Engineered Cytochrome
c
-Catalyzed Lactone-Carbene B–H
Insertion
Kai Chen
a
,
Xiongyi Huang
a
,
Shuo-Qing Zhang
b
,
Andrew Z. Zhou
a
,
S. B. Jennifer Kan
a
,
Xin
Hong
b
, and
Frances H. Arnold
a
a
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology,
Pasadena, CA 91125, USA
b
Department of Chemistry, Zhejiang University, Hangzhou, Zhejiang 31007, P. R. of China
Abstract
Previous work has demonstrated that variants of a heme protein,
Rhodothermus marinus
cytochrome
c
(
Rma
cyt
c
), catalyze abiological carbene boron–hydrogen (B–H) bond insertion
with high efficiency and selectivity. Here we investigated this carbon–boron bondforming
chemistry with cyclic, lactone-based carbenes. Using directed evolution, we obtained a
Rma
cyt
c
variant
BOR
LAC
that shows high selectivity and efficiency for B–H insertion of 5- and 6-
membered lactone carbenes (up to 24,500 total turnovers and 97.1:2.9 enantiomeric ratio). The
enzyme shows low activity with a 7-membered lactone carbene. Computational studies revealed a
highly twisted geometry of the 7membered lactone carbene intermediate relative to 5- and 6-
membered ones. Directed evolution of cytochrome
c
together with computational characterization
of key iron-carbene intermediates has allowed us to expand the scope of enzymatic carbene B–H
insertion to produce new lactone-based organoborons.
Graphical Abstract
Keywords
cytochrome
c
; carbene; organoboron; lactones; biocatalyst; directed evolution
Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0037-1611662
.
HHS Public Access
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Published in final edited form as:
Synlett
. 2019 March ; 30(4): 378–382. doi:10.1055/s-0037-1611662.
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The significant role of organoboron chemistries
1
–
3
in synthetic methodologies is exemplified
by alkene hydroboration
4
,
5
and Suzuki cross-coupling,
6
,
7
whose enormous footprints in
synthetic chemistry have been recognized by Nobel Prizes. In addition, organo-boronic acids
or -borates
8
can act as transition-state-analog inhibitors in biological systems and are useful
functionalities in chemotherapeutics
9
and other biologically active molecules.
10
The broad
applications of organoboron compounds have prompted chemists to develop efficient,
selective and modular synthetic platforms for installing boron motifs onto carbon backbones.
One major class of methods for forming carbon– boron (C–B) bonds relies on transition-
metal-catalyzed B–H bond insertion of carbenes (Figure 1A),
11
–
18
as introduced by Curran
and co-workers. Recently, the Arnold laboratory developed the first biocatalytic system for
this transformation using engineered variants of cytochrome
c
from the Gram-negative,
thermohalophilic bacterium
Rhodothermus marinus
(
Rma
cyt
c
).
19
The laboratory-evolved
enzymes exhibited very high efficiency (up to 15,300 turnovers and 6,100 h−1 turnover
frequency) and enantioselectivity (up to 99:1 enantiomeric ratio) with different carbenes and
boranes (Figure 1B).
To expand the catalytic range of this enzymatic C–B bond-forming platform, we have
engineered
Rma
cyt
c
to accept structurally different carbenes. In previous work, we
typically used
α
-ester-substituted diazo compounds as carbene precursors,
19
but we recently
demonstrated that
Rma
cyt
c
mutants can be tuned to use a spectrum of
α
-trifluoromethyl-
α
-
alkyl diazo compounds to furnish a wide array of chiral
α
-trifluoromethylated organoborons
(Figure 1C).
20
We were curious whether cyclic carbene moieties
21
can also be used by
Rma
cyt
c
, despite significant structural differences compared to the acyclic carbenes used in
previous work.
19
–
26
We started this investigation of cyclic carbenes using five-membered lactone diazo
compound
1
(see Figure 3) as the carbene precursor. With such a rigid structure, the putative
iron-porphyrin carbene (IPC) intermediate is expected to have different conformational
properties and potentially distinct electronic features compared to acyclic carbenes, such as
the one derived from
α
-methyl ethyl diazoacetate (Me-EDA). Recent work by our group
revealed the crystal structure of the Me-EDA-derived IPC intermediate
27
in a triple mutant
of
Rma
cyt
c
(V75T M100D M103E (TDE)) evolved in the laboratory for Si–H bond
insertion.
26
Experiments indicated a singlet electronic state of this iron carbene species
IPC1
, which is in line with the computational result that open/closed-shell singlets are the
dominant electronic states (Figure 2).
We used density functional theory (DFT) calculations to assess the electronic states of the
lactone-based
IPC2
(also using the simplified model of the protein IPC).
