Reaction Mechanism of a Nonheme Iron Enzyme Catalyzed Oxidative Cyclization via C-C Bond Formation

Article abstract of DOI:10.1021/acs.orglett.8b03670

A complementary study including design of mechanistic probes, biochemical assays, model analysis, and liquid chromatography coupled mass spectrometry was conducted to establish the reaction mechanism for a nonheme iron enzyme catalyzed (?)-podophyllotoxin

Full text of DOI:10.1021/acs.orglett.8b03670

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Reaction Mechanism of a Nonheme Iron Enzyme Catalyzed
Oxidative Cyclization via C−C Bond Formation
,
†
‡
‡
,‡
Wei-chen Chang,* Zhi-Jie Yang, Yueh-Hua Tu, and Tun-Cheng Chien*
†
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States
Department of Chemistry, National Taiwan Normal University, Taipei 11677, Taiwan
‡
*
S Supporting Information
ABSTRACT: A complementary study including design of
mechanistic probes, biochemical assays, model analysis, and liquid
chromatography coupled mass spectrometry was conducted to
establish the reaction mechanism for a nonheme iron enzyme
catalyzed (−)-podophyllotoxin formation. Our results indicate that
the originally proposed hydroxylated intermediate is unlikely to be
involved in this reaction. Instead, the formation of benzylic radical/
carbocation intermediate can be utilized to trigger the C−C bond
formation to construct the C-ring of (−)-podophyllotoxin.
nzyme catalyzed oxidative cyclization through stereo- and
regio-specific C−H bond activation represents an effective
from the plant Podophyllum, which is followed by chemical
15
E
elaboration to produce etoposide. Recently, the biosynthetic
way to construct molecule complexity in natural product
biosynthesis as found in amaryllidaceae alkaloids, etoposide,
matacycloprodigiosin, menaquinone, morphine, nogalamycin,
pathway of (−)-podophyllotoxin has been established using a
4
transcriptome mining approach. In this pathway, 2-ODD, a
member of the mononuclear nonheme iron and 2-oxoglutarate
(Fe/2OG) enzyme family, is found to catalyze a novel
oxidative cyclization (Figure 1).
1
−8
premarineosin, streptide, and streptorubin.
Proteins con-
taining a cofactor or a transition metal center, such as
mononuclear nonheme iron, cytochrome P-450, Rieske-type
cluster, or [4Fe−4S] and S-adenosylmethionine (SAM), are
The Fe/2OG enzyme family is composed of a large number
of members and catalyzes versatile reactions to regulate
16,17
9
−12
primary and secondary metabolite production.
To the
known to catalyze this type of transformation.
From a
best of our knowledge, compared to other reaction types, such
as hydroxylation, desaturation, halogenation, epoxidation, and
mechanistic point of view, despite the diverse substrate
structures, a consensus pathway involving substrate radical
generation followed by C−C bond formation has been
9
,16,17
endoperoxdation,
oxidative cyclization via a C−C bond
9
−12
formation catalyzed by 2-ODD represents an unexplored
reaction type in Fe/2OG enzyme catalysis. Elucidating the 2-
ODD catalyzed reaction pathway and investigating the
corresponding mechanism will provide insight into under-
standing this unique transformation.
suggested.
Although a few possible pathways for the C−
C bond formation have been proposed, their mechanisms
remain to be further explored. Herein, we investigate the
plausible mechanism of a nonheme iron enzyme, 2-
oxoglutarate/Fe(II)-dependent dioxygenase (2-ODD), which
catalyzes the oxidative cyclization for the core structure in
constructing (−)-podophyllotoxin, an important precursor in
producing etoposide (Figure 1).
In the majority of Fe/2OG enzyme catalyzed reactions, a
consensus step involving hydrogen atom transfer (HAT) by
the Fe(IV)-oxo species to form the substrate radical has been
17,18
repeatedly utilized.
The reaction is then followed by
Etoposide is listed on the World Health Organization list of
diverse but well-controlled reaction outcomes. For example, in
hydroxylation, subsequent to the HAT step, the substrate
radical reacts with the Fe(III)−OH intermediate to form the
C−O bond and generates the Fe(II) to complete the catalysis.
essential medicine and is used in chemotherapy regimens for
13,14
various malignancies.
