Regio- and Enantio-selective Chemo-enzymatic C?H-Lactonization of Decanoic Acid to (S)-δ-Decalactone

Article abstract of DOI:10.1002/anie.201901242

The conversion of saturated fatty acids to high value chiral hydroxy-acids and lactones poses a number of synthetic challenges: the activation of unreactive C?H bonds and the need for regio- and stereoselectivity. Here the first example of a wild-type cytochrome P450 monooxygenase (CYP116B46 from Tepidiphilus thermophilus) capable of enantio- and regioselective C5 hydroxylation of decanoic acid 1 to (S)-5-hydroxydecanoic acid 2 is reported. Subsequent lactonization yields (S)-δ-decalactone 3, a high value fragrance compound, with greater than 90 % ee. Docking studies provide a rationale for the high regio- and enantioselectivity of the reaction.

Full text of DOI:10.1002/anie.201901242

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Accepted Article
Title: Regio- and Enantio-selective Chemo-enzymatic C-H-lactonization
of Decanoic Acid to (S)-δ-Decalactone.
Authors: Jack Manning, Michele Tavanti, Joanne Porter, Nico Kress,
Sam De Visser, Nicholas Turner, and Sabine Flitsch
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201901242
Angew. Chem. 10.1002/ange.201901242
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Regio- and Enantio-selective Chemo-enzymatic C-H-lactonization
of Decanoic Acid to (S)-δ-Decalactone.
a
a
a
a
b
Jack Manning , Michele Tavanti , Joanne L. Porter , Nico Kress , Sam P. De Visser , Nicholas J.
a
a
Turner and Sabine L. Flitsch *
Abstract: The conversion of saturated fatty acids to high value
chiral hydroxy-acids and lactones poses a number of synthetic
challenges: the activation of unreactive C-H bonds and the need
for regio- and stereoselectivity. Here we report the first example
of a wild type cytochrome P450 monooxygenase (CYP116B46
from Tepidiphilus thermophilus) capable of enantio- and
regioselective C5 hydroxylation of decanoic acid 1 to (S)-5-
hydroxydecanoic acid 2. Subsequent lactonization yielded (S)-δ-
decalactone 3, a high value fragrance compound, with > 90 % ee.
Docking studies provide a rationale for the high regio- and
enantioselectivity of the reaction.
regioselective ‘mid-chain’ C5 and C6 hydroxylation (γ- and δ-
positions, respectively) of fatty acids, and additional
stereoselectivity, is still highly challenging.[19] Yet, γ- and δ-
hydroxylated fatty acids are important intermediates for the
production of the corresponding lactones via intramolecular
esterification. These lactones find a variety of commercial uses
within flavourings and fragrances,[20] in particular δ-decalactone
(3), a common fragrance compound, with the (S)-enantiomer
presenting a significantly more intense creamy peach note
compared to the (R)-isomer, which is found preferentially in
Nature.[21,22] A number of stereoselective biocatalytic methods
have been reported for these lactones including use of Baeyer–
Villiger monooxygenases,[23] alcohol dehydrogenases and
biocatalytic cascades, respectively.[24,25] However, all reported
Hydroxy fatty acids (HFAs) are a group of naturally derived
compounds that are ubiquitous in Nature. They find a wide range
of diverse applications in many high value chemical industries
such as flavourings, fragrances, food supplements and
pharmaceuticals.[1–4] The relationship between hydroxyl and
carboxylic acid functional groups often has a marked effect in their
properties. For example, α-hydroxyacids are used in many
cosmetics and have been shown to possess anti-ageing
properties whereas short chain β-hydroxyacids have been shown
to display antimicrobial properties.[5,6] Hence, there is a need for
selective synthetic methods towards HFAs, ideally by direct
oxidation of readily available fatty acids. The chemical
oxyfunctionalisation of non-activated C-H bonds in fatty acids is
one of the most demanding chemical reactions, because of the
additional need for regio-selective targeting of one of many
methylene groups and demands on enantioselectivity on highly
synthetic methods start from functionalized materials including
[26–29]
11-hydroxy palmitic acid, coriolic acid and Massoia lactone.
