Synthesis and characterization of cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA

Article abstract of DOI:10.1016/j.ab.2010.02.026

The measurement of acyl-CoA dehydrogenase activities is an essential part of the investigation of patients with suspected defects in fatty acid oxidation. Multiple methods are available for the synthesis of the substrates used for measuring acyl-CoA dehydrogenase activities; however, the yields are low and the products are used without purification. In addition, the reported characterization of acyl-CoAs focuses on the CoA moiety, not on the acyl group. Here we describe the synthesis of three medium-chain acyl-CoAs from mixed anhydrides of the fatty acids using an aqueous-organic solvent mixture optimized to obtain the highest yield. First, cis-4-decenoic acid and 2,6-dimethylheptanoic acid were prepared (3-phenylpropionic acid is commercially available). These were characterized by gas chromatography/mass spectrometry (GC/MS), ¹H nuclear magnetic resonance (NMR), and ¹³C NMR. Then cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA were synthesized. These were then purified by ion exchange solid-phase extraction using 2-(2-pyridyl)ethyl-functionalized silica gel, followed by reversed-phase semipreparative high-performance liquid chromatography with ultraviolet detection (HPLC-UV). The purified acyl-CoAs were characterized by analytical HPLC-UV followed by data-dependent tandem mass spectrometry (MS/MS) analysis on the largest responding MS mass (HPLC-UV-MS-MS/MS) and ¹³C NMR. The yields of the purified acyl-CoAs were between 75% and 78% based on coenzyme A trilithium salt (CoASH). Acyl-CoA dehydrogenase activities were measured in rat skeletal muscle mitochondria using, as substrates, the synthesized cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA. These results were compared with the results using our standard substrates butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA.

Full text of DOI:10.1016/j.ab.2010.02.026

Analytical Biochemistry 401 (2010) 114–124
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Synthesis and characterization of cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA,
and 2,6-dimethylheptanoyl-CoA
Hany F. Sobhi a,b, Paul E. Minkler a,b, Charles L. Hoppel a,b,c,
*
a Center for Mitochondrial Diseases, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA
b Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA
c Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The measurement of acyl-CoA dehydrogenase activities is an essential part of the investigation of patients
with suspected defects in fatty acid oxidation. Multiple methods are available for the synthesis of the
substrates used for measuring acyl-CoA dehydrogenase activities; however, the yields are low and the
products are used without purification. In addition, the reported characterization of acyl-CoAs focuses
on the CoA moiety, not on the acyl group. Here we describe the synthesis of three medium-chain acyl-
CoAs from mixed anhydrides of the fatty acids using an aqueous–organic solvent mixture optimized to
obtain the highest yield. First, cis-4-decenoic acid and 2,6-dimethylheptanoic acid were prepared (3-
phenylpropionic acid is commercially available). These were characterized by gas chromatography/mass
spectrometry (GC/MS), 1H nuclear magnetic resonance (NMR), and 13C NMR. Then cis-4-decenoyl-CoA, 3-
phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA were synthesized. These were then purified by
ion exchange solid-phase extraction using 2-(2-pyridyl)ethyl-functionalized silica gel, followed by
reversed-phase semipreparative high-performance liquid chromatography with ultraviolet detection
(HPLC–UV). The purified acyl-CoAs were characterized by analytical HPLC–UV followed by data-depen-
dent tandem mass spectrometry (MS/MS) analysis on the largest responding MS mass (HPLC–UV–MS–
MS/MS) and 13C NMR. The yields of the purified acyl-CoAs were between 75% and 78% based on coen-
zyme A trilithium salt (CoASH). Acyl-CoA dehydrogenase activities were measured in rat skeletal muscle
mitochondria using, as substrates, the synthesized cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-
dimethylheptanoyl-CoA. These results were compared with the results using our standard substrates
butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA.
Received 28 December 2009
Received in revised form 4 February 2010
Accepted 19 February 2010
Available online 23 February 2010
Keywords:
Acyl-CoA dehydrogenase activity
Semipreparative HPLC
Short-chain acyl-CoA dehydrogenase
(SCAD)
Medium-chain acyl-CoA dehydrogenase
(MCAD)
Long-chain acyl-CoA dehydrogenase (LCAD)
Very-long-chain acyl-CoA dehydrogenase
(VLCAD)
Ó 2010 Elsevier Inc. All rights reserved.