21
,
27
–
30
As we
expected,
IPC2
features a near-planar geometry with a very small dihedral angle d(Fe-C-C-
O) (<13°) in all electronic states, which is very different from the acyclic
IPC1
with d(Fe-C-
C-O) of nearly 90° in singlets. The small dihedral angle renders the singlet states less stable
due to strong repulsion between fully occupied orbitals and thus significantly changes the
energy levels of the electronic states of the IPC.
We wanted to know how the different structural and electronic properties of this intermediate
affect carbene B–H insertion. We thus tested the borylation reaction using the five-
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membered lactone diazo compound
1
and an
N
-heterocyclic carbene (NHC)-stabilized
borane as substrates in the presence of whole
E. coli
bacteria expressing engineered variants
of
Rma
cyt
c
, starting with those obtained during directed evolution for borylation with
acyclic carbene precursors (e.g., Me-EDA) (Figure 3). We were pleasantly surprised to see
that wild-type
Rma
cyt
c
exhibited high efficiency for lactone carbene B–H insertion, with
960 total turnovers (TTN) and 70% gas chromatography (GC) yield. The enantioselectivity,
however, was poor, with only a 68:32 enantiomeric ratio (e.r.). A distal axial-ligand M100D
mutation, previously discovered to facilitate both carbene Si–H and B–H insertion,
19
,
26
improved the yield, but did not improve the enantiocontrol of this reaction. Residue V75 in
an
α
-helix region was previously shown to affect carbene orientation.
19
,
20
Screening of
M100D variants containing mutations at V75 identified M100D V75R as the most selective,
with 93.3:6.7 e.r., whereas V75T/C/K/P/G mutations resulted in poor to moderate
enantioselectivities. An additional M103V mutation led to even more precise stereochemical
control, giving an e.r. of 94.9:5.1 (Figure 3).
To increase the enantioselectivity of lactone-carbene B– H insertion, we subjected the
Rma
cyt
c
V75R M100D M103V (RDV) variant to site-saturation mutagenesis, targeting active-
site amino acid residues which are close to the iron center in wild-type
Rma
cyt
c
(within 10
Å). It is known that the residues residing on the flexible front loop are important for
controlling the structure of the heme pocket, which is presumably the active site for this
novel function (Figure 3A).
27
Consequently, introducing suitable mutations on this loop may
help to orient the iron-carbene intermediate or tune the approach of the borane substrate and
lead to desired enantioselectivity. A double-site-saturation mutagenesis library at residues
M99 and T101 was cloned using the 22-codon-trick protocol
32
and screened as whole-cell
catalysts in four 96-well plates for improved borylation enantioselectivity. Double mutant
M99Q T101Y (
BOR
LAC
) was identified to exhibit higher selectivity (96.3:3.7 e.r.) and good
catalytic efficiency (970 TTN, 80% GC yield).
With an efficient and selective borylating variant
BOR
LAC
in hand, we then assessed the
scope of boranes that this platform can use (Figure 4A). Boranes without stabilizing groups
are highly reactive and unstable in aqueous conditions. Lewis bases, such as ethers, amines,
phosphines and NHCs, are generally used as good stabilizing groups for free boranes.
33
Considering the biocompatibility and cell permeability of the borane reagents, NHC-
stabilized boranes turned out to be suitable candidates for this borylation platform.
34
Indeed,
borane complexes stabilized by NHCs featuring different electronic properties, steric
hindrance and/or lipophilicity all served as good substrates for the target borylation reaction,
furnishing the desired products with up to 1160 TTN and enantioselectivities up to 97.1:2.9
e.r. For instance, fluorine-containing alkyl groups (e.g.,
2c
), which are electron-withdrawing
and usually exhibit very different lipophilicity and hydrophilicity relative to general aliphatic
alkyl groups, were found to be compatible with the whole-cell reaction conditions.
To explore the longevity of the biocatalyst, we tried portionwise addition of the two
substrates. Every 1.5 hours, we added an additional aliquot corresponding to 12.5 mM of
each substrate to the reaction. Over 20 additions, we observed continuous and steadily
increasing product formation, which indicates that the catalyst maintained function over 30
hours (Figure 4C). However, we did notice that enantioselectivities decreased. Covalent
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modification of the protein backbone by carbene species and non-covalent binding of borane
substrate or product to the protein may cause structural changes that compromise
stereocontrol.
35
Further reaction engineering and optimization, such as using a flow system
to maintain consistent concentrations of reagents, may be able to address this selectivity
drop.