[
7]
flexible substrates. In this regard, biocatalysis using P450
monooxygenases (P450s, CYPs) represents an attractive option
for selective C-H bond activation with the added advantage of mild
reaction conditions. Consequently, fatty acid hydroxylation
catalysed by P450s has been widely investigated, but often
suffers from poor regioselectivity and limitation to terminal
positions.[8–12] For example, P450-BM3 has been reported to
hydroxylate unsaturated fatty acids (C10:0-C18:0) at positions ω-
1
, ω-2 and ω-3.[
13,14]
Some elegant studies using genome mining
and protein engineering have sought to address this issue and
resulted in new product profiles.[15–18] Nevertheless, the
[
[
a]
b]
J. Manning, Dr. M. Tavanti, Dr. J. L. Porter, Dr N. Kress, Prof. N. J.
Turner, Prof. S. L. Flitsch.
Manchester Institute of Biotechnology (MIB), School of Chemistry.
The University of Manchester 131 Princess Street, M1 7DN,
Manchester (United Kingdom).
Scheme 1. A) Biocatalytic synthesis of δ-decalactone (3) by regio- and
stereoselective hydroxylation of decanoic acid (1) with CYP116B46 followed by
para-toluenesulfonic acid catalysed lactonization to S-δ-decalactone.
*
E-mail:Sabine.Flitsch@manchester.ac.uk
Dr S. P. De Visser
School of Chemical Engineering and Analytical Science.
The University of Manchester, Oxford Road, Manchester, M13 9PL.
Supporting information for this article is given via a link at the end of
the document
The extraction of Massoia lactone from Cryptocaria massoia
poses environmental concerns,[30] whereas the chemical
synthesis requires transition metal catalysts, peracids and organic
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high activity towards medium chain length fatty acids.[13,35] Initial
screening reactions were performed with 0.4 mM substrate
concentration and cell-free extracts containing 10 µM enzyme as
determined by CO difference spectroscopy. A cofactor recycling
system consisting of glucose and glucose dehydrogenase was
used to regenerate the NADPH cofactor. All P450s showed
activity towards fatty acid substrates, with P450-AT and P450-JT
showing particularly good conversions up to quantitative levels
across the panel of fatty acids (Table 1).
Table 1. Initial screen of fatty acids 1,4 and 5 against a panel of
self-sufficient cytochrome P450 monooxygenases.
Regio-
selectivity [%]
Enzyme
JT Hal1 RhF BM3
SubstrateA
AT AX TB TT
65 18 46
conversion[B]
89
>99
43
46
11
63
28
0
94
57
39
4
31
41
56
3
n.d.
0
[
%]
C7
C6
37 33 39 67
60 40 61 18
Octanoic
acid
(4)
0
Octanoic acid (4) was mainly converted to the C6 and C7 hydroxy
fatty acids with P450-TT and P450-Hal1 showing slight
preference to hydroxylation at C7, whereas P450-AT, P450-TB
and P450-RhF generated the C6 hydroxylated isomer as the main
product. Interestingly, P450-AX, TT and JT were capable of
performing C-H activation at the C5 position more efficiently than
the other enzymes tested, although the C7 and C6 hydroxylated
isomers remained the major products. When presented with
decanoic acid (1), P450-TB and Hal1 formed only the C9 and C8
hydroxy fatty acids. P450-TB hydroxylated decanoic acid
predominantly at the C9 position (79%), whereas the opposite
was observed for P450-Hal1 (65% C8 hydroxylation). P450-BM3
preferentially formed the C9 hydroxylated product (66%), with
minor C8 and C7 hydroxy fatty acids, in accordance with literature
reports.[13] A variety of hydroxylated products were observed
when P450-AT and P450-AX were reacted with decanoic acid:
P450-AT did not show any preferred position for hydroxylation,
whereas P450-AX preferentially formed the C9 hydroxylated
product (48%).
