Mechanistic Investigation of Biocatalytic Heme Carbenoid Si?H Insertions

Article abstract of DOI:10.1002/cctc.201801755

Recent studies reported the development of biocatalytic heme carbenoid Si?H insertions for the selective formation of carbon-silicon bonds, but many mechanistic questions remain unaddressed. To this end, a DFT mechanistic investigation was performed which reveals an Fe^II-based concerted hydride transfer mechanism with early transition state feature. The results from these computational analyses are consistent with experimental data of radical trapping, kinetic isotope effects, and structure-reactivity data using engineered variants of hemoproteins. Detailed geometric and electronic profiles along the heme catalyzed Si?H insertion pathways were provided to help understand the origin of experimental reactivity trends. Quantitative relationships between reaction barriers and some properties such as charge transfer from substrate to heme carbene and Si?H bond length change from reactant to transition state were found. Results suggest catalyst modifications to facilitate the charge transfer from the silane substrate to the carbene, which was determined to be a major electronic driving force of this reaction, should enable the development of improved biocatalysts for Si?H carbene insertion reactions.

Full text of DOI:10.1002/cctc.201801755

Accepted Article
Title: Mechanistic Investigation of Biocatalytic Heme Carbenoid Si-H
Insertions
Authors: Rahul Khade, Ajay L. Chandgude, Rudi Fasan, and Yong
Zhang
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Mechanistic Investigation of Biocatalytic Heme Carbenoid Si-H
Insertions
Rahul L. Khade,a) Ajay L. Chandgude,b) Rudi Fasan,* b) and Yong Zhang* a)
Abstract: Recent studies reported the development of biocatalytic
heme carbenoid Si-H insertions for the selective formation of carbon-
silicon bonds, but many mechanistic questions remain unaddressed.
To this end, a DFT mechanistic investigation was performed to show
an FeII-based concerted hydride transfer mechanism with early
transition state feature. The results from these computational
analyses are consistent with experimental data of radical trapping,
kinetic isotope effects, and structure-reactivity data using engineered
variants of myoglobin (this work) and cytochrome c (previous work).
Detailed geometric and electronic profiles along the heme catalyzed
Si-H insertion pathways were provided to help understand the origin
of experimental reactivity trends. Quantitative relationships between
reaction barriers and some properties such as charge transfer from
substrate to heme carbene and Si-H bond length change from
reactant to transition state were found. Results suggest catalyst
modifications to facilitate the charge transfer from the silane
substrate to the carbene, which was determined to be a major
electronic driving force of this reaction, should enable the
development of improved biocatalysts for Si-H carbene insertion
reactions.
for accomplishing this type of transformations in aqueous
solvents and under mild reaction conditions were reported using
engineered variants of cytochrome c[1c] and myoglobin.[1h] These
systems offer several attractive features for sustainable
chemistry due to the accessibility and biocompatibility of these
iron-based metalloprotein biocatalysts, along with their
promising catalytic activity in these transformations (>1,500-
8,000 catalytic turnovers).[1c, 1h]
Despite this progress, many mechanistic questions remain
unanswered. For instance, what is the major electronic driving
force for this reaction? What is the origin of the experimentally
observed substrate and carbene reactivity trends? What kind of
roles do conformation and spin state play on the transition state
(TS)? What factors are critical to catalyst design? Computational
work on Si-H insertion is scarce[8] and detailed analyses of
geometric or electronic property changes along these metal-
carbenoid Si-H insertion pathways have been missing.
To address these mechanistic questions,
a quantum
chemical investigation of heme-carbenoid Si-H insertions was
performed here, using basically the same computational
approach in recent accurate predictions of iron porphyrin
carbenes’ (IPCs) experimental X-ray crystal structures,
Mössbauer and NMR properties, and their experimental
formation, cyclopropanation, and C-H insertion reactivity and
selectivity results,[9] see details in Experimental section.
