Aliphatic hydroxylation and epoxidation of capsaicin by cytochrome P450 CYP505X

Article abstract of DOI:10.1016/j.tet.2018.08.049

Microbial cytochrome P450 enzymes (CYPs) are able to mimic the metabolism of human CYPs. One challenge is to identify the respective drug metabolites and to compare substrate specificities to those of the human enzymes. In this study, a class VIII self-sufficient CYP from Aspergillus fumigatus (CYP505X) and variants of this enzyme were heterologously expressed in E. coli. The substrate scope of the variants was determined using active pharmaceutical ingredients (APIs) and (hetero)cyclic compounds. Capsaicin – the active compound in chili peppers – was oxidized most efficiently (4.36 μM/min) in a whole cell mediated biotransformation. The products were isolated, purified and their structures elucidated by 1D and 2D NMR. The two major metabolites showed modifications on the lipophilic side chain. Specifically, capsaicin was hydroxylated at position 8 to give (E)-8-hydroxy-N-(4-hydroxy-3-methoxybenzyl)-8-methylnon-6-enamide and epoxidized at the double bond to give N-(4-hydroxy-3-methoxybenzyl)-5-(3-isopropyloxiran-2-yl)-pentanamide.

Full text of DOI:10.1016/j.tet.2018.08.049

Accepted Manuscript
Aliphatic hydroxylation and epoxidation of capsaicin by cytochrome P450 CYP505X
Anna K. Migglautsch, Melissa Willim, Bettina Schweda, Anton Glieder, Rolf
Breinbauer, Margit Winkler
PII:
S0040-4020(18)31032-9
10.1016/j.tet.2018.08.049
TET 29769
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 11 July 2018
Revised Date: 22 August 2018
Accepted Date: 28 August 2018
Please cite this article as: Migglautsch AK, Willim M, Schweda B, Glieder A, Breinbauer R, Winkler
M, Aliphatic hydroxylation and epoxidation of capsaicin by cytochrome P450 CYP505X, Tetrahedron
(2018), doi: 10.1016/j.tet.2018.08.049.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Aliphatic hydroxylation and epoxidation of capsaicin by Cytochrome P450
CYP505X
a
a
c
b
Anna K. Migglautsch , Melissa Willim , Bettina Schweda , Anton Glieder , Rolf
Breinbauera, b, Margit Winkler b*
aInstitute of Organic Chemistry, Graz University of Technology, NAWI Graz, 8010 Graz, Austria
bAustrian Centre of Industrial Biotechnology (ACIB), Petersgasse 14/4, 8010 Graz, Austria
cbisy e.U., Hofstaetten, Austria
*Corresponding author
E-mail address: margit.winkler@acib.at
Key-words:
Cytochrome P450
Capsaicin
Hydroxylation
Epoxidation
API Metabolism
Abstract
Microbial cytochrome P450 enzymes (CYPs) are able to mimic the metabolism of human CYPs.
One challenge is to identify the respective drug metabolites and to compare substrate
specificities to those of the human enzymes. In this study, a class VIII self-sufficient CYP from
Aspergillus fumigatus (CYP505X) and variants of this enzyme were heterologously expressed in
E. coli. The substrate scope of the variants was determined using active pharmaceutical
ingredients (APIs) and (hetero)cyclic compounds. Capsaicin – the active compound in chili
peppers – was oxidized most efficiently (4.36 µM/min) in a whole cell mediated
biotransformation. The products were isolated, purified and their structures elucidated by 1D
and 2D NMR. The two major metabolites showed modifications on the lipophilic side chain.
Specifically, capsaicin was hydroxylated at position 8 to give (E)-8-Hydroxy-N-(4-hydroxy-3-
methoxybenzyl)-8-methylnon-6-enamide and epoxidized at the double bond to give N-(4-
Hydroxy-3-methoxybenzyl)-5-(3-isopropyloxiran-2-yl)-pentanamide.
