Enzymatic Oxidative Tandem Decarboxylation of Dioic Acids to Terminal Dienes

Article abstract of DOI:10.1002/ejoc.201600358

The biocatalytic oxidative tandem decarboxylation of C₇–C₁₈dicarboxylic acids to terminal C₅–C₁₆dienes was catalyzed by the P450 monooxygenase OleT with conversions up to 29 % for 1,11-dodecadiene (0.49 g L^–1). The sequential nature of the cascade was proven by the fact that decarboxylation of intermediate C₆–C₁₁ω-alkenoic acids and heptanedioic acid exclusively gave nonconjugated 1,4-pentadiene; scale-up allowed the isolation of 1,15-hexadecadiene and 1,11-dodecadiene; the system represents a short and green route to terminal dienes from renewable dicarboxylic acids.

Full text of DOI:10.1002/ejoc.201600358

DOI: 10.1002/ejoc.201600358
Communication
Dienes from Diacids
Enzymatic Oxidative Tandem Decarboxylation of Dioic Acids to
Terminal Dienes
Alexander Dennig,[a] Sara Kurakin,[b] Miriam Kuhn,[b] Andela Dordic,[a] Mélanie Hall,[b] and
Kurt Faber*[b]
Abstract: The biocatalytic oxidative tandem decarboxylation of of intermediate C6–C11 ω-alkenoic acids and heptanedioic acid
C7–C18 dicarboxylic acids to terminal C5–C16 dienes was cata- exclusively gave nonconjugated 1,4-pentadiene; scale-up al-
lyzed by the P450 monooxygenase OleT with conversions up to lowed the isolation of 1,15-hexadecadiene and 1,11-dodecadi-
29 % for 1,11-dodecadiene (0.49 g L–1). The sequential nature ene; the system represents a short and green route to terminal
of the cascade was proven by the fact that decarboxylation dienes from renewable dicarboxylic acids.
Introduction
E. coli and electron-transfer proteins Fdr/FldA) led to mixtures
of 1-alkenes (4–48 mg L–1 d–1),[9a,9e] and dienes were only de-
tected in their terminal/internal forms (e.g., 1,10-heptadecadi-
ene).[7,9e] Other promising 1-alkene-forming enzymes are the
non-heme di-iron monooxygenase UndA,[11] which is limited by
a narrow substrate scope and low total turnover numbers
(TTNs), and ferulic acid decarboxylases,[12] which require α,ꢀ-
unsaturated acids as substrates. On the other hand, aliphatic
diacids are widely distributed in various metabolic pathways[13]
and hence represent a renewable basis for the synthesis of ter-
minal dienes. Alternatively, enzymatic ω-oxidation of FAs yields
C6–C22 diacids in industrially relevant quantities (>100 g L–1).[14]
Further sources of diacids (e.g., adipic acid) are polyester waste
materials.[2c]
To reposition the chemical economy, novel metal-free synthetic
routes to chemical building blocks from renewable C sources
are demanded.[1,2] Among the primary platform chemicals,
short- and medium-chain 1-alkenes (obtained by steam crack-
ing of crude oil at >800 °C)[3] are of outstanding economic im-
portance.[4] In contrast, terminal dienes (>C4) cannot be ob-
tained from steam cracking, the Shell higher olefin process
(SHOP) process,[4b] or the chemical decarboxylation of fatty ac-
ids (FAs),[3,5] but they are synthesized by cross-metathesis cleav-
age of cycloalkenes or polyenes with lower alkenes, such as
ethylene (ethenolysis).[6] As dienes represent essential building
blocks for synthetic rubbers, co-crosslinkers, and starting mate-
rials for macrocycle synthesis, alternative routes are highly de-
sired.[1b,2d] A sustainable synthetic route to terminal dienes from
dicarboxylic acids has so far not been reported. In 2011, Rude
et al. reported the first direct enzymatic oxidative decarboxyl-
ation of FAs by the P450 monooxygenase OleT to yield long-
chain 1-alkenes[7] through a not-yet-elucidated mechanism.[8]
By employing whole cells, cell-free extracts, or purified enzyme
preparations,[7–9] the reaction proceeds either with H2O2 or O2
as the oxidant. By using purified OleT in combination with the
putidaredoxin electron-transfer system CamAB,[10] efficient
NADH (NAD = nicotinamide adenine dinucleotide) regeneration
Results and Discussion
To explore the synthetic potential and substrate scope of the
P450 monooxygenase OleT, we investigated the direct tandem
decarboxylation of dicarboxylic acids to terminal dienes
(Scheme 1). As saturated FAs are initially regarded as the natural
substrates of OleT, it was unclear to what extent terminal modi-
fication of the substrate would influence the overall catalytic
performance. Initial experiments with the use of 5a (10 mM)
and the OleT-CamAB-FDH reaction system[9c] and O2 as the
oxidant revealed the formation of 5c with 12.4 % conversion.
