Elucidating the Reaction Pathway of Decarboxylation-Assisted Olefination Catalyzed by a Mononuclear Non-Heme Iron Enzyme

Article abstract of DOI:10.1021/jacs.8b10077

Installation of olefins into molecules is a key transformation in organic synthesis. The recently discovered decarboxylation-assisted olefination in the biosynthesis of rhabduscin by a mononuclear non-heme iron enzyme (P.IsnB) represents a novel approach in olefin construction. This method is commonly employed in natural product biosynthesis. Herein, we demonstrate that a ferryl intermediate is used for C-H activation at the benzylic position of the substrate. We further establish that P.IsnB reactivity can be switched from olefination to hydroxylation using electron-withdrawing groups appended on the phenyl moiety of the analogues. These experimental observations imply that a pathway involving an initial C-H activation followed by a benzylic carbocation species or by electron transfer coupled β-scission is likely utilized to complete C=C bond formation.

Full text of DOI:10.1021/jacs.8b10077

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Communication
Elucidating reaction pathway of decarboxylation-assisted
olefination catalyzed by a mononuclear non-heme iron enzyme
Cheng-Ping Yu, Yijie Tang, Lide Cha, Sergey Milikisiyants, Tatyana
I. Smirnova, Alex I. Smirnov, Yisong Guo, and Wei-chen Chang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10077 • Publication Date (Web): 30 Oct 2018
Downloaded from http://pubs.acs.org on October 31, 2018
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Elucidating reaction pathway of decarboxylation-assisted
olefination catalyzed by a mononuclear non-heme iron
enzyme
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Cheng-Ping Yu,ǁ,† Yijie Tang,ǁ,‡ Lide Cha,† Sergey Milikisiyants,† Tatyana I. Smirnova,† Alex I.
Smirnov,†,* Yisong Guo,‡,* Wei-chen Chang†,*
†Department of Chemistry, North Carolina State University, Raleigh, NC 27695. ‡Department of Chemistry, Carnegie
Mellon University, Pittsburgh, PA 15213.
Supporting Information Placeholder
will shed lights on the factors that direct and govern the
reaction outcomes. Herein, we employ a complementary
approach including mechanistic probe design, transient
kinetics, CW EPR, 2D pulsed EPR hyperfine sublevel
correlation spectroscopy (HYSCORE), Mössbauer and LC-
UV/MS to elucidate the plausible pathways of this novel
reaction. Different from the originally proposed hydroxylation
and CO2 elimination pathway,12, 13 our results suggest that an
alternate pathway involving a benzylic carbocation or electron
transfer coupled decarboxylation is likely to be utilized to
trigger the olefination (Scheme 1).
ABSTRACT: Installation of olefins into molecule is a key
transformation in organic synthesis. The recently discovered
decarboxylation-assisted olefination in the biosynthesis of
rhabduscin by a mononuclear non-heme iron enzyme (P.IsnB)
represents a novel approach in olefin construction. This method is
commonly employed in natural product biosynthesis. Herein, we
demonstrate that a ferryl intermediate is used for C-H activation at
the benzylic position of the substrate. We further establish that
P.IsnB reactivity can be switched from olefination to
hydroxylation using electron-withdrawing groups appended on
the phenyl moiety of the analogs. These experimental
observations imply that a pathway involving an initial C-H
activation followed by a benzylic carbocation species or by
electron transfer coupled β-scission is likely utilized to complete
C=C bond formation.
R
R
X
a
H
-
R
H
CO2
H
O2
O
Asp
/Glu
R
O
FeIV
-
succ
X= OH,
Cl, Br, I
and etc.
