Key words
cytochrome
c
- carbene - organoboron - lactones - biocatalyst - directed evolution
The significant role of organoboron chemistries[1]
[2]
[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 [1]A),[11]
[12]
[13]
[14]
[15]
[16]
[17]
[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 [1]B).
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
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 [1]C).[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]
[20]
[21]
[22]
[23]
[24]
[25]
[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]
[28]
[29]
[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.
Figure 2 Electronic states of acyclic IPC1 and cyclic IPC2 intermediates. The Gibbs free energies were obtained at the B3LYP-D3(BJ)/def2-TZVPP//B3LYP/def2-SVP
level. OSS = open-shell singlet, CSS = closed-shell singlet
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-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]).
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 quadruplicate: 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. Enantiomeric 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, 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 [3]A).[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 (BORLAC
) 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
[4]A). 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.
Figure 4 A. Scope of lactone-carbene B–H insertion with BORLAC
. Reactions were conducted in quadruplicate: suspension of E. coli expressing 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 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
(OD600 = 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)
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 [4]C). However, we did notice that enantioselectivities 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 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 BORLAC
(Figure [4]A). 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
[4]B). However, with one additional carbon in the ring, the 7-membered lactone-carbene
behaved in a completely different manner: BORLAC
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 lactone-type 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 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 carbene for B–H insertion;
further engineering using BORLAC
as a starting template could well lead to variants with improved reactivity on this
7-membered lactone carbene.
Figure 5 Comparison of 5-, 6- and 7-membered lactone-based IPCs. The Gibbs free energies were
obtained at the B3LYP-D3(BJ)/def2-TZVPP//B3LYP/def2-SVP level
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 carbene-transfer
reactions.