After product extraction and derivatization of the remaining
acid(s) in the reaction medium, α- and ꢀ-hydroxy diacids were
found as side products (in total 38 %, Table 1), whereas interme-
diate 5b was not found (see the Supporting Information). How-
ever, α- and ꢀ-hydroxy derivatives of 5b were detected. To ex-
plore the substrate scope, 10 dicarboxylic acids (i.e., com-
and atmospheric O2 as the oxidant allowed the synthesis of 1-
[9c]
alkenes ranging from C3 to C21
with product titers close to
1 g L–1. In contrast, in vivo production by using whole cells (e.g.,
[a] Austrian Centre of Industrial Biotechnology (ACIB), c/o Department of
Chemistry, Organic & Bioorganic Chemistry, University of Graz,
Heinrichstrasse 28, 8010 Graz, Austria
[b] Department of Chemistry, Organic & Bioorganic Chemistry, University of
Graz,
pounds 1a–10a, 10 mM) were subjected to the OleT-CamAB-
FDH reaction system (Scheme 1, Route 1; Table 1).
Terminal dienes 1c–10c were detected with up to 29 % con-
version at a 0.06 mol-% catalyst loading. The highest conversion
was obtained with 3a, which yielded up to 29 % of 3c; this
Heinrichstrasse 28, 8010 Graz, Austria
E-mail: Kurt.Faber@Uni-Graz.at
http://biocatalysis.uni-graz.at
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201600358.
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Communication
Scheme 1. Biocatalytic routes to terminal dienes through oxidative decarboxylation of (di)carboxylic acids by using P450 monooxygenase OleT. Route 1:
Tandem decarboxylation of dicarboxylic acids (C18–C7) to dienes with the OleT-CamAB-FDH cascade[9c] and O2 as oxidant. Route 2: Decarboxylation of ω-
alkenoic acids (C11–C6) with the OleT-CamAB-FDH cascade and O2 as oxidant. Route 3: Tandem decarboxylation of C14 dioic acid with OleT and H2O2 as
oxidant; 2- and 3-hydroxy diacid/ω-alkenoic acid side products are shown in brackets.
Table 1. Decarboxylation of dicarboxylic acids with the OleT-CamAB-FDH cascade at room temperature and at 4 °C.[a]
Entry
Substrate Diene [m
M]
TOF [h–1 [b]
]
TTN
r.t./4 °C
Diene [%]
r.t./4 °C
α-OH + ꢀ-OH diacids [%][c]
r.t./4 °C
α-OH + ꢀ-OH ω-enoic acids [%][c]
r.t./4 °C
r.t./4 °C
1
2
3
4
5
6
7
8
9
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
n.d./n.d.
1.12/0.74
2.93/1.63
0.31/0.95
1.24/1.22
0/2.05
n.d.
18
73
36
91
n.d.
n.d.
n.d.
n.d.
n.d.
n.d./n.d.