1. Introduction
Capsaicin (1) (8-methyl-N-vanillyl-6-nonenamide, Scheme 1) is the major pungent component of
chilli peppers. Among other capsaicinoids, 1 is especially well known for its sensory irritant
properties, and therefore finds application in pepper sprays[1] and in crop protection agents.[2]
As an active pharmaceutical ingredient, 1 is used in heat wraps for pain relief. Moreover,
capsaicin has substantial therapeutic significance including antioxidant, anticancer and anti-
inflammatory activities and is in Phase III clinical trials as an agent for the treatment of arthritis,
postoperative pain, acute and chronic neuropathic and musculoskeletal pain.[3] In the drug
development process, authentic standards of human drug metabolites are needed for structure
elucidation and toxicological tests. The challenge is to identify the molecular structure of these
metabolites and to prepare significant amounts of each compound (milligram to gram
quantities). For capsaicin(oids), human metabolites were recently identified.[4] A typical method
to identify the metabolites is LC-MS/MS analysis of samples from incubations with disrupted
tissue samples (e.g. human liver homogenate) or microsomal preparations thereof,[5] or
recombinant drug metabolizing enzyme containing bactosomes[6] or supersomes.[7] Increasingly,
human enzymes become available as recombinant catalysts, and their application in preparative
syntheses has already been demonstrated for several metabolites of APIs.[8] However, many
human enzymes are more complex in comparison to microbial counterparts, they are less easily
accessible from microbial host systems and they may show less activity and stability. In case of
cytochrome P450 enzymes (CYPs), human enzymes are membrane associated. Like other CYPs
1

ACCEPTED MANUSCRIPT
they rely on an electron transport system that provides the electrons required for oxygen
activation and substrate oxidation.[9] The redox partner of all drug and xenobiotic metabolizing
CYPs is the NADPH-cytochrome P450 reductase (CPR). Microbial CYPs may be used as mimics of
human CYPs.[10] CYP505X from Aspergillus fumigatus (AfCYP505X), for example, is a self-
sufficient class VIII CYP, which is characterized by the fusion of the reductase domain to the CYP
domain.[11] From the overall structure, it is similar to the well investigated CYP102 from Bacillus
megaterium, commonly known as CYP BM3.[12] The advantage of self-sufficient CYPs is on the
one hand their intrinsically balanced ratio of CYP to redox partner of 1:1 and on the other hand
their soluble nature, which typically leads to higher expression levels as compared to membrane
associated CYPs. Herein, we investigated wild-type AfCYP505X and a number of variants thereof
heterologously expressed in E. coli. The resulting biocatalysts were evaluated for their potential
to oxidize APIs and heterocyclic molecules with the aim to provide alternatives to the repertoire
of oxidative human enzymes.
2. Results and Discussion
2.1 Screening of CYP505X for oxidation of APIs and heterocycles
Wild-type AfCYP505X and ten rationally designed variants were heterologously expressed in E.
coli and used as lyophilized whole cell biocatalysts. A substrate profile was established using
active pharmaceutical ingredients (APIs) and (hetero)cyclic compounds as listed in 4.1 as the
substrates. Chlorzoxazone (2), Clopidogrel (3), Estriol (4), Ibuprofen (5), Metoprolol (6) 2-
naphthol (7), Progesterone (8) and Tolbutamide (9) were oxidized by CYP505X variants (Tab.
1). Notably, wild-type CYP505X only oxidized capsaicin and chlorzoxazone (Tab.
1, entry 1),
whereas a variant with five mutations (Tab. , entry 9) was capable to oxidize all nine
1
compounds to some extent. Of all tested compounds, capsaicin – the active compound in chili
peppers which is frequently used as an API in heat wraps for pain relief – was oxidized most
efficiently. According to mass spectrometry, two or more different products were formed and
we asked whether CYP505X produces human-like metabolites of 1 or different compounds.
Tab. 1: Results of screening CYP505X variants on their ability to oxidize APIs. Conversions were calculated by area
normalization and represent the average of at least two separate measurements.
Conversion (%)
entry CYP505X variants
1a
2
7
2
0
0
3
0
0
0
0
0
0
0
0
5
2
0
4
0
0
0
0
1
0
0
0
2
0
0
5
6
0
0
0
0
0
0
0
0
1
7
0
0
0
12
0
0
0
0
6
2
0
8
9
0
1
CYP505X bsy01 (wild-type)
85 a
72 a
7
0
0
2
CYP505X bsy02
CYP505X bsy03
CYP505X bsy04
CYP505X bsy05
CYP505X bsy06
CYP505X bsy07
CYP505X bsy08
CYP505X bsy09
CYP505X bsy10
CYP505X bsy11
0
0
0
3
<1
<1
<1
1
0
0
4
20 a
0
0
5
28 a <1
28 a 10
10 a <1
0
<1
1
6
0
7
1
0
1
8
5
<1
1
0
<1
1
9
53 a 25
54 a 13
15 a
12 a
11 a
0
10
11
19 a <1
2
17 a
0
1
0
0
aHPLC-MS indicated the formation of 2-4 products
2

ACCEPTED MANUSCRIPT
2.2 Synthesis of capsaicin
Capsaicin was produced via a four-step-synthesis, which is outlined in Scheme 1. The first three
steps were carried out according to Kaga et al. starting from 6-bromohexanoic acid 10.[13] 10
was transformed to the corresponding Wittig salt 11, which was then deprotonated using
potassium tert-butoxide in dry THF and converted with isobutyraldehyde to the elongated acid
12. In the following step, the acid, which is mainly present in its Z-form was isomerized. Fatty
acids are known to isomerize upon treatment with HNO2, generated in situ from NaNO2 and a
suitable acid.[14–17] An aqueous mixture of HNO3 and NaNO2 was used to obtain 13 in an E/Z ratio
of 9:1 according to NMR analysis. Finally, TBTU-mediated amide coupling of acid 13 with
vanillylamine produced capsaicin (1) in the same isomer ratio (E/Z 9:1) as a viscous oil, which
crystallized under high-vacuum and freezing in liquid nitrogen. Eventually, 1 was furnished as
an off-white power in 51 % overall yield.[18]
Scheme 1: Synthetic route to Capsaicin
2.3 Characterization of whole cell biotransformations of capsaicin
Capsaicin oxidation by wild-type AfCYP505X was studied further using a resting cell approach.
