the
reaction between Fe(II) and dioxygen at non-cryogenic
temperatures.86-87 In conclusion, we have succeeded in the
efficient modeling of biological tryptophan 2,3-dioxygena-
tion using synthetic heme-based superoxo models, how-
ever, further interrogations are warranted for definitive
mechanistic conclusions, and/or for fully comprehending
the identity of key intermediates, and unique substrate
specificities observed in this study that draw close resem-
blance to TDO/IDO enzymes.
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EXPERIMENTAL SECTION
H2(TMPP) were purchased from commercial sources. The
syntheses of H2(F20TPP),88 H2(F20TPP)-d8,89 and 1,3-dime-
thylindole90 were carried out according to previously pub-
lished methods. Metalation of the porphyrinates to generate
Materials and Methods. All commercially available
chemicals were purchased at the highest available purity,
and used as received unless otherwise stated. Air-sensitive
compounds were handled either under an argon atmos-
phere using standard Schlenk techniques, or in an Mbraun
Unilab Pro SP (<1 ppm O2, <1 ppm H2O) nitrogen-filled
glovebox. All organic solvents were purchased at HPLC-
grade or better. DCM and THF were degassed (by bubbling
argon gas for 30 min at room temperature) and dried (by
passing through a 60 cm alumina column) using an Inert
Pure Solv MD 5 (2018) solvent purification system. These
solvents were then stored in amber glass bottles inside the
glovebox over 4 Å molecular sieves at least for 72 hrs prior
to use. The purity of O2 gas used was >99%, and was further
dried by passage through a 12” column containing drierite
and 5 Å molecular sieves. Benchtop UV-vis experiments
were carried out using an Agilent Cary 60 spectrophotome-
ter equipped with a liquid nitrogen chilled Unisoku Cool-
Spek UV USP-203-B cryostat. A 2 mm path length quartz cell
cuvette modified with an extended glass neck with a female
14/19 joint and stopcock was used to perform all UV-vis ex-
periments. Resonance Raman samples were excited at
406.7 nm, using a Coherent I90C-K Kr+ ion laser, while the
sample was immersed in a liquid nitrogen cooled (77 K)
EPR finger dewar (Wilmad). Power was ~4 mW at the sam-
ple. Data were recorded while rotating the sample manually
to minimize photodecomposition. The spectra were rec-
orded using a Spex 1877 CP triple monochromator with a
600 groves/mm holographic spectrograph grating, and de-
tected by an Andor Newton CCD cooled to -80 °C. Spectra
were calibrated on the energy axis to citric acid. The reso-
nance Raman data was processed using the spectroscopy
software SpectraGryph version 1.2 (Dr. Friedrich Menges
Software-Entwicklung, Oberstdorf, Germany) and Origin
2019b software. 1H NMR and 13C NMR spectra at room tem-
perature were recorded on a Bruker Avance III-HD 500 MHz
[(TMPP)FeIIICl],
d8)FeIIICl],
and
[(F20TPP)FeIIICl],
the subsequent
and
reduction
[(F20TPP-
to
[(THF)2(Por)FeII] complexes were carried out by following
previously reported procedures.81 [{(TPP)FeIII}2(µ-O)] and
‘naked’ [(THF)2(TPP)FeIII]SbF6 compounds were synthe-
sized as previously reported.91-93
Formation of the Heme Superoxo Complexes,
[(B)(Por)FeIII(O2–•)], where B = THF or Im; Por = the por-
phyrinate supporting ligand. Generation of the superoxo
complexes, [(B)(Por)FeIII(O2–•)], was carried out following
a literature-adapted procedure.70-71, 73, 81 In a typical exper-
iment,
a 10 µM 9:1 DCM:THF solution (1 mL) of
[(THF)2(Por)FeII] was added into a 2 mm pathlength
Schlenk cuvette inside the glovebox, and was sealed using a
rubber septum. Upon cooling down inside the UV-vis cryo-
stat stabilized at –80 °C, this solution was bubbled with dry
dioxygen gas using a needle, and the complete formation of
the superoxide complexes, [(THF)(Por)FeIII(O2–•)], was
monitored by UV-vis spectroscopy. Excess O2(g) was re-
moved by three vacuum/Ar purge cycles, and the subse-
quent addition of 2 equiv of 4-methylimidazole (Im; in 50
µL of 9:1 DCM:THF) resulted in the 6-coordinate superoxide
complexes, [(Im)(Por)FeIII(O2–•)] (Figures 2A and S1).
Resonance Raman Sample Preparation. In a typical ex-
periment, 300 µL of the ferrous heme complex,
[(THF)2(Por)FeII] (2 mM in 9:1 DCM:THF), was placed in a
9 mm NMR tube, and was sealed with a rubber septum. Fol-
lowing cooling down to −80 °C, dry O2(g) (or 18O2(g)) was bub-
bled through the solution mixture using a three-way gas-
tight syringe in order to generate the corresponding super-
oxide
complex,
[(B)(Por)FeIII(O2–•)].