O
O
(succ)
FeII
His
CO2
(2OG)
O
FeIII
O
His
succ, FeII
O
O
CO2
O
b
NC
CN
HN
O
O
O
OH
OH
Members of the non-heme iron enzyme family are known to
catalyze oxidative transformations responsible for both
primary and secondary metabolite production.1 Examples
include hydroxylation, halogenation, epoxidation, and
desaturation.2-4 Iron(II) and 2-oxoglutarate dependent
(Fe/2OG) enzymes, a subclass of this enzyme family, carry
out reactions by using a reactive ferryl (Fe(IV)=O) as the key
intermedaite.3, 5 In the last decade, the Fe(IV)=O species along
with intermediates involved in hydroxylation and halogenation
have been extensively studied (Figure 1a).6-8 In contrast,
reaction mechanisms for other biologically and chemically
intriguing transformations, such as decarboxylation-assisted
olefination, have not been thoroughly investigated.
NC
HO
IsnB
HO
2
1
rhabduscin
Figure 1. a) Proposed pathway of Fe/2OG enzyme catalyzed
reactions. b) P.IsnB catalyzed decarboxylation-assisted
olefination found in rhabduscin biosynthesis.
Scheme 1. Possible pathways account for P.IsnB catalyzed
olefination; products and probes employed in this study (1-9).
+H+
OH
O
OH2 O
-CO2, -OH2
i
H
O
O
R
R
O
O
R
NC
NC
FeII
R
O
R
O
NC
NC
HO
O
OH
CN
O
FeIII
FeIV
FeIII
OOR
R
O
ii
NC
-CO2
R
O
HO
FeII
A majority of the identified biosynthetic pathways leading
to vinyl isonitriles, such as found in hapalindoles, ambiguine,
and rhabduscin, utilize a conserved approach to produce the
vinyl isonitrile group.9,10 Among these pathways, Fe/2OG
enzymes install an olefin group via loss of CO2 and hydride
removal.11 Compared to other reactions types, this reaction
represents a unique approach of constructing olefins. Recently,
P.IsnB, an Fe/2OG enzyme, was reported to catalyze the
installation of an olefin group in the isonitrile-containing
tyrosine 1. Subsequently, the vinyl isonitrile intermediate 2
would then be subjected to further modifications to generate
rhabduscin (Figure 1b).10 Elucidating the mechanism will
provide fundamental understanding of this transformation, and
NC
O
NC
OH
O
O
2
6
7
X = OH,
1
3
4
X = OH,
O
H,
F,
H,
F,
NC
NC
X
9
8
CF3,
5
X
CF3,
To study P.IsnB-catalyzed reaction, substrate (1), its
deuterium labelled analog at the benzylic position (D-1), and
the product standard (2) were synthesized (see SI for
procedures). P.IsnB was heterologously expressed in E. coli
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and purified (Figure S23). Activity of P.IsnB was tested by
stopped-flow absorption spectroscopy (SF-Abs) to monitor the
reaction after rapid mixing of the anaerobic
elucidate the interactions between the substrate and the non-
heme iron centers.15-18 HYSCORE Q-band (34 GHz)
spectrum of the NO treated P.IsnB•Fe(II)•2OG•D-1 complex
was obtained at a magnetic field corresponding to the
maximum peak intensity of the echo detected field-swept
EPR spectrum (effective g = 3.97, magnetic field of 6,094 G,
Figure 3 and S30) at T = 3.1 K. This field position
corresponds to the g  4 resonance in X-band (9 GHz) CW
EPR spectrum (Figure S29) attributed to the substrate bound
P.IsnB complex. The most pronounced features observed in
the (+, +) quadrant HYSCORE spectrum are due to deuterons
at the benzylic position located in relative proximity to the Fe
center (Figure 3A). While the splitting along the main
diagonal is caused by the quadrupolar interaction, an
extension along the anti-diagonal is due to electron-nuclear
hyperfine interaction of < 1 MHz in amplitude. Such small
coupling is indicative of Fe-2H interspin distances longer than
the typical bond length, thus, justifying point-dipole
approximation for the analysis. Figure 3B shows the optimal
simulation with the following hyperfine interaction
parameters: electron-nuclear hyperfine tensor A = [0.9±0.3, -
0.45±0.15, -0.45±0.15] MHz, quadrupolar parameter K =
0.05±0.05 MHz and quadrupolar asymmetry parameter η =
0.3±0.3. By using point-dipole approximation, A can be
converted to the distance between the spin center of the Fe-
NO unit and deuterons of D-1, yielding r = 3.78±0.5Å. This
value is comparable to the distances revealed in other Fe/2OG
enzymes by HYSCORE and ESEEM techniques16-18 and
provides an additional support of the benzylic C-H activation
during P.IsnB catalysis.