After harvesting, the cell pellet was washed twice with buffer to remove CYP inhibiting
compounds.[19] ‘Intact’ frozen and thawed cells in combination with citrate and NADP+ were used
to support cofactor regeneration.[20,21] The reaction progress was monitored by HPLC-MS.
Therefore, separate analytical reactions on the 1 mL scale were started and three of the
reactions were terminated at each given time point as depicted in Fig. 1. The first emerging and
finally most prominent product according to the UV chromatogram showed a mass signal of 304
in the mass selective detector in positive mode, suggesting that the product might be M1, M4 or
M9 (Fig. 2) as described by Reilley et al.[4] A second metabolite with a mass of 322 emerged,
indicating insertion of an oxygen, for example on the aromatic ring or the lipophilic side chain
(M5/M7 and M2/M3, as defined by Reilley et al.[4] respectively; Fig 2). The third product
emerged in minor amounts and showed a mass signal of 338, indicating the consecutive
insertion of two oxygen atoms. To identify appropriate conditions that would lead to high
metabolite titers, 0.1-25 mM of 1 were used as the substrate concentration. Fig. 3 indicates that
the initial reaction rates with substrate concentrations ≥2.5 mM – 10 mM were highly similar.
Specifically, the activities were 4.07 µM/min at 2.5 mM of 1, 4.36 µM/min at 5 mM and 4.29
µM/min at 10 mM. The highest total amount of metabolites was observed with 10 mM substrate.
However, 10 mM substrate concentration did not only show slightly lower initial rate activity
but also led to only 43% consumption of 1 after 24 h, whereas it was 79% for 5 mM and 90% for
2.5 mM. This concentration was therefore chosen for preparative scale reactions as described in
2.4.
3

ACCEPTED MANUSCRIPT
100
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
time (h)
Fig. 1: Time dependent oxidation of capsaicin catalyzed by resting cells of E. coli DH5α expressing A. fum. CYP505X
wild-type. Blue circles: capsaicin (molecular mass: 305 g/mol); Red triangles: Metabolite 1a (molecular mass: 303
g/mol); Green squares: Metabolite 1b (molecular mass: 321 g/mol); Black dashes: Metabolite 1c (molecular mass:
337 g/mol)
Fig. 2. Selected phase I metabolites as proposed by Reilley et al.[4] Figure adapted from Fig. 7 in Ref [4]
.
4

ACCEPTED MANUSCRIPT
100
90
80
70
60
50
40
30
20
10
0
0
5
10
15
20
time (h)
Fig. 3. Total of oxidation products of different capsaicin (1) concentrations catalyzed by resting cells of E. coli DH5α
expressing A. fum. CYP505X wild-type. Red circles: 0.1 mM; Pink plus: 0.5 mM; light blue squares: 1 mM; Green
diamonds: 2.5 mM; black triangles: 5 mM; Dark blue circles 10 mM; Orange crosses: 25 mM. Each time point
represents biological duplicates of technical triplicates. Error bars were omitted for clarity.
2.4 Capsaicin metabolite preparation
On the multi-mg scale, two major (1a and 1b) and one minor (1c) metabolite were formed (Fig.
1 and Scheme 2). The reaction did not proceed any further after approximately 16 h and was
terminated after 19 h. The two major metabolites could be isolated in 14 and 13% yield,
respectively, whereas the amount of 1c was negligible and could not be used for structure
elucidation. Unreacted capsaicin was re-isolated in 37% yield. According to NMR, 1a resembles
metabolite M3 (Fig. 2) that is formed by human CYP3A4 and 2C8. As a tertiary alcohol, the
compound is prone to dehydration, which may explain the compounds mass peak at 304 upon
HPLC-MS detection. 1b is a capsaicinoid that has not yet been explicitly associated with CYP
activity, however, it was observed as a metabolite when 1 was incubated in the presence of
Penicillium janthinellum AS 3.510.[22] The cytotoxic potential against cancer cells has been
evaluated using synthetic 1b, and oxidant based action in human breast carcinoma cells was
ascribed to this compound.[23] CYP505X used herein was annotated as a putative fatty acid
hydroxylase. This may explain the high activity of the wild-type enzyme for the hydroxylation of
the lipophilic side-chain of capsaicin.
