Mining the Cinnabaramide Biosynthetic Pathway
mixture of the starting alcohol and the desired corresponding alde-
hyde (1:1), and it was used without further purification for the next
step.
solution (3m, 2ꢃ15 mL). The aqueous layer was acidified with HCl
(10%) to pH 2 and extracted with Et2O (3ꢃ25 mL). The new organ-
ic layer was dried over MgSO4 and the solvent was removed under
reduced pressure. The crude reaction product was purified by flash
column chromatography (silica gel, cyclohexane/ethyl acetate) to
yield (2E,4E)-octa-2,4-dienoic acid as reaction product. Any at-
tempts to avoid HCl elimination and to obtain the desired (E)-5-
chlorooct-2-enoic acid failed (Figures S2 and S3).
(E)-8-Chlorooct-2-enoic acid: The mixture of 6-chlorohexanol and 6-
chlorohexanal (1:1, 17.8 g, about 7.9 g/66 mmol of the aldehyde),
together with malonic acid (8.25 g, 79.2 mmol) in dry pyridine
(18 mL), was stirred at 308C for 4 d, at 608C for 1 h, and finally at
958C for 1 h. The reaction mixture was poured into ice/6m HCl so-
lution (100 mL) and extracted with Et2O (3ꢃ50 mL). The organic
layer was extracted with NaOH solution (3m, 2ꢃ25 mL). The aque-
ous layer was acidified with HCl (10%) to pH 2 and extracted with
Et2O (3ꢃ50 mL). The new organic layer was dried over MgSO4 and
the solvent was removed under reduced pressure to yield the
desired compound (10.8 g), which was used without further purifi-
cation.
Analytical methods and structure elucidation: HPLC-MS was per-
formed with a HPLC-DAD system (Agilent 1100) coupled to an HCT
ultra ESI-MS ion trap apparatus (Bruker Daltonics) operating in pos-
itive ionization mode. Separation was achieved by use of a Luna
RP-C18 column with a solvent system consisting of a water to ace-
tonitrile gradient. For separation of cinnabaramides and their deriv-
atives and of oct-2-enoyl-SNAC and 2-carboxyl-octenoyl-SNAC
esters, a solvent system based on water (A)/acetonitrile (B) contain-
ing formic acid (0.1%) was used. Gradient: 0–2 min 5% B; 2–
22 min linear from 5% to 95% B; 22–25 min isocratic at 95% B;
25–27 min linear from 95–5% B.
Corresponding NAC thioester: EDAC (3.12 g, 16.3 mmol) was added
to a solution of (E)-8-chlorooct-2-enoic acid (1.9 g, 10.8 mmol) and
N-acetylcysteamine (1.64 g, 12.9 mmol) in dry DMF (108 mL). The
reaction mixture was stirred at RT overnight. HCl solution (10%,
50 mL) was added, and the mixture was diluted with water
(100 mL) and extracted with CH2Cl2 (2ꢃ150 mL). The organic layer
was washed with brine, dried over MgSO4, and concentrated to
dryness. The product was purified by flash column chromatogra-
phy (silica gel, cyclohexane/ethyl acetate) and afterward by prepa-
rative HPLC (H2O/acetonitrile). The desired compound (460 mg,
about 15%) was obtained (Figures S2 and S3).
High-resolution measurements were performed with an Accela
UPLC-system (Thermo-Fisher) coupled to an LTQ-Orbitrap (linear
trap-FT-Orbitrap combination) operating in positive ionization
mode with a Waters BEH-C18 column with a solvent system con-
sisting of a water (A)/acetonitrile (B) gradient containing formic
acid (0.1%).
NMR spectra were recorded in [D6]DMSO with a Bruker DRX 500
spectrometer at 303 K, operating at 500.13 MHz proton frequency.