Immediately
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following the heme superoxide formation, the solutions using a Hamilton gas-tight syringe, and was quickly mixed
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were frozen in liquid N2, and the tubes were flame-sealed.
Spectroscopic Reactivity Studies of [(B)(Por)FeIII(O2
with Ar bubbling. The tube was then transferred into the
cryostat of the NMR spectrometer held at –55 °C (Figure 4,
black). The major NMR peak observed at –55 °C was cen-
tered at 3.2 ppm, indicating the formation of a ferryl inter-
mediate81 (Note: the feature at 93.2 ppm is indicative of the
presence of a minor paramagnetic component). The final
heme product consists of µ-oxo bridged diferric compound
centered at 14.4 ppm, along with a minor monomeric
iron(III) hydroxy species with δpyrrole = 80.2 ppm (Figure 4,
pink).97-98
–
•)] with Indole Substrates. The indole substrates (1 mM,
50 µL) were added into the 2 mm pathlength cuvette con-
taining [(B)(Por)FeIII(O2–•)] (10 µM, 1 mL) in 9:1 DCM:THF
using a Hamilton gas-tight syringe at –80 °C, and the cuvette
contents were quickly mixed with Ar bubbling. No spectral
changes were observed at –80 °C. Then, the temperature of
the cryostat was raised up to –40 °C (Figures 3, S3, S5 and
S6) or –55 °C (Figure S4), upon which, the reactions with the
indole substrates were initiated, and the corresponding
spectral changes were recorded. The self-decay of the su-
peroxide complexes was also monitored at –40 °C without
the addition of any indole substrates (Figure S8).
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The corresponding authentic ferryl intermediate,
[(THF)(F20TPP-d8)FeIV(O)], was prepared by adding 1
equiv of m-CPBA to a solution of [(THF)2(F20TPP-d8)FeII] in
9:1 DCM:THF, and the NMR data was collected at –55 °C
(Figure 4, green; δpyrrole = 3.2 ppm); the heme superoxide
starting complex, [(THF)(F20TPP-d8)FeIII(O2–•)] (Figure 4,
red; δpyrrole = 8.8 ppm), was generated by bubbling dry O2(g)
into a solution of [(THF)2(F20TPP-d8)FeII] (Figure 4, blue;
δpyrrole = 89.2 ppm) in 9:1 DCM:THF, and the data was col-
lected at –80 °C (Figure 4).
Kinetic Analysis of Indole Dioxygenation. For variable
substrate concentration kinetic analyses, different indole
concentrations were utilized, and the aforementioned
methodology followed to initiate the reaction. The kinetic
data was analyzed utilizing the initial rate method, where
the first 20% of the data was fit to obtain the reaction rates
(Figure 6).
Generation of the Authentic [(Im)(Por)FeIV(O)] Inter-
mediates. [(Im)(Por)FeIV(O)] (ferryl) intermediates were
prepared following a previously published method by Kar-
lin and co-workers.77 In a typical procedure, 1 equiv of m-
CPBA was added into a 10 µM solution of [(THF)2(Por)FeII]
in 9:1 DCM:THF (1mL) at −40 °C to yield [(Por)FeIV(O)] ad-
ducts; successive addition of 2 equiv of Im resulted in the
Triphenylphosphine (PPh3) Oxidation Studies. PPh3
oxidation to O=PPh3 was primarily analyzed by 31P NMR
spectroscopy. Sample preparation for 31P NMR experiments
was carried out as follows:
a
solution of
[(THF)(TPP)FeIII(O2–•)] was prepared in a 25 mL Schlenk
flask using 10 mg of [(THF)2(TPP)FeII] and O2(g) at –80 °C
in 9:1 DCM:THF (5 mL). Then, 2.5 mL of 3-methylindole
(200 mg, 1.5 mM) and 2.5 mL of PPh3 (400 mg, 1.5 mM)
were added in. This mixture was stirred at –40 °C for 2 hrs.
The resultant solution was dried under vacuum, and the
residue was redissolved in CDCl3 for NMR analysis (Figure
S11). Three control experiments were carried out following
similar procedures, but without the addition of (1)
[(THF)2(TPP)FeII], (2) O2(g) and (3) 3-methylindole. The re-
sults of these control experiments are summarized in Fig-
ures S12 – S14. UV-vis spectroscopic changes for the reac-
tion between [(B)(TPP)FeIII(O2–•)] (where B = THF or Im)
and 3-methylindole was monitored in the presence of 100
equiv of PPh3, in order to confirm that the formation of the
(ferryl) intermediate does occur in the presence of PPh3.