373/245
978/542
104/317
413/406
n.d./684
20/13
4/29
38/34
4/146
66/70
54/69
48/51
24/44
44/43
0/48
9/9[d]
25/21[d]
0/0[d]
11/6
1
2
2
9
46/31[d]
41/43
51/37
38/37
25/19
18/20
74[e]/39
n.d./n.d.
n.d./0
26[e]/13
n.d./n.d.
n.d./0
0.06/<0.05[f]
<0.05[f]/0.09
0.12/0.1
≥99[g]/≥99[g]
≥99[g]/≥99
≥99[g]/≥99[g]
≥99[g,h]/≥99[g,h]
n.d./n.d.
n.d./n.d.
n.d./n.d.
n.d./n.d.
10
<0.05/0.4
[a] General reaction conditions: Unless stated otherwise, all reactions were performed in 4 mL glass vials closed with a polytetrafluoroethylene (PTFE) septum
by using potassium phosphate buffer (KPi, pH 7.5, 0.1 ) containing 6 μ purified OleT, 0.05 U CamAB, 2 U FDH, 1200 U catalase (for H2O2 removal), 100 m
ammonium formate, 0.4 m substrate, and 5 % EtOH (v/v) in a final volume of 1 mL at r.t. (24 h and 170 rpm shaking) or at 4 °C (72 h and
NAD+, 10 m
M
M
M
M
M
100 rpm stirring). Reference data for the assignment of side products can be found in the Supporting Information; n.d.: not determined. [b] Turnover frequency
(TOF) calculated from the product amount after 1 h reaction time. [c] % GC area of products (α,ω-diene + α-/ꢀ-hydroxy acids = 100 % GC area). [d] Additional
minor side products were detected during conversion of diacids 1a and 2a; 5 % DMSO (v/v) was used as co-solvent. [e] No diene was formed; therefore, the
ratio between hydroxy diacid/hydroxy enoic acid peak areas is given. [f] Detection limit 50 μM. [g] Volatile fraction analyzed. [h] 1-Heptene was detected in
the volatile fraction; TOF (product extraction and quantification after 1 h reaction time) and TTN (product extraction and quantification after 24 or 72 h
reaction time) were calculated on the basis of quantified diene products. Volatile dienes 6c–10c were quantified by headspace GC–MS analysis by using
commercial standards for calibration (see the Supporting Information).
corresponds to a product titer of 0.49 mg mL–1 (or 0.49 g L–1) ductivity at very low concentrations (>2.5 %). Interestingly,
and a TTN of 978. Remarkably, the corresponding saturated FA DMSO was tolerated from 0 to 20 % (v/v), albeit at significantly
with identical chain length (C14:0) gave only 5.5 % conversion decreased productivities (TTN max. 445) relative to the other
under similar reaction conditions.[9c] Lowering the concentra- co-solvents. The selectivity for the formation of dienes (defined
tion of 3a to 5 and 2 m
and 8.9-fold, respectively (5 m
2 m : 19 % conversion, TTN 110), which indicated a strong de- identified in the volatile fraction starting from 7a–10a. Besides
M
decreased the TTN of OleT by 3.1- as the amount of diene formed relative to that of all reaction
M
: 18 % conversion; TTN 312; products) ranged between 0 and 99 %: >99 % of 7c–10c was
M
pendence of OleT activity/productivity on the concentration of α- and ꢀ-hydroxy diacids (7–74 % of total products), minor
the diacid substrate. To address the low solubility of long-chain amounts of α- and ꢀ-hydroxy ω-alkenoic acids, presumably aris-
fatty acid/diacid substrates (>C14), we performed a co-solvent ing from hydroxylation of the ω-alkenoic acids, were detected
study by using 10 mM stearic acid as the model substrate and (6–26 % of total products; Tables 1 and 2, also see the Support-
six water-miscible organic solvents (Figure 1). OleT displayed ing Information). In the case of 6a, only hydroxy acid products
the highest TTN (1028 to 1817) in the presence of EtOH, DMF, were detected in a ratio of 74:26 [(α/ꢀ-hydroxy diacid)/(α/ꢀ-
and dioxane with an optimal concentration of 5 % (v/v). In con- hydroxy ω-alkenoic acid); Table 1, entry 6; r.t. value],[15] which
trast, THF and propan-2-ol led to a significant decrease in pro- indicated that mono-decarboxylation had occurred.