Scheme 2: Biooxidation of 1
3. Conclusion
Self-sufficient Cytochrome P450 enzyme CYP505X from Aspergillus fumigatus AF293 was
evaluated as a biocatalyst for the biooxidation of APIs and heterocyclic compounds. Whereas a
variant with five mutations in the amino acid sequence was able to oxidize 9 xenobiotics, the
wild-type CYP505X was most efficient for the metabolization of capsaicin using a resting cell
biotransformation approach. This reaction was studied in detail and suitable substrate
5

ACCEPTED MANUSCRIPT
concentration (5 mM) and reaction time (<24 h) were determined in view of upscaling the
reaction to preparative scale. Two products were isolated, purified by preparative HPLC and
their chemical structures determined by NMR (Supporting Information). One metabolite was a
product hydroxylated at the ω-1 position of the aliphatic chain of capsaicin, which has
previously been described as metabolites of human CYP 3A4 and 2C8. The second metabolite
was characterized by epoxidation of the double bond in the aliphatic chain. This compound has
not been described as a human CYP metabolite, but it was observed upon incubation with fungal
cells, possibly as a product of the action of a microbial CYP.
4. Experimental section
4.1 Strains and chemicals
Escherichia coli DH5α harboring the pMS470_CYP505-M1 plasmid was used. The gene of interest
codes for the wild-type protein sequence of Aspergillus fumigatus Af293 CYP505X with the
protein accession number EAL92660. Ten variants (designated pMS470_CYP505-bsy01 to
pMS470_CYP505-bsy011) with up to seven amino acid mutations were similarly expressed in E.
coli DH5α or E. coli BL21 (DE3).
Capsaicin (1) was synthesized as described in section 4.5 or purchased from Sigma Aldrich
(Order Nr. M2028, ≥95% purity). Benzydamine hydrochloride, Caffeine, Chlorzoxazone (2),
Clopidogrel (3) bisulfate, Dextrometorphane, Sodium diclofenac, Ethionamide, Flumetsulam,
Harmine, Hydrocortisol, Lidocain, Metoprolol (6) tartrate, 2-naphthol (7), Naproxen,
Phenacetine, Piperine, Progesterone (8), Propranolol hydrochloride, quinazoline, Salbutamol
hemisulfate, Testosterone, Tolbutamide (9), and Trifluoperazine dihydrochloride were
purchased from Sigma Aldrich/Fluka. Estriol (4) Nebivolol hydrochloride, Simvastatine,
Famciclovir and Ibuprofen (5), were obtained from TCI (Tokyo, Japan) and Vardenafil
hydrochloride trihydrate from USP (Rockville, USA). Moclobemide was ordered from AvaChem
Scientific (San Antonio, USA). DMF was purchased as absolute solvent from Acros Organics.
Toluene (Sigma Aldrich, 99.7%) was dried in an aluminum oxide column under inert conditions
and stored in Schlenk bottle over 4A molecule sieves under argon atmosphere. Tetrahydrofuran
was dried at reflux temperature under argon atmosphere over sodium until benzophenone
indicated dryness by a deep blue color. All applied starting materials were commercially
available from Alfa Aesar and Sigma Aldrich and were used as received. Silica gel
1
13
chromatography was performed with Acros Organics silica gel 60 (35-70 µM). H and C NMR
13
spectra were recorded on a Bruker AVANCE III 300 spectrometer (1H: 300.36 MHz; C: 75.53
MHz) and chemical shifts are referenced to residual protonated solvent signals as internal
standard. Melting points were determined with the apparatus “Mel-Temp®“ from
Electrothermal with an integrated microscopical support. GDH.002 was obtained from DSM
Innovative Synthesis BV.
TB Medium consists of 12 g tryptone, 24 g yeast extract, 4 mL glycerol in 900 mL of ddH2O. TB-
buffer consists of 23.1 g KH2PO4 and 125.4 g K2HPO4 in 1 L of ddH2O (pH 7.6). Trace salt solution
consists of 2.45 g Fe(III)citrate, 0.121g ZnCl2, 0.2 g CoCl2·6 H2O, 0.1 g NaMoO4·2 H2O, 0.127 g
CuCl2·2 H2O and 0.05 g H3BO3 per 100 mL.
4.2 Preparation of resting-cell biocatalyst
Pre culture: One cell colony was used to inoculate 30 mL of TB-media supplemented with 100
mg/L ampicillin. The cells were grown at 37 °C on an orbital shaker at 120 rpm overnight.
Main culture: 900 mL of TB-medium, 100 mL of TB-buffer, 250 μL trace salt solution, 125 μL 1M
MgCl2 and 1 mL ampicillin 100 mg/L were mixed. 400 mL of this culture medium in 2 L
Erlenmayer flask without baffles were inoculated to OD600 of 0.05. The cells were agitated at 37
°C in an orbital shaker at 120 rpm until an OD600 of 0.6-0.8 was reached. The flasks were
incubated at 4 °C for 1 h. 1 mM IPTG was added to induce protein expression in an orbital shaker
at 28 °C and 120 rpm for 24 h.