Medium-chain acyl-CoA dehydrogenase (MCAD)1 deficiency is
recognized as a potentially lethal inborn error of metabolism during
childhood [1]. The use of metabolic data, such as elevation of blood
octanoylcarnitine coupled with enzymatic and mutational analysis,
is used for the diagnosis of MCAD deficiency [2]. From a metabolic
viewpoint, 3-phenylpropionyl-CoA has been identified as a specific
substrate for MCAD [3] and the glycine conjugate 3-phenylpropio-
nylglycine has been established as a pathognomonic marker in urine
from patients affected with MCAD deficiency [4]. cis-4-Decenoic acid
also is a marker for MCAD deficiency [5,6]. Metabolically, MCAD
effectively accepts cis-4-decenoyl-CoA as
a substrate, whereas
long-chain acyl-CoA dehydrogenase (LCAD) does not [7]. 2,6-Dim-
ethylheptanoyl-CoA is a selective substrate for LCAD but not for
very-long-chain acyl-CoA dehydrogenase (VLCAD) [8].
* Corresponding author. Address: Department of Pharmacology Medicine, Case
Western Reserve University School of Medicine, Cleveland, OH 44106, USA. Fax: +1
216 368 5162.
Several methods have been described to synthesize fatty acyl-
CoA substrates for use in the enzymatic assessment of acyl-CoA
dehydrogenase activity. These synthetic approaches involve acyl-
ating free coenzyme A trilithium salt (CoASH) using commercially
available fatty acid derivatives such as symmetric anhydrides [9],
mixed anhydrides of ethylhydrogen carbonate [10], acid chlorides
[11], N-hydroxysuccinimide esters [12,13], and acyl imidazoles
[14,15]. Due to the low solubility of CoASH in most organic sol-
vents, the acylation has been performed in aqueous solvents at
pH 7.5 to 8.0 [16]. However, under these conditions, poor solubility
of the acylating reagents or the occurrence of various side reactions
E-mail address: charles.hoppel@case.edu (C.L. Hoppel).
Abbreviations used: MCAD, medium-chain acyl-CoA dehydrogenase; LCAD, long-
1
chain acyl-CoA dehydrogenase; VLCAD, very-long-chain acyl-CoA dehydrogenase;
CoASH, coenzyme A trilithium salt; SPE, solid-phase extraction; HPLC–UV, high-
performance liquid chromatography with ultraviolet detection; HPLC–UV–MS–MS/
MS, HPLC–UV followed by data-dependent tandem mass spectrometry analysis on the
largest responding mass spectrometry mass; NMR, nuclear magnetic resonance; GC/
MS, gas chromatography/mass spectrometry; RT, room temperature; DMF, dimeth-
ylformamide; BSTFA/TMCS, bis(trimethylsilyl) trifluoroacetamide/trimethylchlorosi-
lane; TMS, trimethylsilane; THF, tetrahydrofuran; TIC, total ion current; SCAD, short-
chain acyl-CoA dehydrogenase.
0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2010.02.026

Synthesis and characterization of acyl-CoAs / H.F. Sobhi et al. / Anal. Biochem. 401 (2010) 114–124
115
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.

116
Synthesis and characterization of acyl-CoAs / H.F. Sobhi et al. / Anal. Biochem. 401 (2010) 114–124
Pyridine / Stirring at RT for 18 h
+
CH3
SO2Cl
OH
6-Methyl-2-heptanol
O
S
p-Toluenesulfonylchloride
O
CH3
O
O
S
H2SO4 / DMF ,
KCN , Heat for 3 h at 100 C
O
CH3
C
N
º
O
2,6-Dimethylheptanoyl nitrile
2-Methylsulfonate-6-methyl heptane
Hydrolysis / NaOH
C
N
COOH
2,6-Dimethylheptanoic acid
2,6-Dimethylheptanoyl nitrile
Scheme 2. Synthesis of 2,6-dimethylheptanoic acid.