Reproducing experimentally observed structure-activity trends
and kinetic isotope effects (KIEs), our computational analyses
provide key insights into the basic mechanism of heme-
catalyzed Si-H carbene insertion and into the impact of structural
changes at the level of the substrate, carbene, and heme
catalyst on this reactivity.
Introduction
Artificial hemoproteins have been proven useful for promoting a
broad range of carbene-mediated transformations, including
carbene insertion into X-H bonds (X = N, S, Si, C, B),[1]
cyclopropanations,[2] aldehyde olefinations,[3] and sigmatropic
rearrangements.[4] Among these reactions, the synthesis of
organosilicon compounds find important applications in the area
of material science[5] and medicinal chemistry.[6] While synthetic
methodologies for the transition metal-catalyzed carbene
insertion into Si-H bonds have been reported,[7] these protocols
require organic solvents and are typically characterized by
limited catalytic turnovers (<100). Recently, biocatalytic methods
Results and Discussion
Basic mechanism
[a]
[b]
Dr. R. L. Khade, Prof. Dr. Y. Zhang
Department of Chemistry and Chemical Biology
Stevens Institute of Technology
1 Castle Point on Hudson, Hoboken, NJ 07030 (USA)
E-mail: yong.zhang@stevens.edu
Dr. A. L. Chandgude, Prof. Dr. R. Fasan
Department of Chemistry
University of Rochester
120 Trustee Road, Rochester, NY 14627 (USA)
E-mail: rfasan@ur.rochester.edu
Since the previously reported active biocatalysts for this reaction
(i.e., cytochrome c and myoglobin) both contain a histidine-
ligated heme cofactor,[1c, 1h] [Fe(Por)(His)(C(Me)CO2Et)] was first
investigated to model the core part of these biocatalysts in the
Si-H carbene insertion reaction 1 of Scheme 1. Por is a non-
substituted porphyrin, His is modeled as 5-methylimidazole as
done previously,[9b-d] and C(Me)CO2Et is the carbene group
derived from the corresponding diazo ester reagent.[1c] This
carbene structure was also confirmed in a recent X-ray
crystallographic study.[8c] Since there is no prior computational
Supporting information for this article is given via a link at the end of
the document.
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study of effects of substrates, carbenes, and catalysts on heme
carbenoid Si-H insertions, this work focuses on the Si-H insertion
reactivities of the core part of the protein due to these effects.
for the radical mechanism, the initial broken-symmetry setup of
FeIII (S=1/2), carbene (S=-1/2), H (S=1/2) from the Si-H bond,
and the remaining substrate (S=-1/2) for the overall singlet
transition state was optimized the same as FeII-based concerted
TS(1) described above. Similarly the initial broken-symmetry
setup of FeIII (S=5/2), carbene (S=-1/2), H (S=1/2), and the
remaining substrate (S=-1/2) for the overall quintet radical-based
transition state was also optimized to the same quintet concerted
TS(1) mentioned above. In contrast with these results at singlet
and quintet levels, the triplet TS(1) has obvious radical feature
with 1.092, 0.558, and 0.377 e spin densities for Fe, the carbene
α-carbon atom, and Si, respectively. However, it has the highest
Gibbs free energy of activation, with G‡ of 12.81 kcal/mol
larger than that of the lowest energy TS(1), see Table S4.
Scheme 1. Si-H insertion pathways in reactions 1-5.
Both a concerted mechanism as proposed for other metal
c, 8b, 10]
carbenoid Si-H insertions[7b,
and a stepwise radical
mechanism reminiscent of native heme enzymatic reactions[11]
were investigated for reaction 1 to explore the basic mechanism,
with details in Supporting Information (SI) and key results
discussed here.
Comparisons of different mechanisms, conformations, and
spin states. We first studied the concerted transition state.
Because electronic state and conformation may influence the
mechanism, such effects were examined first to identify the most
favorable conformations and spin states, see SI for details.
