Enzymatic Regio- And Enantioselective C-H Oxyfunctionalization of Fatty Acids

Letter

Enzymatic Regio- and Enantioselective C−H Oxyfunctionalization of

Fatty Acids

Hao Chen, Mengfei Huang, Wenliang Yan, Wen-Ju Bai,* and Xiqing Wang*

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sı Supporting Information

ABSTRACT: Directed evolution of a P450 hydroxylase (P450_B_S_β

)

achieves an engineered enzyme that is able to catalyze C−H

oxyfunctionalization of fatty acids (FAs) in a highly regio- and

enantioselective fashion (>20:1 Cβ/Cα and > 99% ee in all cases).

The biocatalyst displays high reactivity (TON up to 1540), takes

inexpensive H O as oxidant, and converts C11−C18 saturated

FAs as well as naturally derived unsaturated oleic and linoleic acids

to optically pure β-hydroxy FAs. Merging biocatalysis with chemical transformation, we further oﬀer a chemoenzymatic strategy to

access valuable FA derivatives bearing 1,3-diol, β-amino, β-lactone, and β-lactam functionalities in either enantiomeric form.

Molecular docking studies provide a rationale for the regio- and enantioselectivity of this reaction.

KEYWORDS: protein engineering, biocatalysis, cytochrome P450 enzymes, β-hydroxylation, fatty acids

aturally occurring β-hydroxy fatty acids (β-OH FAs)

are an important class of compounds, demonstrating

extremely challenging: nonasymmetric regioselective functional-

ization of unactivated C−H bonds is elusive, let alone its

enantioselective variant. Synthetic methods often use transition

metal complexes and require innately diﬀerentiated C−H bonds

within a substrate or a previously installed directing group to

attractive biological activity (Figure 1). For example, β-OH

decanoic acid (myrmicacin) is a natural herbicide, and β-OH

C10−C14 FAs exhibit antifungal properties. When incorpo-

rated with amino acids or carbohydrates, β-OH FAs create

more natural products with marvelous structural complexity

and biological interest. Serratamolide, a cyclic peptide, shows

satisfy regioselectivity, neither of which is applicable to FAs.

An enzyme instead embeds the substrate and the oxidation

agent within its structurally well-deﬁned active site and therefore

oﬀers a unique solution to regio- and enantioselectively oxidize

potential for developing antibiotic agents. Additionally, lipid A

is a component of an endotoxin, playing a physiological role in

human immune responses to the infection of Gram-negative

small molecules. In this respect, notable progress on bio-

catalytic hydroxylation of FAs using cytochrome P450s has

been made, but regioselectivity remains a key issue, and the

requirement of redox partners and electron-donating cofactors

bacteria. β-OH FAs can also serve as synthetic precursors to

the resulting β-amino, β-lactone, and β-lactam FA derivatives

that possess diverse bioactivities. For instance, rhodopeptin

further complicates the practical application. Excellent regio-

selectivity is only seen at Cα- (e.g., P450_S_P_α), Cω- (e.g., P450_B_M₃

and CYP153A), and recently realized Cδ-positions (e.g.,

and dieseloleiT6-6 display antifungal and antibiotic proper-

ties, respectively. Thus, β-OH FAs and derivatives are popular

targets for synthetic and pharmacological studies. Not surprisingly,

medicinal investigation of lipstatin, a potent natural inhibitor of

pancreatic lipases, has led to the discovery of orlistat, an FDA-

P450 ) with speciﬁc FAs. As to β-C-H oxyfunctionalization,

P450_B_S_βfrom Bacillus subtilis is the ﬁrst discovered enzyme that

can hydroxylate myristic acid, albeit giving a poor regiose-

approved drug for obesity treatment. In addition, polymer-

lectivity (1:1.5 Cα/Cβ). P450_C_L_Afrom Clostridium acetobu-

ization of β-OH FAs provides polyhydroxyalkanoates as a type

tyliticum can also fulﬁll this goal, but regioselectivity is inclined

of biodegradable and biocompatible plastics.