6

ACCEPTED MANUSCRIPT
Cell harvest: The cell suspension was centrifuged for 15 min at 4 °C and 4,000 rpm in a Beckman
Coulter Avanti J-20 centrifuge using a JA10 rotor. The wet cell pellet was washed twice with 50
mM PS-buffer and centrifuged each time for 20 min at 14,000 rpm in an Eppendorf centrifuge
5810R. The cell pellet was resuspended in about 10 mL 100 mM PS-buffer and stored at -20°C.
4.3 Biooxidation assay
Frozen cells were thawed and suspended in PS buffer (100 mM potassium phosphate, pH 7.4,
8.5% w/v sucrose) in 24-well plates. To 825 µL of cell suspension, 50 µL of trisodium citrate
(1 M in ddH2O), 50 µL of NADP+ (1 mM in ddH2O), 10 µL of MgCl2 (1 M in ddH2O), 10 µL of
glucose (1 M in ddH2O), and 5 µL GDH (10 mg mL-1 in ddH2O). The reaction was started by the
addition of 50 µL of capsaicin (100 mM in DMSO). The final cell density at 600 nm (OD600) was
100 in a total volume of 1 mL. The plate was sealed with gas permeable adhesive seal and
incubated at 30 °C and 120 rpm in an orbital shaker, typically for 16 h. The reactions were
terminated by the addition of 1 mL of MeOH/ACN 1:1 v/v. After mixing, 1000 µL of the reaction
mix was transferred into 1.5 mL Eppendorf vessels. Solids were removed by centrifugation for
5 min at 4 °C at 13,400 rpm in a tabletop centrifuge. 200 µL of the supernatant were transferred
into wells of polypropylene microtiter plates and the conversion was determined by HPLC-MS
measurement. These were performed on a 1200 HPLC Series equipped with G1379B degasser,
G1312B binary pump, SL G1367C HiP-ALS SL autosampler, G1314C VWD SL UV detector,
G1316B TCC SL column oven and G1956B mass selective detector (MSD) with a Kinetex 50x4.6
mm; 2.6u; C18; 100 Å HPLC column (Phenomenex) equipped with a UHPLC C18 Security Guard
ULTRA cartridge (Phenomenex). The mobile phases were 0.1% v/v formic acid in water, and
acetonitrile (ACN) at a flow rate of 1.00 mL/min. A stepwise gradient was used at 25 °C: 0% ACN
(0-1 min), 0-100% ACN (1-4 min), 100% ACN (4-5 min) and 0% ACN (5-6 min) re-equilibration.
The compounds were detected at 254 nm (DAD). The mass spectrometer was operated in
positive electrospray ionization mode. The drying gas flow was 12 L/min at 350 °C, and the
capillary voltage was 3 kV. Selected-ion monitoring or full-scan product ion spectra were
collected. MS spectra supported data evaluation. For calculations, peak areas of the UV
chromatograms recorded at 254 nm were used. Retention times were 3.5 min for 1c, 3.7 min for
1a, 4.0 min for 1b and 4.4 min for 1.
4.4 Kinetic characterization
Time dependence of capsaicin oxidation was monitored as described in 4.3. The concentration of
1 in the reaction was 5 mM. For every time point, three parallel reactions were started and
terminated individually. Four of 8 time points were carried out in technical triplicates and the
other four in biological duplicates of technical triplicates. Error bars represent standard
deviations.
To determine the optimal substrate concentration for a scale up, time dependence of capsaicin
oxidation was monitored in two independent experimental series as described in 4.3, however,
different concentrations of 1 were used (0.2-25 mM). For every time point, three parallel
reactions were started and terminated individually.
4.5 Capsaicin synthesis
3.5.2. (6-Carboxyhexyl)triphenylphosphonium bromide (11)
In a 100 mL triple-necked round-bottom flask 5.01 g (25.7 mmol, 1.0 eq.) 6-bromohexanoic acid
and 6.71 g (25.6 mmol, 1.0 eq.) triphenylphosphine were dissolved in 30 mL dry toluene. The
reaction mixture was heated to 120 °C and stirred for 17 h. After cooling to room temperature,
the product was collected by filtration and washed with toluene (2 x 20 mL) and Et2O (2 x
20 mL) before drying under reduced pressure. 11 was obtained as colorless powder in
1
quantitative yield. m.p. = 201 – 205°C; H-NMR (300.36 MHz; CDCl3) δ = 9.12 (s, 1H), 7.74 (m,
7

ACCEPTED MANUSCRIPT
15H), 3.60 (s, 2H), 2.34 (s, 2H), 1.64 (s, 6H); 13C-NMR (75.53 MHz; CDCl3) δ = 175.9, 135.1, 133.7,
133.6, 130.7, 130.5, 34.27, 29.6, 24.0, 21.9, 22.2.