Overall, the conformation effect was found to be small. For
example, the Gibbs free energy differences for different
conformations of TS(1) (the reaction number is in parenthesis)
(see Figure S1) are within ~ 0.3 kcal/mol, indicating a weak steric
interaction due to the long C…Si distance (~3.1 Å).
Scheme 2. Radical trap (a) and kinetic isotope effect experiments (b) for
Mb(H64V,V68A)-catalyzed Si-H carbene insertion reaction.
In previous work, an engineered active site variant of sperm
whale myoglobin, Mb(H64V,V68A), was determined to catalyze
a
Si—H
carbene
insertion
reaction
between
In contrast, the spin state effect on mechanism is much
larger. Here, we focused on both singlet and quintet transition
states because the iron-containing product is of quintet ground
state,[12] which is different from the reactant’s singlet ground state
for various IPCs.[13] These experimental ground states of
reactants and products were reproduced in our calculations.[9]
Regarding concerted TS(1), the FeII-based quintet transition
state has Fe spin density of 3.885 e and carbene’s carbon spin
density of only -0.165 e. It has significantly higher energy (G‡
of 10.27 kcal/mol) than the FeII-based closed-shell singlet (Table
S4). In contrast, the third TS spin state with S=1 is of the highest
energy, by G‡ of 12.81 kcal/mol (Table S4). These results
support the singlet TS(1) as the most favorable spin state, and
therefore there is no spin crossover from the singlet reactant.
The results obtained for the other reactions considered here
(Scheme 1) also indicated that the singlet state TS is the most
favorable transition state (see SI for details).
dimethyl(phenyl)silane (1a) and ethyl -diazoacetate (2a) with
up to 1,545 total turnovers.[1h] To probe experimentally the
occurrence of a radical mechanism in this hemoprotein-
catalyzed Si-H insertion process, the reaction was performed in
the absence and in the presence of a large excess (100 mM) of
the spin trap reagent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).
As shown in Scheme 2a, the addition of DMPO did not have a
significant impact on the yield of the Mb(H64V,V68A)-catalyzed
Si-H insertion reaction (52 ± 5% vs. 45 ± 7%), suggesting the
absence of a radical intermediate. To gain further insights into
the mechanism of this reaction, kinetic isotope effect (KIE)
experiments were performed using a deuterated form of the
dimethyl(phenyl)silane
substrate
(d-1a).
Competition
experiments with equimolar amount of 1a and d-1a yielded a
positive KIE (kH/kD) corresponding to 1.19 ± 0.05 (Scheme 2b
and Figure S4). A similar KIE value of 1.3 ± 0.2 was also
obtained by comparing the rate of parallel reactions with the
protiated vs. the deuterated silane substrate (Figure S5). These
results indicate that the cleavage of the Si—H bond is part of the
Compared with such FeII-based concerted transition states,
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rate-limiting step of the reaction. Importantly, using the concerted
TS(1), the calculated KIE at room temperature for reaction 1 and
other reactions discussed later (2 and 3) using the same catalyst
are 1.19, 1.26, and 1.31, respectively, thus showing an excellent
agreement between the computational predictions and the
experimental data obtained with the Mb variant. The KIE values
obtained for the hemoprotein-catalyzed reaction fall in the same
range of those reported for Rh- and Ir-carbenoid Si-H insertions
(1.08-1.6).[7b, 10]
Si-H insertion, these parameters were examined here to gain
further insights into the mechanism of this reaction.