Currently, production of etoposide
requires isolation of its alycone, (−)-podophyllotoxin (1),
1
9
This step is also known as OH group rebound. Recently,
discovery of Fe/2OG enzyme catalyzed endoperoxide and
olefin installation suggests that an alternative pathway
involving a radical or a cation species can be used to trigger
2
0,21
CC or C−O bond formation.
Therefore, in the 2-ODD
catalyzed reaction, a few different pathways can be envisioned
Received: November 16, 2018
Figure 1. An Fe/2OG enzyme, 2-ODD, catalyzes oxidative cyclization
found in the etoposide biosynthetic pathway.
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b03670
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
subsequent to the substrate radical formation to construct the
designated C−C bond (Scheme 1).
depicted. In these pathways, the reaction can proceed through
radical induced cyclization followed by a second HAT
(
pathway III) or cation induced Friedel−Crafts-type cycliza-
tion accompanied by proton removal (pathway IV) to
complete the reaction. In order to elucidate the above-
mentioned pathways, we designed mechanistic probes (6−9,
Scheme 1) to examine the 2-ODD reaction in vitro and
establish the plausible 2-ODD reaction mechanism.
Scheme 1. (Top) Proposed Reaction Pathways to Account
for 2-ODD Catalyzed Cyclization; (Bottom) Mechanistic
Probes (6−9) Used in This Study
a
To study the 2-ODD catalyzed reaction, N-terminal hexa-
histidine tagged 2-ODD was heterologously expressed and
purified (see Supporting Information for details). The
synthesis of mechanistic probes, including yatein and
deoxypodophyllotoxin analogs, started with β-piperonyl-γ-
butyrolactone (3-(3,4-methylenedioxybenzyl)butyrolactone,
1
1) as a common precursor. Lactone 11 was prepared from
22−24
piperonal and dimethyl succinate by literature procedures.
Subjecting 11 to the reaction with lithium diisopropylamide
LDA) at −78 °C followed by alkylation of the resulting
butyrolactone enolate with benzyl bromide or benzyl-α,α-D
(
2
bromide afforded the yatein analog 6 or its 7′,7′-D analog
2
2
3−27
7
.
The reaction of the corresponding enolate with
benzaldehyde gave the diastereomeric mixture of 7′-hydroxy-
23,28,29
yatein analog 9.
Treatment of 9 with trifluoroacetic acid
promoted the intramolecular Friedel−Crafts-type ring closure
to form the epimeric mixtures of deoxypodophyllotoxin analog
23,28,30
1
0 (Scheme 2A).
To prepare 8, the reaction of 11 in
a
Scheme 2. Preparation of 6−10 Used in This Study
a
Tested compounds 6−8 are a pair of enantiomers, and 9 and 10 are
mixtures of racemic diastereomers.
sodium deuteroxide solution (NaOD in D O) furnished α,α-
2
dideuteration with concomitant saponification. Upon relacto-
nization in acidic condition, 12 was obtained in a good yield
with 90% D-incorporation. Subsequent α-benzylation provided
a
In pathway I, a quinone methide (3) serves as the key intermediate.
An alternative desaturated intermediate can be utilized in pathway II.
In pathways III and IV, a benzylic radical (2) or carbocation (5) is
used to trigger C−C bond formation, respectively.
8
with 85% D-retention (Scheme 2B). It is worth mentioning
that all the compounds were prepared as racemic forms for
subsequent studies.
Having 2-ODD, substrate analogs, and the proposed product
in hand, we first incubated 2-ODD with Fe(II), 2OG, substrate
In pathway I, subsequent to substrate radical (2) formation
and OH rebound at the benzylic position, C−C bond
formation is triggered via the quinone methide intermediate
analog 6, and O , with the molar ratio of 2-ODD/Fe(II)/6/
2
2OG being 1.2:1:5:10 and with a final concentration of 250
μM of 6. Compared to the product standard (10), liquid
chromatography coupled mass spectrometry (LC-MS) analysis
of the 2-ODD reaction yielded a peak with the same retention
time and an identical m/z value to that of 10. Additionally, a
peak with the retention time and m/z value that matched the
hydroxylated standard (9) was also detected (Figure 2a). In
(3), which is formed by the para-methoxy group facilitated
hydroxyl group removal. Aromatization via deprotonation
4
completes the reaction to produce 1. Alternatively,
dehydration can also occur through a pathway involving C9-
proton removal of intermediate (4). The reaction then
proceeds analogously to form the C−C bond (pathway II).