These spectra are summarized in Figure S10.
formation
of
Im
ligated
ferryl
derivatives,
[(Im)(Por)FeIV(O)] (Scheme 2). These complexes were
characterized by UV-vis spectroscopy (Figures 3C and S9)
and NMR spectroscopy, and are in excellent agreement with
the literature reports.78, 81, 94-96 Figures 3C and S9 presents a
comparison between the UV-vis features of authentic
[(Im)(Por)FeIV(O)] complexes and those of the corre-
sponding indole oxidation reaction intermediates.
Synthesis and Characterization of [(F20TPP-d8)FeIIICl]
and
[(THF)2(F20TPP-d8)FeII].
The
synthesis
of
H2(F20TPP)-d8 ligand was carried out according to litera-
ture procedures.89 Generation of [(F20TPP-d8)FeIIICl] and
the subsequent reduction to [(THF)2(F20TPP-d8)FeII] were
carried out by following previously reported methods.81
Bulk Reactions and Characterization of Organic Prod-
ucts. The bulk dioxygenation of all three substrates using
[(THF)(TPP)FeIII(O2–•)] was carried out by a generalized
procedure as follows: A 100 mL Schlenk flask containing
[(THF)2(TPP)FeII] (100 mg, 0.15 mM) in 9:1 DCM:THF (20
mL) was cooled down in an acetone bath adjusted to –80 °C.
Upon temperature equilibration, dioxygen gas (or labeled
[(F20TPP-d8)FeIIICl]: UV-vis (0.1 mM in CH2Cl2): 410 (So-
ret;  = 1.2×105 M-1cm-1), 503 ( = 1.5 × 104 M-1cm-1) and 629
( = 7.7 × 103 M-1cm-1) nm; 2H NMR (CHCl3, 298 K): δpyrrole
=
82.5 ppm; ESI-MS: {[(M-Cl)+CH3CN]+} m/z = 1077.55 (calc.
1077.05).
[(THF)2(F20TPP-d8)FeII]: UV-vis (10 µM in 9:1
DCM:THF): 419 (Soret;  = 1.8 × 106 M-1cm-1) and 540 ( =
1.0 × 105 M-1cm-1) nm; 2H NMR (9:1 DCM:THF, 193 K): δpyrrole
= 89.2 ppm.
18
–
O
) was bubbled through to form [(THF)(TPP)FeIII(O2
2(g)
•)]. Then 3-methylindole (20 mg, 0.15 mM; in 5 mL of 9:1
DCM:THF) was added in, and the reaction mixture was
stirred for 2-3 min to mix in the contents. The Schlenk flask
was then quickly transferred to another cold bath at –40 °C,
and the reaction mixture was stirred for 2 hrs. The final re-
action mixture was dried in vacuum, and the final (organic)
product was purified by silica gel column chromatography
using EtOAc: hexane (1:1) as an eluent. (Note: When the re-
action between [(THF)(TPP)FeIII(O2–•)] and 2a was
quenched at different time points (15, 30, 45, 60, 90 min), a
2
Low-temperature H NMR Spectroscopic Studies. For
2
a typical H NMR experiment, 10 mg of [(THF)2(F20TPP-
d8)FeII] (10 mM) was dissolved in 0.5 mL of 9:1 DCM:THF,
and sealed in a 5 mm (outer diameter) NMR tube inside the
glovebox. This tube was then stabilized at –90 °C using a liq-
uid nitrogen/acetone cold bath, followed by the addition of
O2(g) by means of a 9” needle. Substrate (100 equiv of 3-me-
thylindole in 50 µL of the same solvent) was then added
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Pritam Mondal: 0000-0002-7071-1970
Gayan B. Wijeratne: 0000-0001-7609-6406
linear increase of product yield was observed before it started
plateauing between ~60 – 90 min (Figure S25)).
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For LC-MS, an aliquot (20 mL) of each sample was loaded
onto a Phenomenex 4.6 x 250mm, 4μm Hydro-RP, 80 Å re-
verse-phase column (Torrance, CA). A linear gradient of 2-
50% acetonitrile in 0.1% formic acid was utilized at a rate
of 250 μL/min for 14 mins, then 50-98% acetonitrile-0.1%
formic acid was used up to 17.5 min using an Shimadzu
Prominence HPLC (Columbia, MD). The column was washed
with 98% acetonitrile-0.1% formic acid for 0.5 min, and
then re-equilibrated with 2% acetonitrile-0.1% formic acid
for 7 min between each sample.