Given that the 5-membered cyclic lactone-carbene worked so well for this biocatalytic B–H
insertion, we wondered whether other cyclic carbenes, particularly lactone-carbenes with
different ring sizes, would also be accepted. The 6- and 7-membered lactone diazos
4
and
6
were readily prepared from the corresponding lactones and used as cyclic carbene precursors
for testing B–H insertion using variant
BOR
LAC
(Figure 4A). The 6-membered lactone-
carbene showed high reactivities (up to 1190 TTN and 96.1:3.9 e.r.) for the desired
borylation with three different borane substrates. Additionally, enzymatic borylation with
both 5- and 6-membered lactone carbenes was readily scalable to millimole level, affording
the desired products in high isolated yields (Figure 4B). However, with one additional
carbon in the ring, the 7-membered lactone-carbene behaved in a completely different
manner:
BOR
LAC
exhibited only low activity with this carbene precursor (<50 TTN).
To understand the origins of this dramatic impact of ring size on
Rma
cyt
c
-catalyzed
lactone-carbene B–H insertion chemistry, we employed DFT calculations to compare the
structures and the electronic properties of three lactonetype IPCs. Unlike 5-membered
lactone-based
IPC2
, which takes on a rigid planar structure, 6-membered lactone-carbene
IPC3
showed a slightly flexible structure with a dihedral angle d(Fe-C-C-O) of 60° in the
ground OSS state, while its triplet state still possesses a near-planar geometry (Figure 5).
However, 7-membered lactone
IPC4
takes on highly twisted conformations with d(Fe-C-C-
O) from 47° to 73° in all electronic states.
36
It is apparent that none of the electronic states
in
IPC4
shares a similar structure with
IPC2
. This may explain why
BOR
LAC
, which was
evolved for the 5membered lactone carbene, did not exhibit high reactivity towards
borylation with the 7-membered lactone carbene. But this does not necessarily mean that
Rma
cyt
c
cannot be engineered to accommodate the 7-membered lactone carbene for B–H
insertion; further engineering using
BOR
LAC
as a starting template could well lead to
variants with improved reactivity on this 7-membered lactone carbene.
In conclusion, we have expanded the scope of carbenes for biocatalytic B–H insertion
chemistry to include cyclic lactone carbenes.
37
With further development of
Rma
cyt
c
based
biocatalytic platforms, we accessed a range of organoborons at preparative scales and with
unprecedented catalytic efficiencies (up to 24,500 TTN) and high enantioselectivities (up to
97.1:2.9 e.r.). Computational studies provided insights into the conformations and electronic
states of cyclic carbene intermediates with different ring sizes. This mechanistic
understanding together with the new biocatalyst variants identified should promote further
expansion of carbene scope for borylation and other carbine-transfer reactions.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
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Acknowledgment
A.Z.Z. thanks the Caltech SURF program. Calculations were performed on the high-performance computing
system at the Department of Chemistry, Zhejiang University. We thank R. D. Lewis and R. K. Zhang for helpful
discussions and comments. We also thank the Caltech Mass Spectrometry Laboratory.
Funding Information
Financial support from the NSF Division of Molecular and Cellular Biosciences grant MCB-1513007, National
Natural Science Foundation of China (21702182), Zhejiang University, the Chinese “Thousand Youth Talents
Plan”, and the “Fundamental Research Funds for the Central Universities” is gratefully acknowledged. K.C. thanks
the Resnick Sustainability Institute at Caltech for fellowship support. X.H. is supported by an NIH pathway to
independence award (grant K99GM129419).
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General Procedure
To a 20 mL vial were added a suspension of
E. coli
expressing
Rma
cytochrome
c
variant
BOR
LAC
(5 mL, OD
600
= 15), borane (150 μL of a 400 mM stock solution
in MeCN, 0.06 mmol), lactone diazo compound (150 μL of a 400 mM stock solution in MeCN,
0.06 mmol), and D-glucose (50 mM) in M9-N buffer (pH 7.4) under anaerobic conditions. The
vial was capped and shaken (480 rpm) at room temperature for 18 h. Reactions were set up in
quadruplicate. Upon completion, reactions in replicate were combined and transferred to 50 mL
centrifuge tubes. The reaction vials were washed with water (2 × 2 mL) followed by a mixed
organic solvent (hexane/ethyl acetate = 1:1, 3 × 2 mL). The washing solution was combined with
the reaction mixture in the centrifuge tubes. An additional 15 mL of hexane/ethyl acetate solvent
was added to every tube. The tube was then vortexed (3 × 1 min) and shaken vigorously, and
centrifuged (5,000 × g, 5 min). The organic layer was separated and the aqueous layer was
subjected to three more rounds of extraction. The organic layers were combined, dried over
Na
2
SO
4
and concentrated under reduced pressure. Purification by silica gel column
chromatography with hexane/(ethyl acetate/acetone 3:7) as eluent afforded the desired lactone-
based organoboranes. Enantiomeric ratio (e.r.) was measured by chiral HPLC. TTN was
calculated based on measured protein concentration and isolated product yield. Product
3a
was
obtained as a white solid (41.4 mg, 89%, 1290 TTN). 1H NMR (400 MHz, CDCl
3
):
δ
= 6.83 (s,
2 H), 4.45 (dddd,
J
= 10.7, 8.1, 6.8, 1.0 Hz, 1 H), 4.27 (tq,
J
= 9.0, 1.1 Hz, 1 H), 3.83–3.69 (m, 6
H), 2.53–2.25 (m, 1 H), 2.01–1.93 (m, 1 H), 1.88–1.80 (m, 1 H), 1.79–1.16 (m, 2 H); 13C NMR
(101 MHz, CDCl
3
):
δ
= 187.91, 120.62, 67.86, 36.15, 30.86; 11B NMR (128 MHz, CDCl
3
):
δ
=
−27.08 (t,
J
= 89.6 Hz); HRMS (FAB+):
m
/
z
[(M + H+) – H
2
] calcd for C
9
H
14
O
2
N
2
B:
193.1148; found: 193.1144; Chiral HPLC (Chiralpak IC, 40%
i
-PrOH in hexane, flow rate: 1.5
mL/min, temperature: 32 °C,
λ
= 235 nm):
t
R
(major) = 12.857 min,
t
R
(minor) = 10.991 min;
96.2:3.8 e.r.
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Figure 1.
A
. Catalytic carbene B–H insertion.
B
. A hemeprotein biocatalyst for carbene B–H insertion
based on
Rma
cytochrome
c
.
C
. Carbenes used for biocatalytic carbene B–H insertion
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Figure 2.
Electronic states of acyclic
IPC1
and cyclic
IPC2
intermediates. The Gibbs free energies
were obtained at the B3LYP-D3(BJ)/def2-TZ-VPP//B3LYP/def2-SVP level. OSS = open-
shell singlet, CSS = closed-shell singlet
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Figure 3.
A
. Whole protein structure and active-site structure of wildtype
Rma
cyt
c
(PDB: 3CP5, ref.
31
).
B
. Yields and e.r. values of selected
Rma
cyt
c
variants for B–H insertion. Reactions
were conducted in quadruplicate: suspensions of
E. coli
expressing
Rma
cyt
c
variants
(OD
600
= 20), 10 mM borane
2a
, 10 mM lactone diazo
1
, and 5 vol% acetonitrile in M9-N
buffer (pH 7.4) at room temperature under anaerobic conditions for 18 hours. TTN refers to
the molar ratio of total desired product, as quantified by gas chromatography-mass
spectrometry (GC-MS) using trimethoxybenzene as internal standard, to total heme protein.
Enantiomeric ratio (e.r.) was determined by chiral high-performance liquid chromatography
(HPLC)
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Figure 4.
A
. Scope of lactone-carbene B–H insertion with
BOR
LAC
. Reactions were conducted in
quadruplicate: suspension of
E. coli
expressing
Rma
cyt
c
variant
BOR
LAC
(OD
600
= 20),
10 mM borane
2
, 10 mM lactone diazo
1
,
4
or
6
, and 5 vol% acetonitrile in M9-N buffer (pH
7.4) at room temperature under anaerobic conditions for 18 hours. TTN refers to the total
desired product, as quantified by GC-MS using trimethoxybenzene as internal standard,
divided by total heme protein. Enantiomeric ratio (e.r.) was determined by chiral HPLC.
B
.
Preparative-scale synthesis of organoborons.
Reaction conditions
: suspension of
E. coli
expressing
Rma
cyt
c
variant
BOR
LAC
(OD
600
= 15), 12 mM borane
2a
, 12 mM lactone
diazo
1
or
4
, 50 mM
D
-glucose and 6 vol% acetonitrile in M9-N buffer (pH 7.4) at room
temperature under anaerobic conditions for 18 hours. Reactions were set up in quadruplicate
and organoboron products were isolated from the combined reaction replicates.
C
.
Enzymatic synthesis of organoborons with portionwise addition of substrates. Reactions
were conducted in duplicate using the same reaction conditions as in
A
. above, except for
using 2.5 M solution stocks of borane
2b
and lactone diazo
1
(one portion = 2 μL of each
substrate stock)
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Figure 5.
Comparison of 5-, 6- and 7-membered lactone-based IPCs. The Gibbs free energies were
obtained at the B3LYP-D3(BJ)/def2-TZ-VPP//B3LYP/def2-SVP level
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