ether extracts (45 ml) were evaporated under reduced pressure,
and the residue was dissolved in 30 ml of dimethylformamide
(DMF). This solution was stirred with potassium cyanide (2.6 g)
for 3 h in a silicone oil bath of 100 °C to form 2,6-dimethylhepta-
noyl nitrile. The reaction mixture was then extracted with hexane
(15 ml) and the hexane was evaporated. Fourth, the residue (2,6-
dimethylheptanoyl nitrile) was dissolved in 30 ml of ethanol, to
which was added an NaOH solution (1 g in 25 ml of ethanol), and
this solution was then refluxed for 24 h. For the final step, the eth-
anol was evaporated and the reaction mixture was acidified to pH
4.0 using 1 N HCl followed by extraction (three times with 10 ml of
ethyl ether). The ethyl ether extract was evaporated and the prod-
uct (2,6-dimethylheptanoic acid) was collected.
General method for synthesis of medium-chain acyl-CoAs
Medium-chain acyl-CoAs were synthesized from the mixed
anhydrides of fatty acids by a modification of the procedures of
Goldman and Vagelos [10] and Wieland and Rueff [23]. The general
synthesis is shown in Scheme 3, and the compounds prepared in
this study are shown in Table 1. First, the fatty acid (0.6 mmol)
was mixed with 10 ml of CH2Cl2, followed by the addition of a ter-
tiary amine (1.2 mmol, i.e., triethylamine or 2,4,6-trimethylpyri-
dine (collidine) [see Table 1]). The reaction mixture was stirred
for 30 min under nitrogen and then placed in an ice bath for
10 min. Second, anhydrous ethylchloroformate (1.2 mmol) was
added dropwise over 10 min while stirring under nitrogen at RT,
and stirring was continued for 2 h. Third, the solvent, CH2Cl2,
was removed under reduced pressure until dryness. The residue
(containing the mixed anhydride) was dissolved in 10 ml of anhy-
drous tetrahydrofuran (THF), the undissolved amine salts were
precipitated and removed by filtration, and the filtrate (containing
the mixed anhydride) was collected. Under nitrogen, CoASH
GC/MS
Acid samples (2 Â 10À6 mol) were derivatized with 50
ll of bis-
(trimethylsilyl) trifluoroacetamide and trimethylchlorosilane (BST
FA/TMCS, 99:1, Sigma–Aldrich), and 1 l
l containing 10À8 mol of
(64 lmol) was dissolved in 2 ml water/8 ml THF and the pH was
adjusted to 7.5 with 0.01 M KHCO3. Fourth, the mixed anhydride
of the fatty acid was added to the CoASH solution and the pH
was adjusted to 8.0. This solution was stirred for 30 min under
nitrogen and then acidified to pH 4.5 with 1% HClO4. The THF
was removed under reduced pressure. Finally, the reaction mixture
was acidified with 1 ml of 10% HClO4 and washed three times with
5 ml of ethyl ether to remove any acid present. The reaction mix-
ture containing the synthesized acyl-CoAs was then subjected to
SPE and semipreparative HPLC–UV.
the resulting trimethylsilane (TMS) derivative was injected into a
split/splitter injector set at 280 °C [22]. The capillary column was
30 Â 0.25 mm and 0.25 m (HP-5 ms). The oven temperature wÀas1
l
programmed to change from 80 to 150 °C at a rate of 30 °C min
(2.5 min) and then from 150 to 280 °C at a rate of 5 °C minÀ1
(30.0 min), and finally it was held at 280 °C for 10.0 min. Helium
was used as a carrier gas at a flow rate of 1.5 ml/min, the electron
ionization setting was 70 eV, and the heating source was set
at 230 °C.
O
C
O
C
O
C
O
CH2Cl2 / Tertiary amine
30 min Stirring under nitrogen
O
R
O
+
R
OH
O
Cl
Fatty acid
Ethylchloroformate
Mixed anhydride of the fatty acid
O
C
O
C
O
/ pH = 7.5 ~ 8.0
THF
KHCO3
+
CoASH
C
CoA
O
R
O
R
S
Mixed anydride of the fatty acid
CoenzymeA trilithium salt
Acyl-coenzymeA
Scheme 3. Synthesis of medium-chain acyl-CoAs.

Synthesis and characterization of acyl-CoAs / H.F. Sobhi et al. / Anal. Biochem. 401 (2010) 114–124
117
Table 1
Synthesized medium-chain acyl-CoAs.
