Directed Evolution of Cytochrome
c
for Carbon–Silicon Bond
Formation: Bringing Silicon to Life
S. B. Jennifer Kan
1
,
Russell D. Lewis
1
,
Kai Chen
1
, and
Frances H. Arnold
1,*
1
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
CA 91125, USA
Abstract
Enzymes that catalyze carbon–silicon bond formation are unknown in nature, despite the natural
abundance of both elements. Such enzymes would expand the catalytic repertoire of biology,
enabling living systems to access chemical space previously only open to synthetic chemistry. We
have discovered that heme proteins catalyze the formation of organosilicon compounds under
physiological conditions via carbene insertion into silicon–hydrogen bonds. The reaction proceeds
both
in vitro
and
in vivo
, accommodating a broad range of substrates with high chemo- and
enantioselectivity. Using directed evolution, we enhanced the catalytic function of cytochrome
c
from
Rhodothermus marinus
to achieve more than 15-fold higher turnover than state-of-the-art
synthetic catalysts. This carbon–silicon bond-forming biocatalyst offers an environmentally
friendly and highly efficient route to producing enantiopure organosilicon molecules.
Silicon constitutes almost 30% of the mass of the Earth’s crust, yet no life form is known to
have the ability to forge carbon–silicon bonds (
1
). Despite the absence of organosilicon
compounds in the biological world, synthetic chemistry has enabled us to appreciate the
unique and desirable properties that have led to their broad applications in chemistry and
material science (
2
,
3
). As a biocompatible carbon isostere, silicon can also be used to
optimize and repurpose the pharmaceutical properties of bioactive molecules (
4
,
5
).
The natural supply of silicon may be abundant, but sustainable methods for synthesizing
organosilicon compounds are not (
6
–
8
). Carbon–silicon bond forming methods that
introduce silicon motifs to organic molecules enantioselectively rely on multi-step synthetic
campaigns to prepare and optimize chiral reagents or catalysts; precious metals are also
sometimes needed to achieve the desired activity (
9
–
15
). Synthetic methodologies such as
carbene insertion into silanes can be rendered enantioselective using chiral transition metal
complexes based on rhodium (
11
,
12
), iridium (
13
) and copper (
14
,
15
). These catalysts can
provide optically pure products, but not without limitations: they require halogenated
*
To whom correspondence should be addressed. frances@cheme.caltech.edu.
Supplementary Materials
Materials and Methods
Figs. S1 to S6
Tables S1 to S7
References (1–67)
HHS Public Access
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solvents and sometimes low temperatures to function optimally and have limited turnovers
(<100) (
16
).
Because of their ability to accelerate chemical transformations with exquisite specificity and
selectivity, enzymes are increasingly sought after complements to or even replacements for
chemical synthesis methods (
17
,
18
). Biocatalysts that are fully genetically encoded and
assembled inside of cells are readily tunable using molecular biology techniques. They can
be produced at low cost from renewable resources in microbial systems and perform
catalysis under mild conditions. Although nature does not use enzymes to form carbon–
silicon bonds, the protein machineries of living systems are often “promiscuous”, that is,
capable of catalyzing reactions distinct from their biological functions. Evolution, natural or
in the laboratory, can use these promiscuous functions to generate catalytic novelty (
19
–
21
).
For example, heme proteins can catalyze a variety of non-natural carbene transfer reactions
in aqueous media, including N–H and S–H insertions, which can be greatly enhanced and
made exquisitely selective by directed evolution (
22
–
24
).
We hypothesized that heme proteins might also catalyze carbene insertion into silicon–
hydrogen bonds. Because iron is not known to catalyze this transformation (
25
), we first
examined whether free heme could function as a catalyst in aqueous media. Initial
experiments showed that the reaction between phenyldimethylsilane and ethyl 2-
diazopropanoate (Me-EDA) in neutral buffer (M9-N minimal medium, pH 7.4) at room
temperature gave racemic organosilicon product
3
at very low levels, a total turnover number
(TTN) of 4 (Fig. 1A). No product formation was observed in the absence of heme, and the
organosilicon product was stable under the reaction conditions.