C5
2
27
0
14
0
conversion
49
42 30 83
60
35
65
0
n.d. >99
[
%]
C9
C8
C7
C6
25 48 79
20 17 21
0
0
0
0
66
21
13
0
Decanoic
acid
(1)
15 14
16
24 22
0
0
0
0
0
0
0
10
90
21
51
>99
9
0
0
C5
0
0
0
conversion
90
65 78 68
68
74
26
0
63
88
12
0
>99
51
40
9
[
%]
C11
C10
C9
72 48 90
0
0
12
0
8
0
10
0
5
Dodecanoic
acid
(5)
0
0
C6
17 10
34
0
24
76
12
74
0
0
0
Of particular interest and surprise was the observation that some
of the enzymes tested are capable of forming the mid-chain C6
and C5 hydroxylated products. Hydroxylation of decanoic acid by
P450-AX generated 5-hydroxydecanoic acid amongst the other
regioisomers (22%), whereas P450-AT formed both C6 and C5
hydroxylated products (16% and 24% products, respectively).
P450-JT predominantly targeted the C5 position (51%), whereas
P450-TT displayed excellent regioselectivity for the C5 position
C5
0
0
0
0
0
[
A]. 400 µl reactions performed with cell-free extracts containing 10 µM
-1 + -1
enzyme, 0.4 mM substrate, 1 mg mL NADP , 1 mg mL glucose
dehydrogenase (CDX-901), 10 mg mL- D-glucose, 100 mM sodium
phosphate buffer (pH 8), 30˚C, 24h. [B]. Substrate conversion was
determined based on substrate depletion measured by comparison with
reactions carried out with an empty vector control. [n.d.] not detected.
1
(
90%), with only minor (10%) C6 hydroxylated product formed.
solvents.[31] Therefore, the direct P450-catalysed γ- and δ-
hydroxylation of simple bioavailable fatty acids such as 1
represents a highly attractive alternative to access lactones such
as 3, in particular when targeting single enantiomers (Scheme 1).
Here we report the screening and identification of P450
monooxygenases capable of generating hydroxy derivatives of
fatty acids at mid-chain positions. To our surprise, one of the
enzymes (P450-TT) was found to catalyse the production of (S)-
Indeed, this observed regioselectivity for wild-type P450-TT is
significantly higher than reported previously for engineered P450-
BM3 variants (42% and 19% for C5 and C6 regioisomers
respectively). Using dodecanoic acid (5) as substrate, P450-TB,
Hal1 and RhF again exclusively formed C11 and C10 hydroxy
fatty acids and P450-BM3 preferentially targeted the C11 position
[19]
(
51%). P450-AT formed a mixture of products, including C6
hydroxylated dodecanoic acid (17%). Again, P450-TT and P450-
JT displayed the best regioselectivity for C-H bonds in the middle
of the fatty acid chain, forming more than 70% C5 hydroxylated
product.
5-hydroxydecanoic acid (2) with high regio- and enantio-
selectivity, leading to the fragrance lactone (3) by subsequent
chemical lactonization.
In the first instance, a panel of recently reported class VII P450s
was screened with saturated fatty acids of chain lengths 8, 10 and
1
We were especially interested in the excellent regioselectivity for
the challenging C5 hydroxylation of decanoic acid to 5-hydroxy
decanoic acid displayed by P450-TT, because this reactivity
2 carbon atoms (1, 4, 5 respectively) (Table 1).[32–34] Class VII
P450s (such as CYP116B) are self-sufficient monooxygenases,
which had not previously been investigated with fatty acids in such
a systematic manner. The better-studied P450-BM3 (member of
the other self-sufficient class VIII P450s) was included in the
assembled P450 panel for comparison, due to its well established
creates precursor 2 for the valuable δ-decalactone (3). Using the
[36]
recently solved crystal structure of P450-TT,
we performed
molecular docking simulation of decanoic acid into the heme
domain of P450-TT (Figure 1). The energetically most favoured
simulation creates a reasonable binding pose for promoting
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substrate hydroxylation.[37] This simulation suggests that a ‘U-
shaped’ conformation of decanoic acid above the heme prosthetic
group favours the regioselective C5 hydroxylation, which is in
accordance with previous reports of P450-mediated hydroxylation
of fatty acids in the centre of the fatty acid chain.[38] Furthermore,
the formation of the (S)-hydroxy fatty acid enantiomer is favoured
by this model. The substrate carboxy group seems to be
positioned by polar interactions to residues T202 and E277
substrate loadings (5 and 20 mM, Table S4). The identity of
isolated product 3 was confirmed by mass spectrometry and
nuclear magnetic resonance spectroscopy (see Supporting
Information). The product was analysed by chiral normal-phase
HPLC (Figures 2 and S14) which confirmed formation of the (S)-
δ-decalactone with over 90% enantiomeric excess in broad
agreement with the molecular docking predictions (Figure 1).