more to the Cα-site. Although P450_M_Pfrom Methylobacte-

Because of the signiﬁcance of β-OH FAs in natural product

synthesis, drug discovery, and biomaterials, their preparation has

been pursued. Popular chemical methods include asymmetric

aldol reaction of linear aldehydes and asymmetric hydro-

genation of β-keto esters, during which transition metals and

intricate catalytic ligands or stoichiometric chiral auxiliaries are

rium populi shows preference for the Cβ-position, hydrox-

ylation also occurs at Cα-, Cγ-, Cδ-, and Cε-sites together with

-alkene production. In addition, OleT_J_Efrom Jeotgalicoccus

Received: July 22, 2021

Revised: August 6, 2021

Published: August 12, 2021

usually required. Alcohol dehydrogenases may also asym-

metrically reduce β-keto esters, providing enzymatic access to

chiral β-hydroxy esters. However, β-C-H oxyfunctionalization

of bioavailable FAs represents a direct and step-economic way

to synthesize chiral β-OH FAs. Albeit ideal, this approach is

2021 American Chemical Society

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Letter

Figure 1. Bioactive fatty acid derivatives bearing various β-hydroxy and β-amino functionalities.

sp. ATTC 8456 yields 1-alkenes as the major product. Hence,

highly regioselective β-C-H oxyfunctionalization of FAs remains

unresolved.

used to quantify the conversion and product distribution using

authentic standards as references. The mutated gene rendering

a variant most selective at the Cβ-site was used as the template

for the next round of saturation mutagenesis. Gladly, P450_B_S_β

variants targeting residues R73, V74, L78, Q85, V170, A246,

F289, G290, M400, and P401 showed higher β-hydroxylation

selectivity than the wild-type (>5% increment) (Table S1).

Among them, an isoleucine substitution at residue G290

ampliﬁed the β-hydroxylation product most (30% → 66%),

beneﬁting from suppression of both α-OH myristic acid and

1-tridecene formations (46%+24% → 15%+19%) alongside

the reactivity improvement (63% → 90%) (Figure 2b). The

bulkier lipophilic side chain introduced by the isoleucine likely

altered the hydrophobic interaction with the aliphatic chain of

myristic acid and consequently positioned the substrate’s

β-methylene more approachable to Compound I (Figure 2a).

The G290 residue of P450_B_S_βin the FA α-hydroxylase, P450_S_P_α

(from Sphingomonas paucimobilis), is F288, and unsurprisingly,

the P450_B_S_β-₂G290F variant shifted the hydroxylation toward

As a peroxygenase, P450_B_S_βforms the iron(IV)-oxo heme

π-cation radical intermediate (Compound I) from inexpensive

H O (peroxide shunt pathway), representing an advantage

over typical P450s that require redox partners and electron-

donating cofactors to activate O . This highly reactive species

then abstracts Cα/Cβ hydrogen atoms of FAs, followed by

either an ·OH rebound step to give the respective α/β-OH FAs

or a C−C bond scission to yield 1-alkene. Previous search for

improved regioselective P450 variants with hydrophobic

amino acid substitutions (Ala, Phe, Val, Leu, Ile, and Trp) and

combination of beneﬁcial mutations neither realized a universally

satisfying regioselectivity (β-OH/(α-OH+β-OH)<90%) that is

suﬃcient for preparing chiral β-hydroxy FAs nor rationalized the

stereochemical outcome of the transformation from a mechanistic

perspective. We envisioned that directed evolution of P450_B_S_β

could possibly remodel its catalytic pathway to favor a pre-

dominant Cβ-H abstraction and concurrently shut down the

C−C bond scission route, thereby achieving the desired highly

regio- and enantioselective β-hydroxylation of fatty acids.

Herein, we report our ﬁndings.