We next investigated whether heme proteins could catalyze the same carbon–silicon bond-
forming reaction. Screening a panel of cytochrome P450 and myoglobin variants, we
observed product formation with more turnovers compared to the hemin and hemin with
bovine serum albumin (BSA) controls, but with negligible enantioinduction (Table S4).
Interestingly, cytochrome
c
from
Rhodothermus marinus
(
Rma
cyt
c
), a gram-negative,
thermohalophilic bacterium from submarine hot springs in Iceland (
26
), catalyzed the
reaction with 97%
ee
, indicating the reaction took place in an environment where the protein
exerted excellent stereocontrol. Bacterial cytochromes
c
are well-studied, functionally
conserved electron-transfer proteins that are not known to have any catalytic function in
living systems (
27
). Other bacterial and eukaryotic cytochrome
c
proteins also catalyzed the
reaction, but with lower selectivities. We thus chose
Rma
cyt
c
as the platform for evolving a
carbon–silicon bond-forming enzyme.
The crystal structure of wild-type
Rma
cyt
c
(PDB ID: 3CP5;
26
) reveals that the heme
prosthetic group resides in a hydrophobic pocket, with the iron axially coordinated to a
proximal His (H49) and a distal Met (M100), the latter of which is located on a loop (Figs.
1B and 1C). The distal Met, common in cytochrome
c
proteins, is coordinately labile (
28
,
29
). We hypothesized that M100 must be displaced upon iron-carbenoid formation, and that
mutation of this amino acid could facilitate formation of this adventitious “active site” and
yield an improved carbon–silicon bond-forming biocatalyst. Therefore, a variant library
made by site-saturation mutagenesis of M100 was cloned and recombinantly expressed in
E.
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coli
. After protein expression, the bacterial cells were heat-treated (75 °C for 10 min) before
screening in the presence of phenyldimethylsilane (10 mM), Me-EDA (10 mM) and sodium
dithionite (Na
2
S
2
O
4
10 mM) as a reducing agent, at room temperature under anaerobic
conditions. The M100D mutation stood out as highly activating: this first-generation mutant
provided chiral organosilicon
3
as a single enantiomer in 550 TTN, a 12-fold improvement
over the wild-type protein (Fig. 1D).
Amino acid residues V75 and M103 reside close (within 7Å) to the iron heme center in
wild-type
Rma
cyt
c
. Sequential site-saturation mutagenesis at these positions in the M100D
mutant led to the discovery of triple mutant V75T M100D M103E, which catalyzed carbon–
silicon bond formation in >1500 turnovers and >99%
ee
. This level of activity is more than
15 times the total turnovers reported for the best synthetic catalysts for this class of reaction
(
16
). As stand-alone mutations, both V75T and M103E are activating for wild-type
Rma
cyt
c
and the beneficial effects increase with each combination (Table S5). Comparison of the
initial reaction rates established that each round of evolution enhanced the rate: relative to
the wild-type protein, the evolved triple mutant catalyzes the reaction >7-fold faster, with
turnover frequency (TOF) of 46 min
−1
(Fig. 1E).
Assaying the new enzyme against a panel of silicon and diazo reagents, we found that the
mutations were broadly activating for enantioselective carbon–silicon bond formation. The
reaction substrate scope was surveyed using heat-treated lysates of
E. coli
expressing
Rma
cyt
c
V75T M100D M103E under saturating conditions for both silane and diazo ester to
determine TTN. Whereas many natural enzymes excel at catalyzing reactions on only their
native substrates and little else (especially primary metabolic enzymes), the triple mutant
catalyzed the formation of twenty silicon-containing products, most of which were obtained
cleanly as single enantiomers, showcasing the broad substrate scope of this reaction using
just a single variant of the enzyme (Fig. 2). The reaction accepts both electron-rich and
electron-deficient silicon reagents, accommodating a variety of functional groups including
ethers, aryl halides, alkyl halides, esters and amides (
5–10
). Silicon reagents based on
naphthalenes or heteroarenes (
11–13
) as well as vinyldialkyl- and trialkylsilanes could also
serve as silicon donors (
14
,
15
,
18
). In addition, diazo compounds other than Me-EDA could
be used for carbon–silicon bond formation (
16
,
17
) (
16
).