Absolute configuration of the lactone product was assigned using
previously reported methods (see supporting information).
(
Figure 1), while multiple hydrophobic interactions promote the U-
shape binding of the carbon chain. In particular, residues L92,
A270 and V273 (Figure 1) appear to sandwich the substrate’s tail.
Given that the enzyme pocket does not contain an obvious
counterion to the carboxylate, as observed in P450-BM3,
substrate docking was performed both with the protonated as well
as in its deprotonated substrate (Figure S1). Although both
simulations resulted in a similar U-shaped conformation, it is
interesting to note that only the binding pose of the protonated
substrate places the pro-S C-5 hydrogen closest to the iron centre
in agreement with the experimentally observed selectivity.
Figure 2. Chiral normal-phase HPLC trace for the isolated δ-decalactone 3 after
preparative scale biotransformation of decanoic acid 1
In summary, our work provides evidence that class VII P450s are
useful catalysts for generating a variety of hydroxy-carboxylic
acids directly from the parent acids. Both terminal and ‘mid-chain’
C-H activation was observed, with the latter leading to important
products such as the valuable fragrance compound (S)-δ-
decalactone 3 with remarkable regio- and stereo-selectivity. The
wild-type enzymes identified here represent a valuable addition to
the protein scaffolds that can be employed as starting points for
further protein engineering to generate optimised biocatalysts for
chiral lactone synthesis.
Figure 1. Docking structure (VINA approach as implemented in the YASARA
software) of decanoic acid (white) in P450-TT (PDB 6GII). The figure shows the
distance (dotted line) between the hydrogen at the C-5 pro-S hydrogen and the
compound I oxygen (black ball). of the heme prosthetic group (black) and the
potential reaction trajectory (angle).
The research leading to these results received funding from the
Centre of Excellence for Biocatalysis, Biotransformations and
Biocatalytic manufacture (CoEBio3) and the European Union’s
Seventh Framework Programme for research, technological
development and demonstration under grant agreement no.
Next, the biotransformation of P450-TT and decanoic acid was
studied in more detail to achieve a set of conditions optimal for
the formation of 3. The best conversion value (68%) was obtained
when 10 μM enzyme was incubated with 2 mM substrate at 40°C
613849 (BIOOX), ERC (788231-ProgrES-ERC-2017-ADG).
Additionally, we would like to thank Mrs. Rehana Sung for the
valuable analytical support.
(
Figure S3c). Spontaneous lactonization of 5-hydroxy decanoic
acid to the corresponding lactone was indeed observed after GC
analysis, however this accounted for only ~6.5% of substrate
converted (Figure S12). Some slight increase in substrate
conversion was observed when 30% decane was added to the
biotransformation (Table S3). Using the optimized conditions, the
reaction between decanoic acid and P450-TT was then
performed on a preparative scale to confirm the product identity
and determine product stereochemistry, since the stereoisomers
are not commercially available. Preparative scale reactions were
carried out at larger volumes (10 and 100 mL) with different
Keywords: Biocatalysis • Cytochrome P450 • Regioselectivity •
Fatty acids • Lactones
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