2a: Yield: 6 mg (24%); 1H NMR (CDCl3, 298 K): δ (ppm) =
11.60 (br, 1H; NH), 8.76 (d, 1H; Ar-H), 8.52 (s, 1H; -CHO),
7.93 (d, 1H; Ar-H), 7.59 (t, 1H; Ar-H), 7.19 (t, 1H; Ar-H), 2.69
(s, 3H; -CH3); 13C NMR (125 MHz, CDCl3, 298 K): δ (ppm) =
202.8, 159.9, 139.8, 135.2, 131.7, 123.1, 122.0, 121.6, 28.57;
FT-IR (ATR, cm-1): 1679, 1642, 1603, 1577, 1510, 1451,
1390, 1308, 1251, 1173, 1017, 767; LC-MS: [M+H]+ m/z =
164.07 (calc. 164.07).
2b: Yield: 8.5 mg (31%); 1H NMR (CDCl3, 298 K): δ (ppm)
= 11.70 (br, 1H; -NH), 8.75 (d, 1H; Ar-H), 7.90 (d, 1H; Ar-H),
7.56 (t, 1H; Ar-H), 7.12 (t, 1H; Ar-H), 2.67 (s, 3H; -CH3), 2.24
(s, 3H; -CH3); 13C NMR (125 MHz, CDCl3, 298 K): δ (ppm) =
202.8, 169.4, 141.0, 135.1, 131.6, 122.3, 121.7, 120.7, 28.6,
25.5; FT-IR (ATR, cm-1): 1686, 1644, 1582, 1516, 1440,
1360, 1300, 1236, 1157, 746; LC-MS: [M+H]+ m/z = 178.08
(calc. 178.09).
2c: Yield: 8 mg (18%); 1H NMR (CDCl3, 298 K): δ (ppm) =
11.40 (br, 1H; -NH), 8.78 (d, 1H; Ar-H), 8.51 (s, 1H; -CHO),
7.93 (d, 1H; Ar-H), 7.62 (t, 1H; Ar-H), 7.21 (t, 1H; Ar-H), 6.54
(d, 1H; N-HAc), 4.98 (br, 1H; C-H), 3.82-3.72 (m, 2H; -CH2),
3.79 (s, 3H; -COOCH3), 2.05 (s, 3H; -NHCOCH3); 13C NMR
(125 MHz, CDCl3, 298 K): δ (ppm) = 202.0, 171.7, 169.9,
159.8, 140.1, 135.9, 131.0, 123.3, 121.7, 120.9, 52.8, 48.2,
41.8, 23.1; FT-IR (ATR, cm-1): 1745, 1697, 1638, 1604, 1580,
1513, 1449, 1430, 1367, 1296, 1201, 984, 751; LC-MS:
[M+H]+ m/z = 293.11 (calc. 293.11).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The University of Alabama at Birmingham (UAB) is gratefully
acknowledged for startup funds provided to G. B. W. Authors
would like to thank Prof. Ed Solomon, and Anex Jose and Au-
gustin Braun of the Solomon Lab at Stanford University for col-
lecting and interpreting the resonance Raman data included in
this study. The authors would also like to thank Prof. Mary El-
len Zvanut (UAB), Dr. Mike Jablonski (UAB), Mayukh Bhadra
(Johns Hopkins University), and Landon Wilson (UAB) for as-
sistance with EPR, low-temperature NMR and mass spectrom-
etry/LC-MS experiments. Purchase of the AB Sciex 5600 Tri-
pleTOF mass spectrometer in the Targeted Metabolomics and
Proteomics Laboratory (TMPL) at UAB came from funds pro-
vided by the NCRR grant: S10 RR027822-01. Funds for the op-
eration of TMPL come in part from the UAB O'Brien Acute Kid-
ney Injury Center (P30 DK079337).
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Characterization of the Final Heme Product in the
Presence and Absence of Imidazole. Authentic
[{(TPP)FeIII}2(µ-O)] and ‘naked’ [(THF)2(TPP)FeIII]SbF6
were
synthesized
as
previously
reported.91-92
[(Im)2(TPP)FeIII]+ was prepared by adding 2.5 equiv of Im
to
a
0.1
mM
9:1
DCM:THF
solution
of
[(THF)2(TPP)FeIII]SbF6 at –40 °C (Figures S23 and S24).
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the
ACS Publications website at http://pubs.acs.org.
1
UV-vis, EPR, LC-MS, resonance Raman, H NMR, and 31P NMR
characterization data related to heme superoxide complexes
and their reactivity products (PDF).
AUTHOR INFORMATION
13. Thackray, S. J.; Efimov, I.; Raven, E. L.; Mowat, C. G., Chapter
12 Mechanism and Function of Tryptophan and Indoleamine
Dioxygenases. In Iron-Containing Enzymes: Versatile Catalysts of
Hydroxylation Reactions in Nature, The Royal Society of Chemistry,
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Corresponding Author
*wijeratne@uab.edu
ORCID
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