Synthesis and characterization of acyl-CoAs / H.F. Sobhi et al. / Anal. Biochem. 401 (2010) 114–124
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often results in low yields. The current article describes a method
for solubilization of CoASH in an aqueous–organic solvent mixture
followed by acylation using an ethylchloroformate mixed anhy-
dride. By this procedure, we synthesized cis-4-decenoyl-CoA, 3-
phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA and used
them as substrates for MCAD and LCAD activity measurements. A
significant feature of this study is that the synthesized acyl-CoAs
were purified by solid-phase extraction (SPE) and semipreparative
high-performance liquid chromatography with ultraviolet detec-
tion (HPLC–UV). The overall procedure results in excellent yields
with high recovery of the acyl-CoAs. Furthermore, the purified
acyl-CoAs are characterized by analytical HPLC–UV followed by
data-dependent tandem mass spectrometry (MS/MS) analysis on
the largest responding mass spectrometry (MS) mass (HPLC–UV–
MS–MS/MS) and nuclear magnetic resonance (NMR).
HP1100 series HPLC system (quaternary pump with degasser and
autosampler) from Agilent Technologies and a 3200 Q TRAP mass
spectrometer from Applied Biosystems (Concord, Ontario, Canada).
Both instruments were controlled and data were collected using
Analyst 1.5 software (Applied Biosystems). For final characteriza-
tion of the synthesized acyl-CoAs, an analytical HPLC–UV–MS–
MS/MS system was used and consisted of an Agilent HPLC device
and an LCQ Deca ion trap mass spectrometer as described previ-
ously [17]. The analytical column used was a Hypersil GOLD col-
umn (250 Â 4.6 mm,
5 lm) purchased from Thermo Fisher
Scientific (Waltham, MA, USA). NMR spectra were obtained with
a 400-MHz Varian Inova spectrometer (Varian, Lake Forest, CA,
USA) or a 600-MHz Varian Inova spectrometer. Fourier transform
data were processed using Master Nova software (version 5.2.5-
4119, Masterlab Research, Bajo, Spain). The acyl-CoA dehydroge-
nase activity measurements were made using a DU 800 UV/visible
spectrophotometer purchased from Beckman Coulter (Fullerton,
CA, USA). The concentrations of synthesized acyl-CoAs were deter-
mined by the method of Ellman [18].
Materials and methods
Chemicals and solvents
Synthesis of cis-4-decenoic acid
CoASH, cis-4-decen-1-al (97% pure), hydrocinnamic acid (3-
phenylpropionic acid), anhydrous ethylchloroformate, ammonium
formate, silver nitrate, thionyl chloride, 6-methyl-2-heptanol, p-
toluene sulfonyl chloride, potassium phosphate, cytochrome c,
potassium cyanide, phenazine ethosulfate, N-ethylmaleimide, and
rotenone were purchased from Sigma–Aldrich (St. Louis, MO,
USA). Butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA trilithium
salts also were purchased from Sigma–Aldrich. Methanol (HPLC
grade) was purchased from Fisher Scientific (Cleveland, OH, USA).
Acetonitrile (Burdick & Jackson brand, HPLC grade) was purchased
from Jade Scientific (Canton, MI, USA). The CDCl3 and D2O used in
NMR experiments were purchased from Cambridge Isotope Labo-
ratories (Andover, MA, USA). Reagent-grade water was prepared
using a Milli-Q system (Millipore, Bedford, MA, USA). 2-(2-Pyri-
dyl)ethyl-functionalized silica gel and empty polypropylene 6-ml
SPE tubes with polyethylene frits were purchased from Sigma–
Aldrich.
cis-4-Decenoic acid was synthesized according to Scheme 1
[19]. First, silver oxide was freshly prepared by the addition of a sil-
ver nitrate solution (3.03 g in 10 ml of water) into a solution of so-
dium hydroxide (2.40 g in 20 ml of water), and the mixture was
stirred for 60 min. Silver oxide was precipitated and collected by fil-
tration, and the filtrate was discarded. Second, the silver oxide was
dissolved in a solution of sodium hydroxide (1.25 g in 15 ml of
water), followed by the dropwise addition of 1.5 ml of cis-4-decen-
1-alover 20 min, and thereaction mixture was stirred for 2 h. During
the reaction, silver is formed and precipitates. The solution was fil-
tered and the pellet was discarded. Third, the filtrate containing
the acid was collected. The product, cis-4-decenoic acid, was ob-
tained by acidification of the filtrate to pH 4.5 with 1 N HCl followed
by extraction (three times with 15 ml of ethyl ether each). The ethyl
ether (45 ml containing the acid) was collected and the aqueous
phase was discarded. Finally, the ethyl ether was removed under re-
duced pressure and cis-4-decenoic acid was collected.
Equipment
The gas chromatography/mass spectrometry (GC/MS) consisted
of an HP6890 GC device, an HP5973 mass spectrometer, and an HP
autosampler purchased from Agilent Technologies (Wilmington,
DE, USA). A BioCAD SPRINT perfusion chromatography system
(PerSeptive Biosystems, Framingham, MA, USA) was configured
to perform semipreparative reversed-phase HPLC [17] and in-
cluded a Gilson model FC-205 fraction collector. The column used
Synthesis of 2,6-dimethylheptanoic acid
2,6-Dimethylheptanoic acid was synthesized according to
Scheme 2 [20,21]. First, 6-methyl-2-heptanol (5.0 g) was added
to pyridine (25 ml), followed by the addition of p-toluene sulfonyl
chloride (8.8 g), and the reaction mixture was stirred for 18 h at
room temperature (RT) under nitrogen. Second, the 2-methylsulfo-
nate-6-methyl-heptane formed was extracted with hexane
(15 ml), and the hexane was removed under reduced pressure.
The residue of 2-methylsulfonate-6-methyl heptane was dissolved
in 100 ml of 3 N sulfuric acid, stirred for 30 min, and extracted
three times with 15 ml of ethyl ether. Third, the combined ethyl
was an Alltima C18 LL (250 Â 22 mm, 5
lm) purchased from All-
tech Associates (Deerfield, IL, USA), and the UV detector was set
to monitor absorbance at 258 nm. For flow injection MS and MS/
MS analysis of the fractions collected from the semipreparative
HPLC–UV, an LC–MS–MS/MS system was used. It consisted of an
2 AgNO3+ 2 NaOH
Ag2O (ppt) + 2 NaNO3 + H2O
O
NaOH
Ag2O
O
H
O H
cis-4-Decen-1-al
cis-4-Decenoic acid
Scheme 1. Synthesis of cis-4-decenoic acid.