The evolved
Rma
cyt
c
exhibits high specificity for carbon–silicon bond formation. Even in
the presence of functional groups that could compete in carbene-transfer reactions,
enzymatic carbon–silicon bond formation proceeded with excellent chemoselectivity. For
example, styrenyl olefins, electron-rich double bonds, and terminal alkynes that are prime
reaction handles for synthetic derivatization are preserved under the reaction conditions,
with no competing cyclopropanation or cyclopropenation activity observed. As a result,
organosilicon products
12–13
and
18
–
20
were afforded with 210 to 5010 turnovers and
excellent stereoselectivities (98 to >99%
ee
). Preferential carbon–silicon bond formation
could also be achieved with substrates bearing free alcohols and primary amines, yielding
silicon-containing phenol
21
(910 TTN, >99%
ee
) and aniline
22
(8210 TTN, >99%
ee
).
This capability removes the need for functional group protection and/or manipulation,
offering a streamlined alternative to transition metal catalysis for incorporating silicon into
small molecules. Indeed, when the same reactants were subjected to rhodium catalysis (1
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mol% Rh
2
(OAc)
4
), O–H and N–H insertions were the predominant reaction pathways, and
copper catalysis (10 mol% Cu(OTf)
2
) gave complex mixtures of products (Table S7).
Tolerance of these highly versatile functionalities in enzymatic carbon–silicon bond-forming
reactions provides opportunities for their downstream processing through metabolic
engineering, bioorthogonal chemistry, and other synthetic endeavours.
We next asked whether all
Rma
cyt
c
variants would catalyze carbon–silicon bond formation
selectively over insertion of the carbene into an N–H bond in the same substrate. We re-
visited the evolutionary lineage and tested all four generations of
Rma
cyt
c
(wild-type,
M100D, V75T M100D and V75T M100D M103E) with Me-EDA and 4-
(dimethylsilyl)aniline (
23
), a reagent that could serve as both nitrogen and silicon donor, to
probe the proteins’ bond-forming preferences. The wild-type cytochrome
c
in fact exhibited
a slight preference for forming amination product
24
over organosilicon product
22
. Even
though silane
23
was not used for screening, and the
Rma
cyt
c
therefore never underwent
direct selection for chemoselectivity, each round of evolution effected a distinct shift from
amination to carbon–silicon bond forming activity (Fig. 3A). This evolutionary path that
focused solely on increasing desired product formation culminated in a catalyst that
channeled the majority of the reactants (97%) through carbon–silicon bond formation (>30-
fold improved with respect to the wild-type), presumably by improving the orientation and
binding of the silicon donor.
Some fungi, bacteria and algae have demonstrated promiscuous capacities to derivatize
organosilicon molecules when these substances were made available to them (
1
). The
possibility ultimately to establish silicon-based biosynthetic pathways led us to investigate
whether the evolved
Rma
cyt
c
could produce organosilicon products
in vivo
.
E. coli
whole
cells (OD
600
= 15) expressing
Rma
cyt
c
V75T M100D M103E in glucose-supplemented
M9-N buffer were given silane
23
(0.1 mmol) and Me-EDA (0.12 mmol) as neat reagents.