The solvent peak was used as internal reference (dH =2.50 ppm,
dC =39.5 ppm). Chemical shifts are given in ppm, coupling con-
stants in Hertz. In the cases of the synthetic precursors used for
the mutasynthesis projects, NMR spectra were obtained with a
Bruker AVANCE spectrometer at 293 K in CDCl3 (300 MHz proton
frequency), which was also used as internal reference (dH =
7.26 ppm). Analytical HPLC-MS was performed with an Agilent 1100
HPLC system coupled with an Agilent DAD detector, an evaporat-
ing light scattering detector (ELSD), and an LCT mass spectrometer
(Micromass, Manchester, UK) operating in positive and negative ESI
modes. A Waters Symmetry C18 column (3.5 mm, 150ꢃ2.1 mm)
and a linear gradient from 0 to 100% MeCN (0.1% HCOOH, flow
rate 0.4 mLminÀ1) in 21 min were used. HR-ESIMS data were ob-
tained with a Bruker MicroTOF instrument, coupled with an HPLC
system as described above and with use of sodium formate as in-
ternal reference. Optical rotation values were measured in MeOH
with a Schmidt+Haensch Polartronic HH8 polarimeter; concentra-
tions are given in g per 100 mL. Structure elucidation of the novel
cinnabaramide derivatives was achieved by thorough interpreta-
tion of 1D and 2D NMR spectra, combined with LC-MS data includ-
ing extracted UV as well as positive and negative mode ESI spectra.
Cinnabaramide A was used for comparison. Additionally, the mo-
lecular formulas and elemental compositions of all cinnabaramide
derivatives were confirmed by high-resolution ESI-MS. The NMR
dataset used consisted of 1D proton and carbon, 1H,1H gCOSY,
1H,13C gHSQC, and 1H,13C gHMBC spectra (Figures S3 and S4).
D) Investigation of the synthesis of (E)-5-chlorooct-2-enoic acid:
Unfortunately, attempts to synthesize (E)-5-chlorooct-2-enoic acid
by a strategy similar to that successfully applied for 6- and 8-
chlorooct-2-enoic acids failed. Spontaneous HCl elimination of the
desired product, leading to the corresponding octa-2,4-dienoic
acid is unavoidable.
Ethyl 3-chlorohexanoate: Benzotriazole (29.5 g) was added to a so-
lution of SOCl2 (18.06 mL) in dry CH2Cl2 (50 mL). More CH2Cl2 was
added until 165.4 mL of a 1.5m solution of SOCl2/benzotriazole
were obtained. This solution was added dropwise to a solution of
ethyl 3-hydroxyhexanoate (15.9 g, 99.2 mmol) in dry CH2Cl2
(500 mL), and the mixture was stirred at RT overnight. The solid
formed was removed by filtration and the filtrate was washed with
water (2ꢃ200 mL), aq. Na2CO3 (10%, 150 mL), and NaHCO3
(150 mL). The organic layer was dried over MgSO4 and concentrat-
ed to dryness. The product was purified by flash column chroma-
tography (silica gel, cyclohexane/ethyl acetate) to give the target
compound (5.5 g). Starting ethyl 3-hydroxyhexanoate (10 g) was
recovered (yield 83%, conversion 37%).
3-Chlorohexanal: A solution of DIBAL-H in CH2Cl2 (1m, 33.8 mL) was
added at À788C to a solution of ethyl 3-chlorohexanoate (5.5 g,
30.8 mmol) in dry CH2Cl2 (28 mL). The reaction mixture was stirred
at this temperature for 1 h, after which it was poured into 40 g ice/
7.5 mL conc. HCl solution. The aqueous layer was extracted with
CH2Cl2 (2ꢃ20 mL). The combined organic layer was dried over
MgSO4 and the solvent was removed under reduced pressure. The
desired product (5 g, theory: 100%=4.14 g) was obtained and
used without further purification.
Inhibition assay with human 20S proteasome: All chemicals were
of reagent grade and buffers were made with deionized distilled
water. Inhibition experiments were performed in HEPES buffer
(25 mm, pH 7.5) containing EDTA (0.5 mm), Triton-X100 (0.05%, v/
v), and SDS (0.001%, w/v). Inhibition was monitored by measuring
the release of AMC-coupled substrate peptides for the C-L, CT-L,
and T-L activities, respectively. Compounds were pre-incubated for
15 min at 378C in serial dilutions from 0.03 to 3000 nm with
(E)-5-Chlorooct-2-enoic acid/(2E,4E)-octa-2,4-dienoic acid: A mixture
of 3-chlorohexanal (4.1 g, about 30 mmol) and malonic acid
(4.54 g, 43.4 mmol) in dry pyridine (10 mL) was stirred at 308C for
4 d, at 608C for 1 h, and finally at 958C for 1 h. The reaction mix-
ture was poured into ice/6m HCl solution (100 mL) and extracted
with Et2O (3ꢃ25 mL). The organic layer was extracted with NaOH
ChemBioChem 2011, 12, 922 – 931
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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