Acyl-CoA
Molecular structure
Tertiary amine
Yield (%)
O
cis-4-Decenoyl-CoA
2,4,6-Trimethylpyridine (collidine)
75.0
78.0
CoA
S
O
3-Phenylpropionyl-CoA
Triethylamine
Triethylamine
CoA
CoA
S
O
2,6-Dimethylheptanoyl-CoA
76.0
S
Purification of synthesized acyl-CoAs using ion exchange SPE
Semipreparative HPLC–UV
The eluents used were 95% 0.1 M ammonium formate in water/
5% methanol (eluent A) and 95% acetonitrile/5% water (eluent B).
The instrument flow rate was 10 ml/min and was programmed
to start with 100% eluent A for 10 min, followed by a gradient of
100% A to 100% B over 30 min, followed by 100% eluent B for
10 min. The UV detector collected data at 258 nm, and eluent frac-
tions were collected at a rate of 1 fraction/0.5 min. Fractions were
characterized by flow injection analysis.
SPE columns (6 ml) were packed with 500 mg of 2-(2-pyri-
dyl)ethyl-functionalized silica gel [24] and rinsed with 10 ml of
the condition solvent (acetonitrile/isopropanol/water/glacial acetic
acid, 9:3:4:4 by volume) using a Visiprep SPE vacuum manifold
(Sigma–Aldrich). The acyl-CoA reaction mixture (ꢀ5 ml) was
added to 5 ml of the conditioning solvent, and then this solution
was applied to the SPE column and the flow-through was dis-
carded. The SPE column was then washed with 10 ml of the condi-
tioning solvent, and this wash also was discarded. The acyl-CoAs
were eluted with 20 ml methanol/250 mM ammonium formate
(4:1 by volume); this eluent was collected and the volume was re-
duced to approximately 5 ml by evaporation under reduced pres-
sure. This entire sample was then injected into the
semipreparative HPLC–UV.
Flow injection analysis by LC–MS–MS/MS
Aliquots (100
were evaporated to dryness in HPLC sample tubes, reconstituted
in 500 l acetonitrile/water (80:20 by volume), and at a flow of
ll) from the semipreparative HPLC–UV fractions
l
Fig. 1. Characterization of synthesized cis-4-decenoic acid. (A) GC/MS chromatogram of the TMS derivative of cis-4-decenoic acid has a single peak at 16.50 min. The MS
spectrum contains the [M+] ion at m/z 242. (B) 13C NMR contains signals at d = 132.13 and 127.07 ppm, consistent with a double bond at carbon 4.

118
Synthesis and characterization of acyl-CoAs / H.F. Sobhi et al. / Anal. Biochem. 401 (2010) 114–124
Table 2
Key spectral features used for characterization.
Acyl-CoA species
13C NMR data (feature that is observed)
Acyl-CoA MS data (feature that is observed)
Acid (d, ppm)
CoA (d, ppm)
MS (m/z)
MS/MS (m/z)
cis-4-Decenoyl-CoA
3-Phenylpropionyl-CoA
132.13,127.07 (C@C)
140.37, 128.81, 128.51,
126.63 (phenyl group)
38.95
132.73, 127.08 (C@C)
140.18, 128.73,128.47,
126.55 (phenyl group)
38.95
918 ([MÀ])
898 ([MÀ])
408, 426 (phosphoadenosine group)
408, 426 (phosphoadenosine group)
2,6-Dimethylheptanoyl-CoA
906 ([MÀ])
408, 426 (phosphoadenosine group)
(C–CH3 carbon 6)
28.01
(C–CH3 carbon 6)
30.65
(C–CH3 carbon 2)
(C–CH3 carbon 2)
200
ll/min (acetonitrile/water, 80:20) were injected (50
l
l) into
1H NMR and 13C NMR
the HP1100 HPLC/3200 Q TRAP mass spectrometer. The instrument
was operated in the ion trap mode, and full-scan MS spectra (m/z
700–1000) were collected for 2 min. For MS/MS analysis, a second
injection was made and the full-scan MS/MS spectra were col-
lected, fragmenting the ion observed in the MS spectra using a col-
lision energy of À70 V.