The enzyme in this whole-cell system catalyzed carbon–silicon bond formation with 3410
turnovers, yielding organosilicon product
22
in 70% isolated yield (>95% yield based on
recovered silane
23
) and 98%
ee
(Fig. 3B). These
in vitro
and
in vivo
examples of carbon–
silicon bond formation using an enzyme and earth-abundant iron affirm the notion that
nature’s protein repertoire is highly evolvable and poised for adaptation: with only a few
mutations, existing proteins can be repurposed to efficiently forge chemical bonds not found
in biology and grant access to areas of chemical space which living systems have not
explored.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was supported in part by the National Science Foundation, Office of Chemical, Bioengineering,
Environmental and Transport Systems SusChEM Initiative (grant CBET-1403077), the Caltech Innovation Initiative
(CI2) Program, and the Jacobs Institute for Molecular Medicine at Caltech. Any opinions, findings, and conclusions
or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of
the funding organizations. R.D.L. is supported by a NIH/NRSA training grant (5 T32 GM07616). We thank A.
Buller, S. Dodani, S. Hammer and C. Prier for helpful discussions and comments on the manuscript, and N. Peck
Kan et al.
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for screening P450 variants. We are grateful to S. Virgil and the Caltech Center for Catalysis and Chemical
Synthesis and to N. Torian and the Caltech Mass Spectrometry Laboratory for generous analytical support, the
Beckman Institute Laser Resource Center (BILRC) at Caltech for use of their CD spectrometer, B. Stoltz for use of
the polarimeter, and H. Gray for providing the pEC86 plasmid. A provisional patent application has been filed
through the California Institute of Technology based on the results presented here. All data necessary to support this
paper’s conclusions are available in the Supplementary Materials.
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Fig. 1. Heme protein-catalyzed carbon-silicon bond formation
(A)
Carbon–silicon bond formation catalyzed by heme and purified heme proteins.
(B)
Surface representation of the heme-binding pocket of wild-type
Rma
cyt
c
(PDB ID: 3CP5).
(C)
“Active site” structure of wild-type
Rma
cyt
c
showing a covalently bound heme
cofactor ligated by axial ligands H49 and M100. Amino acid residues M100, V75 and M103
residing close to the heme iron were subjected to site-saturation mutagenesis.
(D)
Directed
evolution of
Rma
cyt
c
for carbon–silicon bond formation (reaction shown in (
A
)).
Experiments were performed using lysates of
E. coli
expressing
Rma
cyt
c
variant (OD
600
=
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15; heat-treated at 75 °C for 10 min), 10 mM silane, 10 mM diazo ester, 10 mM Na
2
S
2
O
4
, 5
vol% MeCN, M9-N buffer (pH 7.4) at room temperature under anaerobic conditions for 1.5
h. Reactions performed in triplicate. (
E
) Carbon–silicon bond forming rates over four
generations of
Rma
cyt
c
.
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Fig. 2. Scope of
Rma
cyt
c
V75T M100D M103E-catalyzed carbon–silicon bond formation
Standard reaction conditions: lysate of
E. coli
expressing
Rma
cyt
c
V75T M100D M103E
(OD
600
= 1.5; heat-treated at 75 °C for 10 min), 20 mM silane, 10 mM diazo ester, 10 mM
Na
2
S
2
O
4
, 5 vol% MeCN, M9-N buffer (pH 7.4) at room temperature under anaerobic
conditions. Reactions performed in triplicate. [a] OD
600
= 5 lysate. [b] OD
600
= 0.5 lysate.
[c] OD
600
= 15 lysate. [d] 10 mM silane. [e] OD
600
= 0.15 lysate.
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Fig. 3. Chemoselectivity and
in vivo
activity of evolved
Rma
cyt
c
(A)
Chemoselectivity for carbene Si–H insertion over N–H insertion increased dramatically
during directed evolution of
Rma
cyt
c
. Standard reaction conditions as described in Fig. 2.
Reactions performed in duplicate using heat-treated lysates of
E. coli
expressing
Rma
cyt
c
with protein concentration normalized across variants. Product distribution was quantified
after 2 h reaction time (before complete conversion, no double insertion product was
observed under these conditions).
(B)
In vivo
synthesis of organosilicon compound
22
.
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