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Table 2. Decarboxylation of intermediate ω-alkenoic acids to terminal dienes at room temperature and at 4 °C.
Substrate
Concentration
[m
Diene [μ
r.t./4 °C
M]
TTN
r.t./4 °C
Diene [%]
r.t./4 °C
α-OH + ꢀ-OH ω-enoic acids[c] [%]
r.t./4 °C
M
]
5b
5b
6b
6b
7b
8b
9b
10b
4
141/n.d.
0/0
21/0
24/n.d.
0/0
4/0
53/n.d.
57/111
13/32
12/7
70/n.d.
0/0
45/0
47/n.d.
30/n.d.
0/13*
55/3*
53/n.d.
n.d./10*
n.d./0
10
10
2
10
10
10
10
267/n.d.
345/669
77/192
71/34
≥99[a]/≥99[a]
≥99[a]/≥99[a]
≥99[a]/≥99[a]
≥99[a]/≥99[a]
n.d./0
n.d./n.d.
59/1776[b]
10/296
[a] Volatile fraction analyzed. [b] Trace amounts of 1-heptene (derived from trace amounts of octanoic acid in the starting material). [c] % GC area of products
(α,ω-diene + α-/ꢀ-hydroxy acids = 100 % GC area). *GC area compared to GC area of internal standard (0.1 % v/v 1-decanol); n.d.: not determined.
not only on the substrate chain length, type of oxidant, and
source of electrons but also, in particular, on the reaction tem-
perature,[9c] 1a–10a were also converted at 4 °C. Overall, a
lower temperature did not influence the product distribution,
except for 4a. Whereas the conversion of 6a did not show any
diene formation at room temperature (Table 1, entry 6), at 4 °C
it turned out to be the best accepted substrate, which led to a
remarkable conversion of 20.5 % (TTN 684; 0.25 g L–1 of 6c;
Table 1). The shortest diacids converted by OleT were 9a and
10a to yield 9c and 10c (Table 1). Although the conversion
remained low (≈1 %; TTN 34 to 38 for 9a), the selectivity for
diene formation was very high (>99 %). Owing to a radical en-
zyme mechanism, we initially assumed that the C5 diene
formed from heptanedioic acid (10a) and 5-hexenenoic acid
(10b) would be the more stable conjugated 1,3-pentadiene
rather than the nonconjugated 1,4-analogue (see the Support-
ing Information). Much to our surprise, only nonconjugated 1,4-
pentadiene was obtained as the major product. This indicates
that OleT exerts strict control over ꢀ-radical formation followed
by specific α/ꢀ-carbon bond cleavage. The TTN values for 4a,
6a, 8a, and 10a converted at 4 °C were, on average, higher than
those at room temperature (Table 1), which indicates increased
stability of OleT with certain substrates at 4 °C.[9c] The stability
and productivity of P450 monooxygenases are strongly related
to electron-transfer efficiency and O2 activation during forma-
tion of the reactive oxygenating species (compound I),[19] which
determines substrate turnover and catalyst lifetime.[20] Lower-
ing the reaction temperature can significantly alter the produc-
tivity and selectivity of OleT, which was the most prominent for
the conversions of 4a and 6a (Table 1). One explanation could
be that a change in the temperature impacts the redox poten-
tial of the mediator CamB (5 °C = –0.195 V; 25 °C = –0.242 V),[21]
which thereby affects the productivity of OleT.[18–20] Given that
attempts to decarboxylate medium- and short-chain diacids
(i.e., compounds 7a–10a) generated only small amounts of di-
Figure 1. Influence of co-solvents on the catalytic activity of OleT for the
conversion of 10 m stearic acid. TTN values were calculated on the basis of
the formation of 1-heptadecene by using 6 μ of OleT. Experiments were
performed as described in the general reaction setup for decarboxylation
with the OleT-CamAB-FDH system (see the Supporting Information). Reaction
buffer volume was replaced by a given amount of co-solvent.