NMR samples of the synthesized fatty acids were prepared by
dissolving 100 l (50.0 mmol) in 400 l of CDCl3. NMR analysis of
the synthesized acyl-CoAs was performed by dissolving 300
(1.0 mmol) in 200 l of D2O. All NMR spectra were obtained at
l
l
ll
l
ambient temperature and pressure.
Analytical HPLC–UV–MS–MS/MS
Acyl-CoA dehydrogenase activity studies
The eluents were the same as for the semipreparative HPLC–UV.
The gradient was 100% A to 100% B over 60 min at a flow rate of
0.5 ml/min. Simultaneously, UV (259 nm), full-scan MS, and full-
scan MS/MS (data-dependent) data were collected. Data-depen-
dent MS/MS data collection was triggered on the most intense
ion observed in the MS spectrum using a setting of 26 for the rel-
ative collision energy.
Acyl-CoA dehydrogenase activities were measured using, as
substrates, the synthesized and purified cis-4-decenoyl-CoA, 3-
phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA as well as
our standard substrates butyryl-CoA, octanoyl-CoA, and palmi-
toyl-CoA. Rat skeletal muscle mitochondria were prepared and
the acyl-CoA dehydrogenase activities were determined spectro-
photometrically at 37.0 °C with 20 lg of mitochondrial protein in
Fig. 2. Characterization of synthesized 2,6-dimethylheptanoic acid. (A) GC/MS chromatogram of the TMS derivative of 2,6-dimethylheptanoic acid with a retention time of
11.14 min. The MS spectrum contains the [M+] ion at m/z 230. (B) 13C NMR contains signals at d = 38.95 and 28.01 ppm, consistent with two branched methyl groups.

Synthesis and characterization of acyl-CoAs / H.F. Sobhi et al. / Anal. Biochem. 401 (2010) 114–124
119
a 1-ml cuvette as described by Hoppel and coworkers [25]. The
reaction medium contained 34 mM potassium phosphate (pH
7.2), 0.15 mM cytochrome c, 1.5 mM potassium cyanide, 3.0 mM
phenazine ethosulfate, 1.0 mM N-ethylmaleimide, and 0.004 mM
rotenone. The reaction was monitored at 550 nm.
consistent with the presence of the double bond between carbons
4 and 5 in the cis conformation [26] (13C NMR: d = 132.13 and
127.07; 1H NMR: d = 5.43 and 5.35 ppm) (Table 2 and supplemen-
tary material). The GC/MS chromatogram of the TMS acid deriva-
tive shows a single peak at 16.50 min. The MS spectrum contains
fragment ions consistent with cis-4-decenoic acid (m/z 73, 117,
147, and 157), as reported previously [16]. Likewise, 2,6-dimethyl-
heptanoic acid was synthesized. Its yield was 39%, and it was ana-
lyzed by GC/MS. Fig. 2A shows the GC/MS chromatogram and GC/
MS spectrum, and Fig. 2B contains a section of the 13C NMR spec-
trum. The NMR data are consistent with the presence of branched
methyl groups on carbons 2 and 6 [23] (13C NMR: d = 38.95 and
28.01 ppm; 1H NMR: d = 1.18 and 0.89 ppm) (Table 2 and supple-
mentary material). The GC/MS chromatogram of the TMS deriva-
tive of the acid shows a single peak at 11.14 min, and the MS
spectrum contains fragment ions consistent with 2,6-dimethyl-
heptanoic acid (m/z 73, 130, 146, and 159), as reported previously
[15]. The purchased 3-phenylpropionic acid was examined by 13C
NMR, and the spectrum contained the expected signals
(Table 2).
Results and discussion
The goals of this study were to develop a general method for the
synthesis of medium-chain acyl-CoAs from unsaturated and less
common fatty acids, which are not commercially available, and
to establish a procedure for the isolation and purification of highly
purified acyl-CoAs in good yields. The synthesis starts with solubi-
lizing CoASH and the activated mixed anhydride of the medium-
chain fatty acid anhydride in a solvent system of THF/water. This
solvent system decreases the hydrolysis of the mixed anhydride
and maintains the solubility of CoA at RT. The acylation stoichiom-
etry is 20-fold excess of the fatty acid to CoASH; this enhances full
acylation of the CoASH to acyl-CoA.