A large negative charge transfer (QCT: -0.223 e) occurs from
PhMe2SiH to carbene from reactants to TS(1), as shown by the
data in Figure 1A and additional data in Figure S3 and Tables
S7 and S8. As the spin densities of the atoms for the most
favorable singlet TS(1) do not support an electron transfer
mechanism (either coupled or uncoupled with proton transfer),
the large negative charge transfer mentioned above suggests a
hydride transfer feature. This is also the largest charge transfer
in the system, indicating that it may represent a major electronic
driving force for this reaction. The charge transfer direction
shows the electrophilicity of the heme-carbene group, which is
consistent with the experimentally observed lower and higher
reactivity of electron-deficient and electron-rich silane substrates,
respectively, in biocatalytic reactions.[1c] It is also interesting to
note that while Si donates more charge (0.165 e, see data in blue
in Figure 1A) to help the hydride formation for subsequent
charge transfer, it also donates 0.040 e to the remaining part of
substrate as a result of electronegativity difference[16] between C
(2.55) and Si (1.90). Altogether, these effects result in a very
large positive charge for the Si atom at the TS, corresponding to
~1.6 e (Table 1).
Taken together, the results above suggest that the heme
carbene Si-H insertion favors an FeII-based concerted
mechanism, rather than a stepwise radical mechanism. This FeII
feature is consistent with experimental reaction studies where
the starting heme proteins were reduced to the catalytically
competent ferrous form,[1c, 2b, 2e] and experimental spectroscopic
studies using UV/Vis and NMR spectroscopies,[13b,
X-ray
14]
14-15]
crystallography,[13a,
and XANES (directly sensitive to Fe
oxidation state)[15] which show that IPC has a dominant FeII
feature (i.e. closed-shell singlet). The proposed concerted
mechanism is reminiscent of that proposed for rhodium- and
iridium-carbenoid Si-H insertions[7b, c, 8b, 10] and heme carbene
cyclopropanations.[9d]
Major electronic driving force and hydride transfer feature
.
Since charge and geometry changes along the reaction pathway
have not been reported before for any metal-catalyzed carbenoid
A
B
C
D
E
Figure 1. Key geometric parameters at TS (in red), geometry changes (in green) and charge transfers (in blue) from reactants to TS in (A) reaction 1; (B) reaction
2; (C) reaction 3; (D) reactions 4; (E) reaction 5.
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Table 1. Key Energy, Charge, and Geometry Results for Si-H Insertions
B
TS
TS
TTN
∆G‡
(kcal/
mol)
∆Go
(kcal/
QCT
(e)
QSi
(e)
RSiH
(Å)
∆RSiH
(Å)
mol)
1
2
3
4
5
2520
5010
47
9.60
8.40
11.74
10.87
11.24
–62.22
–62.05
–64.02
–76.32
–49.36
-0.223
-0.215
-0.261
-0.246
-0.260
1.573
1.569
1.616
1.594
1.591
1.534
1.531
1.551
1.542
1.547
0.043
0.040
0.060
0.051
0.056
A
Nonsynchronous bond formation and early transition state
features
.
Inspection of the geometric data suggest that the
concerted TS(1) has a nonsynchronous feature. This conclusion
is supported by a more pronounced elongation of the Fe-C bond
by 0.108 Å compared to that of the Si-H bond (0.043 Å) from
reactants to transition state, as illustrated by the data in green in
Figure 1A. In addition, the C-Si bond formation is relatively late
compared to the C-H bond formation as indicated by the longer
C-Si bond length of ~3.1 Å vs. C-H bond length of ~1.7 Å at the
transition state (see data in red in Figure 1A).
Furthermore, the small change of Si-H bond length (∆RSiH
,
~0.05 Å) and the small change (0.4) in the bond angle between
the two carbene substituents at TS(1) compared to those in the
reactants (see Table S7) suggest that TS(1) has an early
transition state character. These features indicate that the
carbene carbon in the transition state is still largely sp2-
hydridized as in the reactant, rather than exhibiting sp3 geometry
as in the product. Indeed, as illustrated in Figure 2A, the key
interactions between the carbene α-carbon and the Si-H bond
involve the carbon p orbital which has  bonding and anti-
bonding interactions with iron’s d orbital in the occupied and
unoccupied frontier molecular orbitals, together with the σ(Si-H)
bond. The p-d bonding interaction in this transition state is
similar to and originates from the p-d bonding interaction in the
IPC reactant (see Figure 2B), which further supports its early
transition state character. The presence of only one interaction
between Fe dπ orbital and the carbene α -carbon pπ orbital and
iron orbital population scheme of (dxy)2(dxz)2(dyz)2(dz2)0(dx2-y2)0
are the same as reported previously for other IPCs.[9a] These
results indicate that the unique p-d bonding pattern in IPC is
maintained in the TS investigated here and the pπ orbital of the
carbene α-carbon interacts with the Si-H bond in this TS,
suggesting an important role of this p-d bonding for catalysis.