M
M
Identification of α/ꢀ-hydroxy ω-enoic acid side products dur-
ing conversion of 1a–6a indicate that dicarboxylic acids un-
dergo tandem decarboxylation via ω-alkenoic acid intermedi-
ates to yield the corresponding terminal dienes (Scheme 1,
Route 1). To prove the practical applicability of the method,
reactions were scaled up to 20 mL to isolate dienes 1c (4.9 mg,
5 % isolated yield) and 3c (6 mg, 7.2 % isolated yield).[16]
In addition to the CamAB electron-transfer system, the de-
carboxylation of 3a was tested with a crude cell-free lysate of
Fdr/FldA from E. coli[9e,17] (not optimized), which resulted in a
fivefold lower conversion to 3c (0.56 mM; TTN 185). This indi-
cates that the class I/ferredoxin-based electron-transfer system
CamAB is more compatible with OleT.[9c,18] With H2O2 as
the oxidant under reaction conditions (supplementation of
enes (max. ≤4 % conversion, 0.4 mM, TTN 146), the decarboxyla-
1.6 m
M
h–1) identical to those used for the P450 peroxygenases
tion of ω-enoic acids was investigated as an alternative route
to terminal dienes. The conversion of six ω-enoic acids (i.e.,
compounds 5b–10b; Scheme 1, Route 2) allowed the synthesis
of 5c–10c (Table 2), which confirmed that ω-alkenoic acids are
[15]
P450Cla and CYPBSꢀ
at elevated catalyst loading (6 μ
; TTN 32) were detected;
the amount was 31-fold lower than that obtained with the Ca-
M OleT),
only trace amounts of 3c (1.4 %, 140 μ
M
mAB system (Table 1), which supports the redefinition of OleT intermediates during the tandem decarboxylation of diacids
as a monooxygenase rather than a peroxygenase.[8,9,9c,9e] As the (Route 1). In addition, the conversions and TTNs for 7b, 8b, and
catalytic performance (TTN and conversion) of OleT depended 10b at 4 °C were higher (up to 17 %, TTN 296) than the conver-
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sions of shorter diacids (i.e., compounds 7a–10a; Table 1 vs. 2), which is (at least) one order of magnitude higher than the 1-
which can be explained by the similarity of the lipophilic ω- alkene titers reported for whole-cell systems: Detailed studies
olefinic terminus with that of the saturated FAs. The highest on reaction/expression optimization and metabolic engineering
to improve whole-cell conversions with OleT have (so far)
not exceeded 48 mg L–1 d–1 in E. coli and have reached only
4 mg L–1 d–1 in S. cerevisiae.[9a,9e] A product titer of up
0.49 g L–1 for 3c can be regarded as a good value for a P450-
catalyzed reaction,[19,22] in particular considering that the ω-
carboxylic acid functionality of a diacid substrate is likely to
impair with the hydrophobic nature of the substrate channel of
OleT.[9b]
conversion and TTN values were achieved for 10b (17 %; TTN
296 at 4 °C), which yielded 10c with >99 % selectivity in the
nonconjugated form. Again, lowering the reaction temperature
allowed an improved conversion and a higher TTN, in particular
for 10b and 7b (6.7 % conversion, TTN 111; Table 2, entry 5).
Nonetheless, the conversion of 7b was still 2 to 2.7-fold lower
than that for the decarboxylation of nonanoic acid at 10 m
M
(18 % 1-octene),[9c] which is indicative of a strong influence of
the ω terminus on substrate conversion by OleT. Decarboxyla-
tion was the main reaction with 5b (70 % diene; 30 % hydroxy-
lated product); however, in the case of 6b (one carbon less),
the selectivity shifted towards hydroxylation (45 % diene, 55 %
hydroxylated products: Table 2). One explanation for this unpre-
dictable change in chemoselectivity (depending on substrate
chain length) is variations in the hydrogen-bonding interactions
in the active site of OleT, which can alter energy barriers for de-
carboxylation/hydroxylation pathways.[8b] Under the standard
reaction conditions, various diacids were converted into termi-
nal dienes without any detectable formation of ω-enoic acid
intermediates (Table 1), whereas decarboxylation of 6a and 5b
to the respective dienes failed at room temperature (Tables 1
and 2). The ω-enoic acid intermediate either undergoes second-
ary decarboxylation to yield the terminal diene or is subject to
hydroxylation to yield the corresponding hydroxy ω-enoic acid
(Table 1, entry 6; Scheme 1, Route 1).