BSβ

the Cα-site. Interestingly, a valine substitution at this site

(G290V) improved 1-alkene production.

The G290I mutant plasmid was then used as the template

for the next round screening at residues L78 and V170, as both

residues contained the second-best hits in the initial screening.

The P450_B_S_β-L78A mutation was reported to aid the β-selectivity

To start with, the iterative saturation mutagenesis strategy

was employed to generate libraries, from which the cell-free

lysates of Escherichia coli expressing P450_B_S_βvariants were used

to catalyze the oxidation of myristic acid (FA C14:0, 1c). The

initial libraries targeted residues that lie within 7 Å from the

substrate in the crystal structure of P450_B_S_β(PDB ID: 1IZO)

Supporting Information, Figure S1). Following product

extraction and esteriﬁcation, gas chromatography (GC) was

in hydroxylation of decanoic acid (67:33 Cβ/Cα). We

discovered that the double variant, P450_B_S_β-L78A/G290I, more

eﬀectively boosted the β-regioselectivity (84:7 Cβ/Cα) with

67% conversion (Figure 2b). No major improvement was

observed from the V170 library alone. Nevertheless, a triple

variant, P450_B_S_β-L78A/V170I/G290I, was identiﬁed to give an

(

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Figure 2. Directed evolution of P450_B_S_βfor β-hydroxylation of myristic acid. (a) Residues involved in the directed evolution shown in the crystal

structure of P450_B_S_β. (b) Product proﬁle and conversion of each round of selection. Reaction conditions: P450_B_S_βvariant (1.0 μM), myristic acid

(

1 mM), H O (2 mM), 0.1 M KPi (pH 7.0), 0.3 M KCl, 0.125% TritonX-100 (v/v), 5% EtOH (v/v) (cosolvent), 1 mL scale, rt, 30 min.

2 2

enhanced β-hydroxylation selectivity (88:6 Cβ/Cα) and better

conversion (67% → 79%) (Figure 2b). This suggests that the

V170I and L78A substitutions act cooperatively to tweak

the substrate conformation. Based on the crystal structure,

these two residues also have the potential to aﬀect the activating

residue R242 that anchors the carboxylate motif of the substrate

Furthermore, the (R)-conﬁguration of the enzymatic products

matches the enantioselective outcome of wild-type P450_B_S_β

Also, regioselectivity was well controlled (>20:1 Cβ/Cα in all

cases), albeit partial formation of 1-alkenes arising from the

largely suppressed C−C bond scission pathway. To demonstrate

the scalability and practicality of our biocatalyst, reactions of

substrates 1b−c were performed using a slightly lower catalyst

loading (0.06 mol %) at 1.0 and 2.5 mmol scales (entries 2−

(

Figure 2a).

The fourth round targeting Q85 and A246 residues reached

[

f‑g]

a quadruple variant, namely P450_B_S_β-L78A/Q85A/V170I/

G290I, which highly regioselectively hydroxylated myristic

acid (92:1 Cβ/Cα) with excellent reactivity (80% conv.) and

therefore concluded our directed evolution eﬀorts (Figure 2b).

The Q85H replacement was found to impact the preference of

the enzymatic pathway between the ·OH rebound and C−C

). 141 mg and 500 mg of β-OH FAs 2b−c were readily

isolated, awaiting their synthetic diversiﬁcations.

To shed light on the incredible regio- and enantioselectivity

of this reaction, the crystal structure of P450_B_S_βwas used as

the template to build the P450_B_S_β-L78A/Q85A/V170I/G290I

structure. Molecular docking simulations of myristic acid into

the active site of the engineered P450_B_S_βwere performed, and

the most energetically favored substrate-binding conformation

is depicted (Figure 3). The distances from the Compound I

oxygen to the nearest Cα-, Cβ-, and Cγ-H atoms (in purple)

are identiﬁed as 4.5, 3.5, and 5.2 Å, respectively, elucidating

the predominant preference for β-regioselectivity. Of the two

Cβ-hydrogen atoms, the pro-R-H (in purple) sits closer to the

Compound I oxygen than the pro-S-H (in cyan) (3.5 vs 5.0 Å),

resulting in the (R)-conﬁguration product. It is worth noting

that the four mutational residues introduced by directed evolution

alter the substrate conformation and consequently bring the

β-methylene closer to the Compound I but push the α-methylene

away from it (Figures S3 −S4). Therefore, this work supports

the view that the second coordination sphere of a protein can

19,26

bond scission routes.