1
2
3
4
5
6
7
8
a
P.IsnB•Fe(II)•2OG•substrate (1 or D-1) complex with
oxygenated buffer at 5 oC. Similar to other characterized
Fe/2OG enzymes, an Fe(II)-2OG metal-to-ligand charge
transfer (MLCT) band centered at 512 nm was observed in the
P.IsnB quaternary complex (Figure S25), which also exhibited
distinct absorption shoulders at 465 and 570 nm.14 When 1
was used, the MLCT band depleted within 0.07 s after O2
mixing and a rapid increase of absorption features at ~ 330 nm
was detected. In contrast, the use of D-1 resulted in an
additional rise of an optical feature at ~ 440 nm that reached a
maximum at ~ 0.05 s and then decayed (Figure 2a, b). In both
cases, the absorption change below 350 nm cannot be
unambiguously assigned to the kinetics of a single species, and
may partially result from product absorption (Figure S26,
ε330nm ~ 1600 M-1cm-1 for 2). Thus, the absorption feature at
440 nm was used to follow the Fe(IV)=O kinetics, while the
identification of the Fe(IV)=O species was verified by
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Mössbauer spectroscopy.
A
three-step kinetic model
(P.IsnB•Fe(II)•2OG•substrate
+
O2 Fe(IV)=O


P.IsnB•Fe(II)•product  P.IsnB•Fe(II)•2OG) reproduced the
time-dependent changes of the 440 and 512 nm absorption
generated in the course of reactions with 1 and D-1 (see SI for
discussions, Figures S27, S28). The decay rate constant for the
P.IsnB quaternary complex, which also represents the rate of
Fe(IV)=O formation is ~ 65 mM-1s-1 for both reactions (1 and
D-1). However, the Fe(IV)=O decay rate constants, k2,
differed significantly. For D-1, k2 is ~ 3 s-1, but for 1, since the
Fe(IV)=O species could not be observed in SF-Abs (the
corresponding 440 nm kinetic trace mainly reflects the decay
of the Fe(II)-2OG MLCT band, Figure 2b) or Mössbauer
experiments (see below), we estimated k2 is larger than 300 s-1.
Thus, the H/D kinetic isotope effect (KIE) of the Fe(IV)=O
decay reaches > 100, which implies the initial C-H activation
site is at the benzylic position of the substrate.
Figure 3. Q-band (34 GHz) HYSCORE spectrum of the
NO•P.IsnB•Fe(II)•2OG•D-1 obtained at magnetic field
corresponding to g ≈ 3.97 resonance in the echo detected field-
swept EPR spectrum and T=3.1 K. (A) The spectral region
showing features resulted from 2H and FeNO interactions, and (B)
the corresponding spectral simulations.
Freeze-quench (FQ) coupled Mössbauer spectroscopy
experiments were also performed (Figure S31 and SI for
discussion). In short, three time-dependent iron species were
observed after rapid mixing of IsnB•Fe(II)•2OG•1 (or D-1)
with oxygenated buffer at various time points: (1) a
quadrupole doublet representing the P.IsnB quaternary
complex (δ = 1.18 mm/s, |ΔEQ| = 2.76 mm/s), (2) another
doublet representing the Fe(IV)=O species (δ = 0.29 mm/s,
|ΔEQ| = 1.06 mm/s), and (3) a third doublet representing the
P.IsnB-Fe(II)-product and/or the P.IsnB-Fe(II)-2OG complex
(δ = 1.28 mm/s, |ΔEQ| = 2.84 mm/s). The Fe(IV)=O doublet
was only observed in the reaction when D-1 was used, but not
with 1 even at quench time of 0.02 s. The failure to detect
Fe(IV)=O species in the reaction with 1 is likely due to the fast
decay of such an intermediate. In addition, Mössbauer data
suggest only ~ 50% of the total P.IsnB quaternary complex is
active. Furthermore, ~ 20-25% of isonitrile-Fe(II) complex
Figure 2.. SF-Abs results of P.IsnB reactions. (a) Change in
absorbance at the indicated reaction times after mixing the
IsnB•Fe(II)•2OG•D-1 with O2. The spectra were obtained by
subtracting the spectrum at 0.002 s. (b) Kinetic traces at 440 nm
used to follow the kinetics of the Fe(IV)=O intermediate in the
reaction using 1 (blue), D-1 (red) and 5 (green). Simulations are
shown in black.