Figure 2. Substrate-concentration-dependent productivity of OleT for the
conversions of 5b and 6b. Experiments were performed in triplicate accord-
ing to the standard reaction protocol at room temperature and 1 mL scale
(see legend of Table 1). Reactions went to completion at ≤2 m
M of 5b and
6b (no substrate recovered). For both substrates, control reactions without
OleT were performed as reference to calculate the recovery of the uncon-
verted substrate; α-OH and ꢀ-OH ω-enoic acids were detected as side prod-
ucts.
The failed decarboxylation of 5b (Table 2, entry 2) prompted
us to investigate the effect of substrate concentration on the
decarboxylation of ω-enoic acids by OleT, which is important
for process design. Conversions were repeated at varying con-
centrations of 5b and 6b (0.1 to 10 m
Interestingly, the decarboxylation of 5b to 5c became feasible
if the substrate concentration was decreased to 6 m (≤1 %
conversion, Figure 2). The optimum productivity was reached
at 4 m 5b, which yielded 0.14 m of 5c (3.5 % conversion;
Figure 2 and Table 2). Similarly, for 6b a higher conversion was
obtained at a 2 m substrate concentration (13.3 %, Figure 2;
Table 2, entry 4), whereas no substrate was recovered (Figure 2)
and α/ꢀ-hydroxy ω-decenoic acids were identified as major
products (55 %). This indicates a dramatic switch from decarb-
oxylation to hydroxylation activity by modification of the ω ter-
minus of the substrate. The results indicate that the conversion
of dioic acids into terminal dienes by OleT proceeds in a step-
wise fashion (Scheme 1, Route 1). For this, the ω-enoic acid
intermediate formed during the first step leaves the substrate
channel and rebinds in an inverted (u-turn) orientation. Sub-
strate concentration, co-solvents, and reaction temperature all
had a significant influence on catalyst productivity and conver-
sions. Inhibition of OleT by unsaturated FAs[9d] has been sug-
gested but has not been investigated in detail. This constitutes
a key for future strategies with the use of whole-cell terminal
alkene production, which is complicated by the hosts own FA
metabolism.[22] So far, free enzyme (purified or cell-free lysates)
M) at room temperature.
Conclusions
M
For the first time, the production of terminal dienes with chain
lengths of C16 to C5 was achieved by sequential oxidative de-
carboxylation of dicarboxylic acids or ω-enoic acids by using
O2 as the sole oxidant catalyzed by the P450 monooxygenase
OleT. The highest productivity of OleT was obtained for tetrade-
canedioic acid (3a), which yielded 1,11-dodecadiene (3c) after
optimization of the substrate concentration, type of oxidant,
and reaction temperature (TTN 978, 29 % conversion,
0.49 g L–1). OleT showed the best performance as a cell-free
catalyst with O2 as the oxidant, and the highly selective forma-
tion of nonconjugated 1,4-pentadiene (rather than the more
stable 1,3-isomer) underlines the advantages of the mild reac-
tion conditions.
M
M
M
Supporting Information (see footnote on the first page of this
article): Experimental details, including catalyst preparation and
analytical data (GC-FID/GC–MS traces and H NMR spectra) for iso-
lated and purified compounds.
1
Acknowledgments
proved to be the most productive reaching up to 1 g L–1 for 1- Funding by the Austrian Ministry of Science, Research and Econ-
alkenes[9c] and 0.49 g L–1 for terminal dienes (Table 1, entry 3), omy (BMWFW), the Ministry for Transport, Innovation and Tech-
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Communication
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Keywords: Biocatalysis · Enzyme catalysis · Carboxylic
acids · Decarboxylation · Dienes
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Received: March 22, 2016
Published Online: June 27, 2016
Eur. J. Org. Chem. 2016, 3473–3477
www.eurjoc.org
3477
© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim