Engineered cytochrome c-Catalyzed lactone-carbene B-H insertion

Article abstract of DOI:10.1055/s-0037-1611662

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 bond-forming 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- A nd 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 7-membered lactone carbene intermediate relative to 5- A nd 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.

Full text of DOI:10.1055/s-0037-1611662

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K. Chen et al.
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Engineered Cytochrome c-Catalyzed Lactone-Carbene B–H Insertion
Kai Chena
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Xiongyi Huanga
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Shuo-Qing Zhangb
Andrew Z. Zhoua
S. B. Jennifer Kana
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Xin Hong*b
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Frances H. Arnold*a
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a Division of Chemistry and Chemical Engineering 210-41, California
Institute of Technology, Pasadena, CA 91125, USA
frances@cheme.caltech.edu
b Department of Chemistry, Zhejiang University, Hangzhou, Zhejiang
31007, P. R. of China
hxchem@zju.edu.cn
Published as part of the 30 Years SYNLETT – Pearl Anniversary Issue
Received: 26.11.2018
Accepted after revision: 05.01.2019
Published online: 14.01.2019
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 transforma-
tion using engineered variants of cytochrome c from the
Gram-negative, thermohalophilic bacterium Rhodothermus
marinus (Rma cyt c).19 The laboratory-evolved enzymes ex-
hibited 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 bo-
ranes (Figure 1B).
DOI: 10.1055/s-0037-1611662; Art ID: st-2018-b0769-l
License terms:
Abstract Previous work has demonstrated that variants of a heme
protein, Rhodothermus marinus cytochrome c (Rma cyt c), catalyze abio-
logical carbene boron–hydrogen (B–H) bond insertion with high effi-
ciency and selectivity. Here we investigated this carbon–boron bond-
forming chemistry with cyclic, lactone-based carbenes. Using directed
evolution, we obtained a Rma cyt c variant BORLAC that shows high se-
lectivity 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 ra-
tio). The enzyme shows low activity with a 7-membered lactone carbe-
ne. Computational studies revealed a highly twisted geometry of the 7-
membered lactone carbene intermediate relative to 5- and 6-mem-
bered ones. Directed evolution of cytochrome c together with compu-
tational 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.
Key words cytochrome c, carbene, organoboron, lactones, biocata-
lyst, directed evolution
The significant role of organoboron chemistries1–3 in
synthetic methodologies is exemplified by alkene hydrobo-
ration4,5 and Suzuki cross-coupling,6,7 whose enormous
footprints in synthetic chemistry have been recognized by
Nobel Prizes. In addition, organo-boronic acids or -borates8
can act as transition-state-analog inhibitors in biological
systems and are useful functionalities in chemotherapeu-
tics9 and other biologically active molecules.10 The broad
applications of organoboron compounds have prompted
chemists to develop efficient, selective and modular syn-
thetic 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
Figure 1 A . Catalytic carbene B–H insertion. B. A hemeprotein biocat-
alyst for carbene B–H insertion based on Rma cytochrome c. C. Carben-
es used for biocatalytic carbene B–H insertion
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 -ester-substituted diazo compounds as car-
bene precursors,19 but we recently demonstrated that Rma
cyt c mutants can be tuned to use a spectrum of -trifluoro-
methyl--alkyl diazo compounds to furnish a wide array of
Georg Thieme Verlag Stuttgart · New York — Synlett 2019, 30, A–E