Kinetic studies of our biocatalyst revealed its

good catalytic performance with a turnover frequency (TOF)

−1

of 46 min (Figure S2 ) and a turnover number (TON) of 1540

for myristic acid. Additionally, this enzyme was most active at

mM H O , displaying good tolerance of H O (Table S2).

The scope of this enzymatic reaction was subsequently

2 2 2 2

investigated (Table 1). Exploiting 0.1 mol % of P450_B_S_β-L78A/

Q85A/V170I/G290I smoothly hydroxylated C11−C18 satu-

rated FAs 1a−e as well as naturally derived unsaturated oleic

and linoleic acids 1f−g, all at preparative scale (0.4 mmol).

Acceptable to good yields (20−85%) were obtained. Myristic

acid 1c displayed the highest reactivity, whereas FAs with a

shorter or longer alkyl chain led to diminished reactivity.

Previous work shows that short-chain FAs have a loose ﬁxation

in the enzyme’s substrate-binding channel and results in low

inﬂuence the product distribution by substrate positioning.

reactivity. In this regard, we found that the decreased enzymatic

activity for long-chain FAs, such as C16:0 and C18:0 (1d−e), was

partially caused by poor aqueous solubility of these substrates.

Lowering the concentration of 1e while maintaining the same

catalyst loading gave higher enzyme turnovers and yields (e.g.,

As shown in Figure 1, bioactive FA derivatives manifest

a broad spectrum of β-functionalities, including β-hydroxy,

β-amino, β-lactone, and β-lactam motifs. Moreover, chiral

1,3-diol scaﬀold is commonly seen in polyketides. As a result, we

presented herein a chemoenzymatic approach to access these

β-functionalities by combining our newly developed biocatalyst

with chemical transformations. With β-OH FAs 2b−c in hand,

we commenced their synthetic diversiﬁcation (Figure 4). Use

of BH ·Me S smoothly reduced the carboxylic acid moiety of

TON_β_‑_O_H= 554 and 55% GC yield at 1 mM [1e ] vs TON_β_‑_O_H

320 and 32% GC yield at 4 mM [1e]) (Table S3 ). Remarkably,

enantiopure β-OH FAs were obtained in all cases. The abosolute

stereochemistry of all products was conﬁrmed either by direct

comparison of optical rotations with those recorded in the literature

or through a chemical transformation to a known compound

followed by comparison of optical rotations (see SI , Table S4).

2c to provide the chiral 1,3-diol 3 in 95% yield (Figure 4, a).

Treatment of 2b with (PyS) formed a pyridyl thioester that

upon exposure to Hg(OMs) cyclized to chiral β-lactone 4 in

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Table 1. β-Hydroxylation of Various FAs by P450_B_S_β-L78A/Q85A/V170I/G290I

Reaction conditions: P450_B_S_β-L78A/Q85A/V170I/G290I (4 μM), substrate (4 mM), H O (5 mM × 2), 0.1 M KPi (pH 7.0), 0.3 M KCl, 0.5%

TritonX-100 (v/v), 5% EtOH (v/v) (cosolvent), 100 mL scale, rt, 45 min. Isolated yield. Determined by chiral HPLC. Determined by crude

H NMR. Determined by GC. 1 mmol (200 mg) scale. 2.5 mmol (570 mg) scale.

Figure 3. Docking structure of myristic acid (green) in P450_B_S_β

L78A/Q85A/V170I/G290I. The distances between the compound I

(

green) oxygen (red ball) and relevant Cα, Cβ, Cγ hydrogen atoms

purple and cyan) are indicated.

Figure 4. Synthetic diversiﬁcation of β-hydroxy fatty acids.

0% yield (Figure 4, b). Subjecting 2c to BnONH in the

participated in a Mitsunobu reaction using DIAD and PPh to

yield a BnO-protected β-lactam. Its successive reduction with

presence of EDC gave a β-OH hydroxamate, which then

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SmI cleaved the N−O bond and released enantiopure β-lactam 5

Author Contributions

(

Figure 4, c). In addition to being directly embedded in bioactive

H.C., M.H., and W.Y. contributed equally to this work. X.W.

and W.-J.B. conceived and designed the project. H.C., M.H.,

and W.Y. performed the experiments and collected the data.

H.C., X.W., and W.-J.B. wrote the manuscript with contri-

butions from all authors.

molecules, β-lactone and β-lactam are also important synthetic

precursors. For example, β-lactone 4 can be directly used to

build fellutamides A−C, which are natural lipopeptide proteasome

inhibitors. Exposure of β-lactam 5 to TMSCl in MeOH

delivered chiral β-amino acid derivative 6 in 90% yield (Figure 4, d).

Notably, the (R)-stereochemistry of β-OH FA 2b, obtained from

our enzymatic reaction, could be stereospeciﬁcally inversed to its

Notes

The authors declare no competing ﬁnancial interest.

(

S)-conﬁguration in a Mitsunobu reaction using p-NO -C H CO H

2 6 4 2

ACKNOWLEDGMENTS

■

as a nucleophile followed by the benzoic ester hydrolysis with

K CO (Figure 4, e). As both enantiomers of β-OH FAs are

X.W. acknowledges the ﬁnancial support from Yangzhou

University, the Interdisciplinary Project from Veterinary Science

of Yangzhou University (Grant No. YZUXK202002), and the

analytical assistance from the Testing Center of Yangzhou

University.

now accessible, the aforementioned 1,3-diol, β-amino, β-lactone,

and β-lactams can be assembled in either enantiomeric form, if

wanted.

In conclusion, directed evolution of a P450_B_S_βhydroxylase

has enabled a highly regio- and enantioselective oxyfunctionaliza-

tion of unactivated C−H bonds of FAs that is elusive through

synthetic methods. This scalable enzymatic transformation hydro-

lylates C11−C18 saturated and naturally derived unsaturated

FAs directly using inexpensive H O as the oxidant. Molecular

docking studies rationalize the regio- and enantioselectivity.

The aﬀorded chiral β-OH FAs further reach a library of FA

derivatives bearing structurally diverse β-functionalities that are

ubiquitous in bioactive molecules. Our work demonstrates a

chemoenzymatic strategy to access diversiﬁed products from

cheap and bioavailable FAs by protein engineering and chemical

elaboration.

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ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acscatal.1c03292 .

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AUTHOR INFORMATION

Corresponding Authors

■

Xiqing Wang − College of Bioscience and Biotechnology,

Yangzhou University, Yangzhou, Jiangsu 225009, China;

orcid.org/0000-0002-6520-6814; Email: xiqingwang@

yzu.edu.cn

Wen-Ju Bai − Department of Chemistry, Stanford University,

Stanford, California 94305, United States; Present

Address: W.-J.B.: Amgen Inc., 1 Amgen Center Drive,

Thousand Oaks, CA 91320, USA ; orcid.org/0000-

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Yangzhou University, Yangzhou, Jiangsu 225009, China

Mengfei Huang − College of Bioscience and Biotechnology,

Yangzhou University, Yangzhou, Jiangsu 225009, China

Wenliang Yan − College of Bioscience and Biotechnology,

Yangzhou University, Yangzhou, Jiangsu 225009, China

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