Additional evidence for the substrate position relative to
the iron center was provided by EPR measurement where
nitric oxide (NO) was used as the O2 surrogate. Addition of
NO to Fe(II) centers in non-heme enzymes typically yields
{FeNO}7 species which the EPR signal could be used to
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were observed, which is likely generated through the direct
Meanwhile, a plausible hydroxylated product with m/z value
of +16 from the substrates (3, 4, and 5) was detected, but the
corresponding species was not observed when 1 was used
(Fig. 4b). Next, the hydroxylated product standard (9) of 3 was
synthesized. LC-MS revealed that 9 has identical elution time
as that of reaction product when 3 was used. Additionally, the
amount of hydroxylated product correlates with electron
withdrawing property of the analogs tested (5 > 4 > 3, (CF3 >
F >H)) (Figure 4b). Two possible scenarios can account for
this observation: (1) due to non-optimum substrate
positioning, the hydroxylated intermediate dissociates from
the active site prior to decarboxylation, or (2) the electron
withdrawing property of the para-substituent redirects the
reaction pathway from olefination to hydroxylation. To
distinguish these possibilities, 9 was incubated with P.IsnB. If
the hydroxylation is the on-pathway intermediate, by
incubating P.IsnB with 9, one should be able to observe
formation of the olefin product 6. However, such a product
was not detected during 20 min of incubation of 9 with P.IsnB
under anaerobic condition (Figure 4c). Thus, hydroxylation is
binding of the isonitrile group to the iron center (Table S2).13b
1
2
3
4
5
6
7
8
LC-UV/MS was used to monitor the P.IsnB reaction. The
product, which has an identical retention time to that of the
synthetic standard 2 was observed in the reaction mixture
containing P.IsnB, O2, 2OG and 1 (Figure 4a). Combined with
SF-Abs and Mössbauer observations, these results suggest that
P.IsnB utilizes an Fe(IV)-oxo species as the key intermediate
to trigger benzylic C-H bond cleavage and installation of a
C=C bond. However, the governing factors that direct reaction
outcome to olefination remain to be elucidated. Subsequent to
C-H activation and substrate radical formation, the reaction
may proceed through a pathway involving a hydroxylated
intermediate or a substrate radical/cation to trigger β-scission
and complete the olefin installation (Scheme 1). To distinguish
these pathways, analogs with a H, F or CF3 (3, 4 or 5, Scheme
1) at the para position were synthesized and subjected to the
P.IsnB-catalyzed reaction. SF-Abs results revealed that all the
analogs showed a similar Fe(II)-2OG MLCT band decay
kinetics when reacting with O2 (Figure S27, ranging between
40-50 mM-1s-1), resembling that of the native substrate (1).
The decay rate constants of the Fe(IV)=O species were
estimated to be 19, 10, and 3 s-1 for 3, 4, and 5, respectively
(Figures 2, S27). Although these analogs show decreased
Fe(IV)=O decay rate than that of the native substrate, the
similar Fe(IV)=O formation rate indicates these analogs are
substrates for P.IsnB. Furthermore, CW EPR measurement of
NO•P.IsnB•Fe(II)•2OG•5 (or 1) showed no obvious
perturbation (Figure S29), thus implying a similar binding
configuration of 5 and 1.