3.5.3. (Z)-8-Methyl-6-nonenoic Acid (12)
In a 250 mL twin-necked round-bottom flask 3.00 g (6.57 mmol, 1.0 eq.) 11 and 1.53 g
(13.89 mmol, 2.0 eq.) KOtBu were dissolved in 80 mL dry THF and cooled to 0 °C using an ice
bath. The reaction mixture turned bright red while the compounds were dissolved. A solution of
720 µL (7.89 mmol, 1.2 eq.) isobutyraldehyde in 5 mL dry THF was slowly added to the cooled
reaction mixture, which turned white immediately. After the addition was completed, the
reaction mixture was warmed to room temperature and stirred for 12 h. The reaction mixture
was quenched by addition of 80 mL H2O at 0 °C. After acidification to pH 2, the reaction mixture
was extracted with EtOAc (3 x 80 mL) and the combined organic layers were washed with brine
(1 x 100 mL) and dried over Na2SO4. The solvent was evaporated under reduced pressure.
Purification via flash chromatography (125 g silica gel, cyclohexane/EtOAc = 4/1 + 0.5% glacial
1
acetic acid, fraction size: 70 mL) gave 12 as a colorless oil (0.99 g, 88 %). H NMR (300.36 MHz;
3
3
CDCl3) δ = 10.51 (s, 1H), 5.30 (m, 2H), 2.57 (q, JHH = 13.6, 6.8 Hz, 1H), 2.36 (t, JHH = 7.4 Hz, 2H),
2.03 (m, JHH = 7.08 Hz, 2H), 1.65 (m, 2H), 1.40 (m, 2H), 0.95 (d, JHH = 6.1 Hz, 6H) 13C NMR
3
3
(75.53 MHz; CDCl3) δ = 180.3, 138.2, 126.7, 34.1, 29.4, 27.0, 26.6, 24.4, 23.3.
3.5.4. (E)-8-Methyl-6-nonenoic Acid (13)
In a 25 mL round-bottom flask with 0.99 g (5.82 mmol, 1.0 eq.) 12, 420 µL (0.145 eq.) 2 M
NaNO2 and 280 µL (0.289 eq.) 6 M HNO3 were added. The reaction mixture was heated to 75 °C
and stirred for 35 min. The yellow reaction mixture was cooled down to room temperature and
diluted with 10 mL Et2O. The organic layer was washed with 10 mL H2O. The aqueous phase was
acidified to a pH value of 2 and extracted with 10 mL Et2O. The combined organic layers were
washed with brine (1 x 10 mL), dried over Na2SO4 and the solvent was evaporated under
reduced pressure. Purification via flash chromatography (50 g silica gel, cyclohexane/EtOAc =
7/1 + 0.5% glacial acetic acid, fraction size: 20 mL) gave 13 as yellowish oil in 82 % yield (0.81
1
3
g). H NMR (300.36 MHz; CDCl3) δ = 5.31 (m, 2H), 2.56, 2.21 (m, 1H), 2.35 (t, JHH = 7.4 Hz, 2H),
2.02 (m, 2H), 1.65 (m, 2H), 1.40 (m, 2H), 0.95 (d, 3JHH = 6.2 Hz, 6H) 13C NMR (75.53 MHz; CDCl3) δ
= 180.1, 138.3, 126.5, 34.1, 32.2, 31.1, 29.1, 24.3, 22.8 (2C)
3.5.5. Capsaicin (1)
A 100 mL round-bottom flask equipped with Schlenk adapter and stirring bar was charged with
750 mg (4.41 mmol, 1.0 eq.) 13 and 20 mL dry DMF were added. After cooling the reaction
mixture to 0 °C, 1.83 mL (13.22 mmol, 3.0 eq.) Et3N, 1.7 g (5.29 mmol, 1.2 eq.) TBTU and 1.0 g
(5.29 mmol, 1.2 eq.) vanillylamine hydrochloride were added successively. The yellowish
suspension was stirred at RT for 2 h. The reaction mixture was quenched by the addition of
100 mL brine. A colorless precipitate formed which dissolved in water by the addition of water.