EMO
Figure 2. Frontier metal-containing molecular orbitals of TS(1) and R1(1). (A)
from low to high are HOMO-2 and LUMO+2 orbitals with contour values of
±0.01 and 0.02 au; (B) from low to high are HOMO-5, HOMO-3, HOMO-2,
LUMO+4, LUMO+9 with contour values of ±0.04 au. The navy blue dashed
line separates the regions of occupied and unoccupied MOs. Red boxes show
p-d interaction.
Substrate and carbene effects
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We next studied the substrate and carbene substituent
effects to provide a better theoretical understanding of the
reactivity trends observed experimentally.[1c] In the cytochrome
c-catalyzed Si-H insertion reactions reported by Arnold and
coworkers, a total number of turnovers (TTN) of 2520 was
for the early TS and thus in the highest energy barrier among
these reactions. In addition, as shown in Figure 1A-C, the major
electronic driving force in all these reactions is the charge
transfer from substrate to carbene. This finding is consistent with
the beneficial effect of the EDG on the silane substrate (reaction
2) toward reducing the barrier compared to the unsubstituted
dimethyl(phenyl)silane substrate (reaction 1), as observed
experimentally.[1c] These results provide insights into the
mechanistic basis for the reactivity trends observed in the
presence of electron-rich and electron-deficient silane
substrates in biocatalytic heme carbene Si-H insertions.[1c]
measured with Ph(Me)2SiH and
a :C(Me)CO2Et carbene
(modelled in reaction 1, Scheme 1). The introduction of an
electron-donating groups (EDG) in the phenyl ring of the silane
substrate was found to enhance the yield of this reaction (e.g.,
4-acetylenyl-Ph)Me2SiH: 5010 TTN (modelled in reaction 2,
Scheme 1). On the other hand, a change of the -substituent on
the carbene from Me to Et was reported to cause a dramatic
decrease in TTN (47) with the electron-rich silane substrate 4-
acetylenyl-Ph)Me2SiH (modelled in reaction 3, Scheme 1).[1c] As
shown in Table 1 and Figure 3, the calculated barriers for the
corresponding reactions 1, 2 and 3 agree very well with the
experimentally observed reactivity trend (i.e., 2 > 1 > 3). These
results indicate that the main reactivity features observed for
these biocatalytic reactions are reproduced by the calculations,
which suggests that the relative experimental reactivity trend of
different substrates and carbenes is influenced by the heme core
part, although the protein environment can also affect the overall
reactivity and stereoselectivity of the biocatalyst through
residues surrounding the heme group.[8c, 17]
Effect of proximal ligand substitutions
Modification of the axial ligand coordinating the heme
cofactor has provided an effective strategy for modulating the
reactivity of hemoproteins as biocatalysts for carbene transfer
2b, c, 2h, 9d]
reactions.[1h,
More recently, we established that
substitution of the conserved histidine axial ligand (His93) with
an Asp residue is well tolerated in the myoglobin scaffold and
results in a functional biocatalyst for olefin cyclopropanation.[18]
To assess the impact of this structural change on the Si-H
insertion reactivity, the activity of Mb(H64V,V68A) and its
proximal
ligand
variants
Mb(H64V,V68A,H93D)
and
Mb(H64V,V68A,H93F) were compared in the model reaction
with dimethyl(phenyl)silane and EDA. As shown by the results in
Table 2, the Asp-containing variant Mb(H64V,V68A,H93D) was
found to exhibit comparable (slightly lower) Si-H insertion
reactivity to the His-containing counterpart, whereas
a
comparatively lower reactivity was observed for the variant
lacking axial coordination (i.e., Mb(H64V,V68A,H93F).
Table 2. Si-H carbene insertion activity of Mb(H64V,V68A) and proximal
ligand variants thereof.a
Proximal
Entry
Protein
Yieldb
TON
Ligand
His
1
2
3
Mb(H64V,V68A)
69%
66%
50%
345
330
250
Mb(H64V,V68A,H93D)
Mb(H64V,V68A,H93F)
Asp
Phe
a Reaction conditions: 10 mM X, 10 mM EDA, 20 μM Mb variant in 50 mM
b
phosphate buffer (pH 7), 1 hour, r.t., in anaerobic chamber. Based on GC
conversion using calibration curves with isolated 3a.
To examine the effect of related proximal ligand
substitutions on the reactivity of the heme carbene toward Si-H
insertion, reaction 4 (where Asp is as the axial ligand) and
reaction 5 (no axial ligand) were computationally compared with
reaction 1. As shown in Table 1, the His-to-Asp substitution is
accompanied by a 1.27 kcal/mol increase in the G‡ of the
reaction (i.e. reaction 4 vs. 1). However, both of these reactions
have a lower G‡ compared to the reaction with no axial ligand
Figure 3. Schematic free energy diagram for Si-H insertion reactions 1-5.
Interestingly, among reactions 1-3 (Scheme 1), the least
efficient reaction 3 (based on TTN) is characterized by the
largest charge and geometry changes (e.g. |QCT|: 3 (0.261 e) >
1 (0.223 e) > 2 (0.215 e); RSiH: 3 (0.060 Å) > 1 (0.043 Å) > 2
(0.040 Å), see Table 1). This results in the largest energy cost
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(reaction 5). For these systems, detailed calculations included in
SI (see Table S4) show that the singlet TS is again the most
favorable TS compared to other spin states, justifying its use for
these analyses. The higher reactivity of histidine- and aspartate-
ligated heme carbene toward Si-H insertion compared to the no
axial ligand case may arise from a weaker Fe-C bond (more
elongated by ~0.05 Å at R1 and by ~0.07 Å at TS, Table S7) due
to their trans effects,[19] thus facilitating carbene transfer. Overall,
the reactivity trend predicted for these reactions (1 > 4 > 5) using
the heme core part with :C(Me)CO2Et is in qualitative agreement
with the reactivity trend observed experimentally with the Mb-
based proximal ligand variants (His >≈ Asp > Phe; Table 2) using
a similar carbene :C(H)CO2Et.
transition state, as supported by (a) a detailed analysis of
different reaction systems, spin state and conformation effects
and (b) an overall good agreement between the computational
predictions with experimental KIEs and proximal ligand effects
obtained with Mb variants here as well as structure-activity
trends for carbene and substrate substituents previously
reported with engineered cytochrome c variants.[1c] Changes in
the geometric properties and charge transfers around the
reaction center along the computed reaction pathways were
helpful to understand the effect of modifications of the substrate,
carbene, and axial ligand on Si-H insertion reactivity. In particular,
the reactivity changes could be associated to effects on the
electronic driving force and early transition state feature, as
indicated by the quantitative correlation between reaction
barriers and geometry/charge properties. Based on these
mechanistic analyses, suggestions were given to help the future
development of efficient biocatalytic methods for Si-H insertion
reactions with challenging substrates.
Insights into structural factors to improve catalytic
reactivity
Interestingly, a good correlation between G‡ and QCT/RSiH
TS
(RSiH also has R2=0.9415) was found across all the Si-H
insertion reactions studied here (see Figure 4). These
relationships further highlight the importance of carbene
electrophilicity in this reaction and the early TS feature, since
smaller charge transfers and geometry changes are associated
with lower energy barriers.
Experimental Section
All calculations were performed using the program Gaussian
09.[20] All models investigated in this work were subject to full
geometry optimizations without any symmetry constraints using
the PCM method[21] with a dielectric constant of 4.0 to simulate
the protein environment effect as done previously[22] in this
computational study of heme carbenoid Si-H insertions, which
focuses on the basic mechanism and core part of the protein.
The frequency analysis was used to verify the nature of the
stationary points on respective potential energy surfaces and to
provide zero-point energy corrected electronic energies (EZPE’s),
enthalpies (H’s), and Gibbs free energies (G’s) at 1 atm and
experimental reaction temperatures, i.e. room temperature (r.t.).
The atomic charges and spin densities reported here are from
the Natural Population Analysis (NPA) and Mulliken schemes
respectively, as implemented in Gaussian 09. Relative Gibbs
free energies and selected geometry, charge, and spin density
results were discussed in the main text, while all absolute values
of electronic energies (E’s), zero-point energy corrected
electronic energies (EZPE’s), enthalpies (H’s), Gibbs free
energies (G’s), key geometric parameters, charges, spin
densities, 3D structures and coordinates of optimized structures
of the most favorable conformations and spin states as well as
other details are in SI.
A
B
Figure 4. G‡ vs. (A) QCT and (B) RSiHTS in Si-H insertions.
These results suggest that the introduction of stronger
electron-withdrawing substituents on the carbene group,
porphyrin, or axial ligand could facilitate the charge transfer from
the silane substrate to the carbene (the major electronic driving
force found here), resulting in improved catalytic performance, in
particular for the more challenging electron-deficient substrates.
These insights may inspire future experimental approaches for
sustainable biocatalytic Si-H insertion reactions.
All calculations were done using a range-separated hybrid
DFT method with dispersion correction, B97XD,[23] based on its
Conclusions
excellent performance on heme carbenes and other catalytic
systems from several methodological studies.[9a-c,
This
24]
B97XD method was found to yield accurate predictions of
various experimental spectroscopic properties, structural
features, and reactivity results of IPCs compared to some other
functionals.[9] The basis set includes the effective core potential
(ECP) basis LanL2DZ[25] for iron and the triple-zeta basis 6-
311G(d) for all other elements, which was found to provide
accurate predictions of various experimental reaction properties
This work provides the first computational and experimental
data to compare different mechanistic possibilities to determine
the basic mechanism of biocatalytic hemoprotein-catalyzed Si-H
insertion reactions, as well as the first theoretical insights into
effects of experimental reactivity trends due to modifications on
substrates, carbenes, and catalysts. Collectively, our results
indicate an FeII-based, concerted, hydride transfer mechanism
involving nonsynchronous Si-C/C-H bond formation and an early
of heme carbenes.[9b-d] The use of
a much larger 6-
311++G(2d,2p) basis for all non-metal atoms was found to yield
similar results for heme carbene reactions[9c] and thus further
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sealed vials, a solution containing sodium dithionite (100 mM
stock solution) in potassium phosphate buffer (KPi 50 mM, pH
7.0) was degassed by bubbling argon into the mixture for 3 min
in a sealed glass crimp vial. A buffered solution containing
myoglobin was degassed in a similar manner in a separate glass
crimp vial. The two solutions were then mixed together via
cannulation. Reactions were initiated by addition of 10 μL of
dimethyl(phenyl)silane (from a 0.5 M stock solution in ethanol),
followed by the addition of 10 μL of EDA (from a 0.5 M stock
solution in ethanol) with a syringe, and the reaction mixture was
stirred for the indicated time at room temperature, under positive
argon pressure. The reactions were analyzed using a Shimadzu
GC-2010 gas chromatograph equipped with an FID detector and
a Chiral Cyclosil-B column (30 m × 0.25 mm × 0.25 μm film).
Separation method: 1 μL injection, injector temp: 200 °C,
detector temp: 300 °C. Gradient: column temperature set at 120
°C for 3 min, then to 150 °C at 0.8 °C/min, then to 245 °C at 25
°C/min. Total run time was 28.60 min. Calibration curve for
quantification of the Si-insertion product was constructed using
authentic standard prepared synthetically as described in the SI.
GC/MS analyses were performed on a Shimadzu GCMS-
QP2010 equipped with a RTXXLB column (30 m × 0.25 mm ×
0.28 μm) and a quadrupole mass analyzer.
support the efficient use of the current basis set here. The use of
an ECP basis for metal here is common in many reaction studies
involving transition metal carbenoids, such as Ir porphyrin
carbene,[7f] Ru porphyrin carbene,[26] Rh carbene.[27] The
advantage of an ECP basis is the inclusion of relativistic effect
basically absent in an all-electron basis set. In addition, it is
available for all transition metals, which may allow direct
comparisons of effects of a vast amount of metal centers. The
alternative use of an all-electron basis for the metal center[9d] was
recently found to yield qualitatively same conclusions of
geometric, electronic, and energetic features for heme carbene
reactions, and therefore supports the use of LanL2DZ basis here,
which may help direct comparisons with late transition metals in
future studies, for which ECP basis is more readily available and
commonly used.
The KIE calculations with tunneling effect correction (KIEW)
were done using the following formulae reported recently:[28]
(
)
/
RT
KIEE = e( −∆G
(1)
(2)
+ ∆G
)
H
D
푄
푄
푡퐻
KIEW= 퐾퐼퐸퐸
×
푡퐷
ℎ휈
푘푇
2
)
Qt (Tunneling correction) = 1 + (
(3)
24
Acknowledgements
where h is Planck’s constant,  is the imaginary frequency of the
transition state, k is Boltzmann’s constant, T is temperature, and
subscripts of H and D indicate hydrogen and deuterium
respectively.
This work was supported by the U.S. National Science
Foundation grant CHE-1300912 to YZ and the U.S. National
Institute of Health grant GM098628 to RF.
Protein Expression. The Mb variants were expressed from
pET-based vectors in E. coli BL21(DE3) as described
previously.[2e, f] After transformation, cells were grown in TB
medium (ampicillin, 100 mg L−1) at 37 °C (200 rpm) until OD600
reached 0.6. Cells were then induced with 0.25 mM isopropyl-β-
D-1-thiogalactopyranoside (IPTG) and 0.3 mM δ-aminolevulinic
acid (ALA). After induction, cultures were shaken at 180 rpm and
27 °C and harvested after 20 h by centrifugation at 4,000 rpm at
4 °C. After cell lysis by sonication, the cell lysate was loaded on
a Ni-NTA column equilibrated with Ni-NTA lysis buffer (50 mM
KPi, 250 mM NaCl, 10 mM histidine, pH 8.0). After washing with
50 mL Ni-NTA lysis buffer and 50 mL of Ni-NTA wash buffer (50
mM KPi, 250 mM, NaCl, 20 mM imidazole, pH 8.0), the protein
was eluted with Ni-NTA elution buffer (50 mM KPi, 250 mM,
NaCl, 250 mM histidine, pH 7.0). The protein solution was buffer
exchanged against potassium phosphate buffer (50 mM, pH
Keywords: biocatalysis • silanes • carbenoids • heme proteins
• density functional calculations
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
An FeII-based concerted hydride
transfer mechanism with
Rahul L. Khade,a) Ajay L. Chandgude,b)
Rudi Fasan,* b) and Yong Zhang* a)
nonsynchronous and early transition
state was found for biocatalytic heme
carbenoid Si-H insertions, with key
insights into effects of substrates,
carbenes, and protein axial ligands to
help understand experimental
structure-reactivity trends and
facilitate design of improved
Page No. – Page No.
Mechanistic Investigation of
Biocatalytic Heme Carbenoid Si-H
Insertions
(bio)catalysts for challenging Si-H
insertion reactions.
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