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unlikely to serve as an intermediate. Furthermore,
a
competition experiment revealed that 9 can inhibit the
formation of 2 (Figure S24), implying that 9 can bind to
P.IsnB. Taken together, these results suggest that the pathway
utilizing a hydroxylated intermediate is unlikely to operate.
Alternatively, a pathway involving a carbocation or electron
transfer coupled decarboxylation may be utilized. When
analogs bearing an electron withdrawing group are used, the
instability of the benzylic cation or the transient species
involved in the later pathway alter the reaction pathway and
the incipient substrate radical is quenched by the Fe(III)-OH
species to produce the hydroxylated product. The 18O labelling
experiment using 18OH2 revealed no 18O incorporation into 9
(Figure S24), thus suggesting OH rebound mechanism for
hydroxylation.
In addition to Fe/2OG enzymes, decarboxylation-assisted
olefination approach has been reported in other enzymatic
transformations where various cofactors, such as thiolate-
heme and [4Fe-4S]-SAM, are involved.19-22 Herein, our results
provide experimental evidence to support a pathway utilizing
C-H activation and followed by a carbocation or by electron
transfer coupled β-scission to trigger olefin installation.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental methods, additional comments on SF-Abs and
Mössbauer analyses, Figures S1-31, Table S1, S2, and references.
AUTHOR INFORMATION
Corresponding Author
Figure 4. a) LC-UV chromatograms of P.IsnB catalyzed
conversion of 1 to 2 in the presence of O2 and 2OG detected at
*aismirno@ncsu.edu
*ysguo@andrew.cmu.edu
*wchang6@ncsu.edu
266. b) LC-MS chromatograms of P.IsnB catalyzed hydroxylation
when substrate analogs were used. The bottom trace represents
the product standard 9. c) LC-UV chromatograms of reacting 9
with P.IsnB anaerobically and the product standard 6 detected at
Author Contributions
266
.
ǁ Cheng-Ping Yu and Yijie Tang contributed equally.
LC-UV/MS analysis revealed that a substantial decrease of
the olefin products (6 and 7) when 3 and 4 were used (Figure
S24), while 5 resulted in no detectable product (8) formation.
Notes
The authors declare no competing financial interests.
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Endogenous Cysteine - Spectroscopic Studies of Site-Specific Cys --> Ser
Mutated Enzymes. Biochemistry 1992, 31, 4602-12.
ACKNOWLEDGMENT
1
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3
4
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7
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16. Muthukumaran, R. B.; Grzyska, P. K.; Hausinger, R. P.; McCracken,
J. Probing the Iron-substrate Orientation for Taurine/alpha-ketoglutarate
dioxygenase Using Deuterium Electron Spin Echo Envelope Modulation
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17. Casey, T. M.; Grzyska, P. K.; Hausinger, R. P.; McCracken, J.
Measuring the Orientation of Taurine in the Active Site of the Non-heme
Fe(II)/alpha-ketoglutarate-dependent Taurine Hydroxylase (TauD) Using
Electron spin Echo Envelope Modulation (ESEEM) spectroscopy. J. Phys.
Chem. B 2013, 117, 10384-94.
18. Martinie, R. J.; Livada, J.; Chang, W.-c.; Green, M. T.; Krebs, C.;
Bollinger, J. M., Jr.; Silakov, A. Experimental Correlation of Substrate
Position with Reaction Outcome in the Aliphatic Halogenase, SyrB2. J.
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19. Grant, J. L.; Hsieh, C. H.; Makris, T. M. Decarboxylation of Fatty
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This work was supported by North Carolina State University,
Carnegie Mellon University, and grants from the National
Institutes of Health (GM127588 to W.-c. C, and Y. G.).
HYSCORE experiments were supported by U.S. DOE Contract
DE-FG02-02ER15354 to A.I.S. EPR instrumentation was
supported by NIH RR023614 and NSF CHE-0840501. We thank
Dr. Peter Thompson for NMR carried out at NCSU METRIC
facility.
9
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FeII
O
HO
FeIII
O
FeIV
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R
O
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O
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O
OR
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R
O
NC
R
O
NC
NC
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