The synthesis protocol resulted in good yields of acyl-CoAs (75–
78% based on CoASH). Semipreparative HPLC–UV chromatograms
are shown in Figs. 3–5. Table 2 shows the key 13C NMR data used
to characterize the synthesized acyl-CoA and shows that they re-
late directly to the 13C NMR data observed in the acid. Also shown
in Table 2 are the MS/MS data generated from the flow injection
analysis. The presence of an acyl-CoA is indicated by the specific
pattern of ions reported (m/z 408 and 426). Fig. 3A shows a semi-
Two of the fatty acids required for this project were not com-
mercially available, and so we synthesized these acids in our labo-
ratory. The key step in the synthesis of cis-4-decenoic acid is the pH
control of the reaction mixture; keeping the pH below 4.0 results in
a yield of 92% and avoids saturation or isomerization. The synthe-
sized acid was characterized by 13C NMR and GC/MS (Fig. 1) (the
full 1H NMR and 13C NMR spectra of the prepared cis-4-decenoic
acid are contained in the supplementary material). NMR data are
Fig. 3. Characterization of cis-4-decenoyl-CoA. (A) Semipreparative HPLC–UV chromatograph (258 nm). The peak between 20 and 21 min is cis-4-decenoyl-CoA. This peak
was collected and characterized. (B) Off-line MS flow injection analysis of the collected peak gives MS response at m/z 918. (C) Full-scan MS/MS flow injection fragmentation
of m/z 918 and the characteristic acyl-CoA fragments of m/z 426 and 408 are shown. (D) 13C NMR spectrum of the peak collected between 20 and 21 min has signal responses
at d = 132.73 and 127.08 ppm. This is consistent with double bond peaks between carbons 4 and 5 (see also Fig. 1B and Table 2).

120
Synthesis and characterization of acyl-CoAs / H.F. Sobhi et al. / Anal. Biochem. 401 (2010) 114–124
preparative HPLC–UV chromatogram of cis-4-decenoyl-CoA, which
eluted at 20.2 min. Fractions 41 and 42 (20.0–21.0 min) were com-
bined and analyzed by flow injection LC–MS–MS/MS. Fig. 3B shows
the MS spectrum containing m/z 918, which is consistent with cis-
4-decenoyl-CoA. Fig. 3C is the MS/MS spectrum generated from
flow injection analysis. The fragments m/z 426 (from the phospho-
adenosine group [27,28]) and m/z 408 (loss of water from the phos-
phoadenosine group) are consistent with CoASH. A section of 13C
NMR of the isolated cis-4-decenoyl-CoA (Fig. 3D) contains spectral
peaks (d = 132.73 and 127.08 ppm) identical to the spectral peaks
observed in cis-4-decenoic acid (Table 2), and these peaks are
consistent with the double bond between carbons 4 and 5 of the
cis-4-decenoic acid (the full 13C NMR spectrum is shown in the
supplementary material).
The synthesis of 3-phenylpropionyl-CoA is greatly aided by our
ability to purchase the acid (3-phenylpropionic acid) rather than
undertake its synthesis (as in the case of cis-4-decenoic acid and
2,6-dimethylheptanoic acid). Table 2 shows the key 13C NMR data
used to characterize synthesized 3-phenylpropionyl-CoA and
shows that the 13C NMR acyl-CoA data relate directly to the 13C
NMR data observed in the acid. Fig. 4A shows a semipreparative
HPLC–UV chromatogram of 3-phenylpropionyl-CoA, which eluted
at 17.8 min. Fraction 36 (17.5–18.0 min) was analyzed by flow
injection LC–MS–MS/MS. Fig. 4B shows the MS spectrum contain-
ing m/z 898, which is consistent with 3-phenylpropionyl-CoA.
Fig. 4C is the MS/MS spectrum generated from flow injection anal-
ysis. The fragments m/z 408 and 426 are consistent with CoASH.
Fractions 25 and 26 (12.0–13.0 min) were identified by 13C NMR
as containing unreacted 3-phenylpropionic acid and by flow injec-
tion MS as containing unreacted CoASH. The 13C NMR of isolate 3-
phenylpropionyl-CoA (Fig. 4D) contains spectral peaks (d = 140.18
and 128.73, 128.47, and 126.55 ppm), consistent with the presence
of a phenyl group. This portion of the spectra is identical to that ob-
served in 3-phenylpropionic acid (Table 2).