In this pathway, the acidity of C -proton is increased by the
the absence of 2OG or O (data not shown), neither substrate
9
2
adjacent carbonyl group and does not involve a quinone
methide intermediate. A third and fourth pathway involving a
radical (2) or a carbocation (5) as the key species can also be
consumption nor product formation could be observed. These
results demonstrate that 2-ODD can catalyze O and 2OG-
2
dependent cyclization using 6 as the substrate. Furthermore,
B
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Letter
31−36
ranges from 20 to 100.
In some cases, the decrease of
HAT efficiency when the deuterium analog is used results in
reduction of substrate consumption and product formation.
Examples include CarC, clavaminate synthase, CytC3, IsnB,
31−33,35,36
and TauD.
Alternatively, in other cases, due to
deuterium incorporation at the designated HAT site, the
Fe(IV)O species now attacks an alternative C−H bond and
prompts a non-native reaction outcome. Thus, the substrate
consumption will not be substantially decreased and alternate
reaction product(s) can be detected. In NapI, SyrB2, and VioC
studies, when deuterated analogs are used, the reactions are
20,37
redirected to non-native hydroxylation.
Compared with
the results obtained using 6 as the substrate, we observe a
similar level of substrate consumption (∼2/3 of substrate
consumption of 6, Figure S20), no cyclized product formation,
and alternative hydroxylation when 7 is used indicating that
the HAT site is likely at the benzylic (7′) position during the
regular reaction. Next, to evaluate the involvement of C −H
9
during the enzyme reaction, 8 was tested with 2-ODD. If C -
9
proton removal occurs during the reaction, we anticipate to
observe, at least partially, deuterium washout during the
reaction when analog 8 is used. As shown in Figure 2c, LC-MS
analysis reveals exclusive deuterium retention in both cyclized
and hydroxylated products (m/z 334.1 → 332.1 and 350.1)
and the ratio of the cyclized product to the hydroxylated
species is not perturbed. This observation suggests that C −H
9
is unlikely to participate in 2-ODD reaction and disfavors
pathway II.
Even though formation of 10 using 6 disfavors the pathway
involving the quinone methide intermediate, due to lack of 2-
ODD structural information, one cannot completely rule out
the possibility of OH group removal with the aid of potential
protein−substrate interaction. To examine the possible role of
the hydroxylated product 9 during 2-ODD reaction, 9 was
incubated with 2-ODD under both anaerobic and aerobic
conditions with a ratio of 9 to 2-ODD of ∼10 to 1. Under
these conditions, no detectable cyclized product 10 could be
observed (Figure 2d). Although we cannot exclude the
possibility that 9 cannot enter the active site of 2-ODD due
to a conformational change during the reaction, our
observation implies that 9 is likely the off-pathway product
and not involved in cyclization. To gain insight into the
possible intermediate and distinguish radical or cation-induced
cyclization during 2-ODD catalysis, nonenzymatic model
studies were carried out using 9 (see Supporting Information
for experimental details). As summarized in Scheme 2A, when
9 was treated with trifluoroacetic acid, 10 was produced as
epimeric mixtures with a ratio of ∼1/1. This reaction most
likely proceeded through trifluoroacetic acid promoted intra-
molecular Friedel−Crafts ring closure via a carbocation
intermediate. Although the normal 2-ODD reaction may be
different from the model study where epimeric mixtures of 10
were obtained, this observation provides a correlation between
carbocation intermediate and cyclization and suggests a
possibility of utilizing a carbocation as the key intermediate
to trigger cyclization.
Figure 2. LC-MS chromatograms of 2-ODD catalyzed reaction using
6
−8 as substrates. (a) Production of 9 and 10 at the expense of 6, O ,
2
and 2OG; (b) alternative hydroxylated product formation when 7 was
2
2
tested; (c) production of H-9 and H-10 when using 8 as the
substrate; (d) no detectable 10 formation under aerobic or anaerobic
conditions when 9 was used. All analytes are detected as their
corresponding sodium ion adduct form using selected ion monitoring.
due to the lack of a para-methoxy group, which is proposed to
trigger elimination of the hydroxyl group (most likely at its
protonated form), 6 is less likely to form the corresponding
quinone methide intermediate as proposed in pathway I. Thus,
production of cyclized product (10) using 6 where the para-
methoxy group is replaced by a proton implies that cyclization
is unlikely to proceed via pathway I.