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chiral -trifluoromethylated organoborons (Figure 1C).20
We were curious whether cyclic carbene moieties21 can
also be used by Rma cyt c, despite significant structural dif-
ferences compared to the acyclic carbenes used in previous
work.19–26
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 an-
gle renders the singlet states less stable due to strong repul-
sion 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 carbe-
ne B–H insertion. We thus tested the borylation reaction
using the five-membered lactone diazo compound 1 and an
N-heterocyclic carbene (NHC)-stabilized borane as sub-
strates in the presence of whole E. coli bacteria expressing
engineered variants of Rma cyt c, starting with those ob-
tained 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 exhib-
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 pu-
tative iron-porphyrin carbene (IPC) intermediate is expect-
ed to have different conformational properties and poten-
tially distinct electronic features compared to acyclic car-
benes, such as the one derived from -methyl ethyl
diazoacetate (Me-EDA). Recent work by our group revealed
the crystal structure of the Me-EDA-derived IPC intermedi-
ate27 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 computa-
tional result that open/closed-shell singlets are the domi-
nant electronic states (Figure 2).
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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ited 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 -helix region was previously shown to affect
carbene orientation.19,20 Screening of M100D variants con-
taining 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 enantioselectivi-
ties. An additional M103V mutation led to even more pre-
cise stereochemical control, giving an e.r. of 94.9:5.1 (Figure 3).
and lead to desired enantioselectivity. A double-site-satura-
tion mutagenesis library at residues M99 and T101 was
cloned using the 22-codon-trick protocol32 and screened as
whole-cell catalysts in four 96-well plates for improved bor-
ylation enantioselectivity. Double mutant M99Q T101Y
) 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
BORLAC in hand, we then assessed the scope of boranes that
this platform can use (Figure 4A). Boranes without stabiliz-
ing groups are highly reactive and unstable in aqueous con-
ditions. 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 bo-
ranes turned out to be suitable candidates for this boryla-
tion platform.34 Indeed, borane complexes stabilized by
NHCs featuring different electronic properties, steric hin-
drance and/or lipophilicity all served as good substrates for
the target borylation reaction, furnishing the desired prod-
ucts 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 ex-
hibit very different lipophilicity and hydrophilicity relative
to general aliphatic alkyl groups, were found to be compati-
ble with the whole-cell reaction conditions.
(BORLAC
To explore the longevity of the biocatalyst, we tried por-
tionwise 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 ob-
served continuous and steadily increasing product forma-
tion, which indicates that the catalyst maintained function
over 30 hours (Figure 4C). However, we did notice that en-
antioselectivities decreased. Covalent 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 us-
ing 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 won-
dered 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 cy-
clic carbene precursors for testing B–H insertion using vari-
ant BORLAC (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 sub-
strates. Additionally, enzymatic borylation with both 5- and
6-membered lactone carbenes was readily scalable to milli-
mole level, affording the desired products in high isolated
yields (Figure 4B). However, with one additional carbon in
Figure 3 A . Whole protein structure and active-site structure of wild-
type 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 qua-
druplicate: suspensions of E. coli expressing Rma cyt c variants (OD600 = 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. Enan-
tiomeric ratio (e.r.) was determined by chiral high-performance liquid
chromatography (HPLC)
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, tar-
geting 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
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bene for B–H insertion; further engineering using BORLAC as
a starting template could well lead to variants with im-
proved reactivity on this 7-membered lactone carbene.
Figure 4 A . Scope of lactone-carbene B–H insertion with BORLAC. Re-
actions were conducted in quadruplicate: suspension of E. coli express-
ing Rma cyt c variant BORLAC (OD600 = 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 re-
fers to the total desired product, as quantified by GC-MS using trime-
thoxybenzene 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 BORLAC (OD600 = 15), 12 mM borane
2a, 12 mM lactone diazo 1 or 4, 50 mM D-glucose and 6 vol% acetoni-
trile in M9-N buffer (pH 7.4) at room temperature under anaerobic con-
ditions for 18 hours. Reactions were set up in quadruplicate and
organoboron products were isolated from the combined reaction repli-
cates. C. Enzymatic synthesis of organoborons with portionwise addi-
tion 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 L of each
substrate stock)
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
the ring, the 7-membered lactone-carbene behaved in a
completely different manner: BORLAC exhibited only low ac-
tivity with this carbene precursor (<50 TTN).
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 orga-
noborons at preparative scales and with unprecedented cat-
alytic efficiencies (up to 24,500 TTN) and high enantiose-
lectivities (up to 97.1:2.9 e.r.). Computational studies pro-
vided insights into the conformations and electronic states
of cyclic carbene intermediates with different ring sizes.
This mechanistic understanding together with the new bio-
catalyst variants identified should promote further expan-
sion of carbene scope for borylation and other carbene-
transfer reactions.
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 lactone-
type IPCs. Unlike 5-membered lactone-based IPC2, which
takes on a rigid planar structure, 6-membered lactone-car-
bene IPC3 showed a slightly flexible structure with a dihe-
dral angle d(Fe-C-C-O) of 60° in the ground OSS state, while
its triplet state still possesses a near-planar geometry (Fig-
ure 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 elec-
tronic states in IPC4 shares a similar structure with IPC2.
This may explain why BORLAC, which was evolved for the 5-
membered 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 car-
Funding Information
Financial support from the NSF Division of Molecular and Cellular
Biosciences grant MCB-1513007, National Natural Science Founda-
tion 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 Res-
nick Sustainability Institute at Caltech for fellowship support. X.H. is
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Letter
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supported by an NIH pathway to independence award (grant
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(25) Chen, K.; Huang, X.; Kan, S. B. J.; Zhang, R. K.; Arnold, F. H.
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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.
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Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0037-1611662.
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To a 20 mL vial were added a suspension of E. coli expressing
Rma cytochrome c variant BORLAC (5 mL, OD600 = 15), borane
(150 L of a 400 mM stock solution in MeCN, 0.06 mmol),
lactone diazo compound (150 L 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 vor-
texed (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 Na2SO4 and con-
centrated under reduced pressure. Purification by silica gel
column chromatography with hexane/(ethyl acetate/acetone
3:7) as eluent afforded the desired lactone-based organo-
boranes. 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, CDCl3):
 = 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, CDCl3):  = 187.91, 120.62, 67.86, 36.15, 30.86;
11B NMR (128 MHz, CDCl3):  = –27.08 (t, J = 89.6 Hz); HRMS
(FAB+): m/z [(M + H+) – H2] calcd for C9H14O2N2B: 193.1148;
found: 193.1144; Chiral HPLC (Chiralpak IC, 40% i-PrOH in
hexane, flow rate: 1.5 mL/min, temperature: 32 °C,  = 235 nm):
tR (major) = 12.857 min, tR (minor) = 10.991 min; 96.2:3.8 e.r.
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