The reaction solution was extracted with EtOAc (3 x 70 mL) and the combined organic layers
were washed with 1 M HCl (1 x 50 mL), saturated NaHCO3-solution (1 x 50 mL), and brine (1 x
50 mL). The combined organic layers were dried over Na2SO4 and the solvent was evaporated
under reduced pressure. The product was purified via flash chromatography (75 g silica gel,
cyclohexane/EtOAc = 2/1, fraction size: 50 mL) yielding a sticky oil which crystallized under
high vacuum and freezing in liquid nitrogen. (583 mg, 82 %) m.p. = 58 – 61°C
3
1H NMR (300.36 MHz; CDCl3) δ = 6.80 (m, 3H), 5.70 (s, 2H), 5.30 (m, 2H), 4.34 (d, JHH = 5.5 Hz,
3
2H), 3.86 (s, 3H), 2.23 (m, 2H), 1.99 (m, 2H), 1.66 (m, 2H), 1.37 (m, 2H), 0.93 (d, JHH = 6.2 Hz,
6H,) 13C NMR (75.53 MHz; CDCl3) δ = 173.0, 146.9, 145.3, 130.5, 138.2, 126.6, 120.9, 114.5,
110.8, 56.1, 43.7, 36.8, 32.4, 31.1, 29.4, 25.4, 22.8
4.6 Preparative scale oxidation of capsaicin
8

ACCEPTED MANUSCRIPT
For the bioconversions, 1 L shaking flasks without baffles were used. The flasks were closed with
cloth to allow oxygen access. Approximately 3 g of wet cells were suspended in 50 mM PS buffer (pH
7.4) in a volume of 165 mL. Successively, 10 mL trisodiumcitrate dihydrate (1 M), 2 mL glucose
(1 M), 1 mL GDH solution (10 mg/mL), 2 mL MgCl2*6 H2O (1 M), 150 mg 1 in 4 mL DMSO and
finally 10 mL NADP+ (1 mM) were added. The reaction mixture was agitated at 120 rpm at 28 °C.
Samples were withdrawn and analyzed periodically. After 19 h, unreacted 1 and the metabolites were
extracted with EtOAc. Centrifugation was used to facilitate phase separation. The combined organic
layers were washed with brine, dried over Na2SO4, and the solvent was removed under reduced
pressure. Compounds were separated via preparative HPLC on a Thermo Scientific UltiMate 3000
system. Detection was accomplished with a Dionex ULtiMate Diode Array Detector. The separations
were carried out on a Macherey-Nagel 125/21 Nucleodur® 100-5 C18EC (125 x 21 mm, 5.0 µm)
column. As eluents acetonitrile and water with 0.01% HCOOH were used. Two methods were used:
Method 1: 0.0 min 40% MeCN, 0.1 – 5.0 min linear increase to 60% MeCN, 5.1 – 7.0 60% MeCN
isocratic, 7.1 – 9.0 min linear increase to 100% MeCN, 9.1 – 11 min 100% MeCN isocratic, 11.1 –
13.0 min linear decrease to 40% MeCN, 13.1 – 15.0 min 40% MeCN isocratic, 12 mL/min, 30 °C.
Method 2: 0.0 min 2% MeCN, 0.1 – 20 min linear increase to 100% MeCN, 20.1 – 22.0 min 100%
MeCN isocratic, 22.1 – 24.0 min linear decrease to 2% MeCN, 12 mL/min, 30 °C.
Metabolite 1a: (E)-8-Hydroxy-N-(4-hydroxy-3-methoxybenzyl)-8-methylnon-6-enamide. Yield:
20.9 mg (0.065 mmol, 13%), colorless oil, C18H27NO4 [321.42 g/mol], tR = 4.66 min (preparative
HPLC).
1H NMR (300.36 MHz; DMSO) δ = 8.83 (bs, 1H), 8.17 (bs, 1H), 6.68 (m, 3H, Harom), 5.46 (m, 2H),
4.42 (s, 1H), 4.14 (d, 3JHH = 5.5 Hz, 2H, CH2-NH), 3.73 (s, 3H, OCH3), 2.11 (t, 3JHH = 6.9 Hz, 2H), 1.95
(m, 2H), 1.52 (m, 2H), 1.31 (m, 2H), 1.14 (s, 6H)
13C NMR (75.53 MHz; DMSO) δ = 171.9, 147.4, 145.4, 139.4, 130.5, 124.9, , 119.7, 115.2, 111.6,
68.8, 55.5, 41.8, 35.2, 31.4, 30.1(2C), 29.0, 25.5.
Metabolite 1b:[23] N-(4-Hydroxy-3-methoxybenzyl)-5-(3-isopropyloxiran-2-yl)-pentanamide.
Yield: 20.1 mg (0.069 mmol, 14%), yellowish oil, C18H27NO4 [321.42 g/mol], tR = 5.18 min
(preparative HPLC).
1H NMR (300.36 MHz; DMSO) δ = 8.47 (bs, 1H), 8.21 (bs, 1H), 6.69 (m, 3H), 4.13 (d, 3JHH = 5.6 Hz,
2H), 3.72 (s, 3H), 2.84-2.67 (m, 1H), 2.42 (m, 1H) 2.11 (t, 3JHH = 6.6 Hz, 2H), 1.55 (m, 2H), 1.39 (m,
5H), 0.89 (m, 6H)
13C NMR (75.53 MHz; DMSO) δ = 171.9, 147.5, 145.5, 130.4, 119.7, 115.2, 111.7, 63.2, 56.7, 55.6,
41.8, 35.3, 31.3, 29.9, 25.3, 25.1, 18.9, 18.2.