Next, to identify the HAT site, analog 7 was tested. Different
from the results obtained using 6, although 7 was consumed at
a similar level (see Figure S20), no obvious cyclized product
(m/z 335.1 → 333.1, 332.1, or 331.1 (−2H, −D−H, −2D))
could be observed. Instead, a peak with the m/z value
corresponding to hydroxylation with retention of two
deuterons (m/z 335.1 → 351.1) was detected (Figure 2b).
The obvious retention time difference of this product to 9
along with both deuterons being retained on the tentative
hydroxylated product suggest that the reaction most likely
occurs at a carbon other than the deuterated position (C7′)
when 7 was used. We reason that observation of alternative
hydroxylation and retardation of cyclization is probably driven
by the intrinsic hydrogen/deuterium (H/D) isotope effect
related to the HAT step. In the majority of characterized Fe/
In summary, combining mechanistic probe design, model
studies, in vitro biochemical assays, and LC-MS analysis of 2-
ODD catalyzed reactions, we explored the plausible reaction
pathways of 2-ODD catalyzed oxidative cyclization. On the
basis of these results, the 2-ODD catalyzed reaction initiates
from the benzylic hydrogen atom abstraction of the substrate.
The reaction is likely followed by benzylic carbocation
2
OG enzymes, when the designated HAT hydrogen atom is
replaced by deuterium, the measured H/D isotope effect
C
DOI: 10.1021/acs.orglett.8b03670
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
formation which is used to trigger C−C bond installation. The
aromatization via deprotonation and reformation of Fe(II)
species completes the reaction. It is also worth noting that
instead of a stepwise mechanism as shown in pathway iv,
Scheme 1, the reaction may proceed through a concerted
mechanism. Formation of the carbocation species requires
ejection of a single electron from the substrate radical to the
Fe(III)−OH species. Experiments to investigate the timing
and the controlling factors that direct the reaction outcome
from canonical hydroxylation into C−C bond installation are
in progress. Alternatively, it is also possible that the use of the
analog has triggered an alternative pathway, unlike the native
pathway, leading to formation of the cyclized product. In
addition, our approach using regioselective deuteron incorpo-
rated analogs coupled with LC-MS analysis provides a simple
and straightforward method to investigate reaction mecha-
nisms that can be applied to other Fe/2OG enzyme catalyzed
reactions.
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ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b03670.
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AUTHOR INFORMATION
Corresponding Authors
■
*
*
2
(
E-mail: wchang6@ncsu.edu (W.-c.C.).
E-mail: tcchien@ntnu.edu.tw (T.-C.C.).
ORCID
enzymes involved in the construction of carbon skeletons of cofactors
and natural products. Nat. Prod. Rep. 2018, 35, 660−94.
(13) Gordaliza, M.; Garcia, P. A.; Miguel del Corral, J. M.; Castro,
M. A.; Gomez-Zurita, M. A. Podophyllotoxin: distribution, sources,
Wei-chen Chang: 0000-0002-2341-9846
Author Contributions
applications and new cytotoxic derivatives. Toxicon 2004, 44, 441−59.
(14) Stahelin, H. F.; von Wartburg, A. The chemical and biological
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
route from podophyllotoxin glucoside to etoposide: ninth Cain
memorial Award lecture. Cancer Res. 1991, 51, 5−15.
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Notes
The authors declare no competing financial interest.
(16) Islam, M. S.; Leissing, T. M.; Chowdhury, R.; Hopkinson, R. J.;
Schofield, C. J. 2-Oxoglutarate-Dependent Oxygenases. Annu. Rev.
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ACKNOWLEDGMENTS
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This work was supported by North Carolina State University,
National Taiwan Normal University, and research grant
MOST 107-2113-M-003-002 from Ministry of Science and
Technology, Taiwan. We thank Dr. Bo Zhang at Penn State
University, Prof. Yisong Guo at Carnegie Mellon University,
and Prof. Christian Melander at University of Notre Dame for
helpful discussion.
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