Acknowledgements
We would like to acknowledge Jörg-Patrick R. Eichmann and Thorsten Bachler for their skillful support in
experimental work. This work received funding from the EU project ROBOX (grant agreement n° 635734) under EU’s
H2020 Programme Research and Innovation actions H2020-LEIT BIO-2014-1 Disclaimer: This publication reflects the
author's view and the Agency is not responsible for any use that may be made of the information it contains. This
work has also been supported by bisy e.U. and the Austrian BMWD, BMVIT, SFG, Standortagentur Tirol, Government
of Lower Austria and Business Agency Vienna through the Austrian FFG-COMET- Funding Program.
References
[1]
[2]
[3]
H. Krishnatreyya, H. Hazarika, A. Saha, P. Chattopadhyay, Eur. J. Pharmacol. 2018, 819, 114–121.
F. E. Dayan, C. L. Cantrell, S. O. Duke, Bioorg. Med. Chem. 2009, 17, 4022–4034.
P. Patowary, M. P. Pathak, K. Zaman, P. S. Raju, P. Chattopadhyay, Biomed. Pharmacother. 2017, 96,
1501–1512.
[4]
[5]
C. A. Reilly, F. Henion, T. S. Bugni, M. Ethirajan, C. Stockmann, K. C. Pramanik, S. K. Srivastava, G. S.
Yost, Chem. Res. Toxicol. 2013, 26, 55–66.
F. P. Guengerich, in Cytochrome P450 - Struct. Mech. Biochem. (Ed.: P.R. Ortiz de Montellano),
9

ACCEPTED MANUSCRIPT
Springer US, Boston, MA, 2015, pp. 523–785.
[6]
M. Kajbaf, R. Longhi, D. Montanari, F. Vinco, M. Rigo, S. Fontana, K. D. Read, Drug Metab. Lett. 2011,
5, 30–39.
[7]
[8]
Y. Chang, W. B. Fang, S.-N. Lin, D. E. Moody, Basic Clin. Pharmacol. Toxicol. 2011, 108, 55–62.
M. Winkler, M. Geier, S. P. Hanlon, B. Nidetzky, A. Glieder, Angew. Chemie Int. Ed. 2018, DOI
10.1002/anie.201800678.
[9]
D. W. Nebert, K. Wikvall, W. L. Miller, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2013, 368, 20120431.
[10] A. Worsch, F. K. Eggimann, M. Girhard, C. J. von Bühler, F. Tieves, R. Czaja, A. Vogel, C. Grumaz, K.
Sohn, S. Lütz, et al., Biotechnol. Bioeng. 2018, DOI 10.1002/bit.26781.
[11] R. Weis, M. Winkler, M. Schittmayer, S. Kambourakis, M. Vink, J. D. Rozzell, A. Glieder, Adv. Synth.
Catal. 2009, 351, 2140–2146.
[12] C. J. C. Whitehouse, S. G. Bell, L.-L. Wong, Chem. Soc. Rev. 2012, 41, 1218–1260.
[13] H. Kaga, M. Miura, K. Orito, J. Org. Chem. 1989, 54, 3477–3478.
[14] C. Litchfield, R. D. Harlow, A. F. Isbell, R. Reiser, J. Am. Oil Chem. Soc. 1965, 42, 73–78.
[15] P. E. Sonnet, J. Org. Chem. 1974, 39, 3793–3794.
[16] J. Wisniak, P. Alfandary, Ind. Eng. Chem. Prod. Res. Dev. 1975, 14, 177–180.
[17] A. Napolitano, L. Panzella, M. Savarese, R. Sacchi, I. Giudicianni, L. Paolillo, M. D’Ischia, Chem. Res.
Toxicol 2004, 17, 1329–1337.
[18] M. Winkler, T. Moraux, H. a Khairy, R. H. Scott, A. M. Z. Slawin, D. O’Hagan, ChemBioChem 2009, 10,
823–8.
[19] A. Fredenhagen, M. Kittelmann, L. Oberer, A. Kuhn, J. Kuhnol, T. Delemonte, R. Aichholz, P. Wang, P.
Atadja, M. D. Shultz, Drug Metab. Dispos. 2012, 40, 1041–1050.
[20] S. P. Hanlon, T. Friedberg, R. Wolf, O. Ghisalba, M. Kittelmann, in Mod. Biooxidation Enzym. React.
Appl. (Eds.: R.D. Schmid, V.B. Urlacher), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany,
2007, pp. 233–252.
[21] S. P. Hanlon, A. Camattari, S. Abad, A. Glieder, M. Kittelmann, S. Lütz, B. Wirz, M. Winkler, Chem.
Commun. 2012, 48, 6001–6003.
[22] X.-L. Xin, Y. Wang, G.-J. Fan, L. Chen, C.-P. Sun, Phytochem. Lett. 2017, 19, 210–214.
[23] A. Lewinska, P. Chochrek, K. Smolag, E. Rawska, M. Wnuk, Redox Rep. 2015, 20, 116–125.
10