Fig. 5A shows the semipreparative HPLC chromatogram of 2,6-
dimethylheptanoyl-CoA, which eluted at 20.1 min. Fraction 41
(20.0–20.5 min) was analyzed by flow injection LC–MS–MS/MS.
Fig. 5B shows the MS spectrum containing m/z 906, which is con-
sistent with 2,6-dimethylheptanoyl-CoA. Fig. 5C is the MS/MS
spectrum generated from flow injection analysis. The fragments
m/z 408 and 426 are consistent with CoASH. These fragment ions
also are present in the flow injection analysis MS/MS spectra for
cis-4-decenoyl-CoA and 3-phenylpropionyl-CoA (Figs. 3C and 4C,
respectively). Fractions 25 and 26 (12.0–13.0 min) showed unre-
acted CoASH. The 13C NMR of the isolated 2,6-dimethylhepta-
noyl-CoA (Fig. 5D) contains spectral peaks (d = 38.95 and
30.65 ppm) identical to the spectral peaks observed in 2,6-dimeth-
ylheptanoic acid (Fig. 2B). These are consistent with the presence
of two branched methyl groups at carbons 2 and 6. Full NMR
spectra are shown in the supplementary material.
Fig. 4. Characterization of 3-phenylpropionyl-CoA. (A) Semipreparative HPLC–UV chromatogram with the 3-phenylpropionyl-CoA peak between 17.5 and 18.0 min. (B) Off-
line MS flow injection analysis of the peak collected between 17.5 and 18.0 min gives a response at m/z 898. (C) Full-scan MS/MS flow injection fragmentation of m/z 898,
producing the characteristic acyl-CoA fragment ions. (D) 13C NMR spectrum contains d = 140.18, 128.73, 128.47, and 126.55 ppm, consistent with four characteristic peaks of
a phenyl group.

Synthesis and characterization of acyl-CoAs / H.F. Sobhi et al. / Anal. Biochem. 401 (2010) 114–124
121
Fig. 5. Characterization of 2,6-dimethylheptanoyl-CoA. (A) Semipreparative HPLC–UV with the 2,6-dimethylheptanoyl-CoA peak between 20.0 and 20.5 min. (B) Off-line MS
flow injection analysis of the peak collected between 20.0 and 20.5 min gives a response at m/z 906. (C) Full-scan MS/MS flow injection fragmentation of m/z 906, producing
the characteristic acyl-CoA fragment ions. (D) 13C NMR spectrum contains d = 38.95 and 30.65 ppm, consistent with two branched methyl groups.
Analytical HPLC–UV–MS–MS/MS
on m/z 898 (Fig. 7D) shows the CoA fragment ions m/z 408 and
426. Fig. 8A shows the 2,6-dimethylheptanoyl-CoA UV chromato-
gram with the peak eluting at 32.4 min; this peak represents
96.5% of the total UV peak area. Fig. 8B shows the MS chromato-
gram that was collected simultaneously. The MS spectrum
(Fig. 8C) shows a mass signal at m/z 906. The full-scan MS/MS spec-
trum on m/z 906 (Fig. 8D) shows the CoA fragment ions m/z 408
and 426.
The purification of the synthesized acyl-CoAs resulted in purity
greater than 95% (based on the UV response of the analytical
HPLC–UV–MS–MS/MS analysis [Figs. 6–8]). Documentation of the
purity of our synthesized acyl-CoA products was performed by
analytical HPLC–UV–MS–MS/MS. Figs. 6–8 show the UV chromato-
grams, MS chromatograms, MS spectra of the acyl-CoA chromato-
graphic peaks, and MS/MS spectra of those same peaks. Fig. 6A
shows the UV chromatogram (at 259 nm) of our purified cis-4-
decenoyl-CoA, which eluted at 34.0 min. There is a second peak
at 35.8 min, which is only 3.2% of the total peak area. There is a
very small peak (0.3% of the total peak area) at 34.6 min. By mass
and chromatographic behavior, this small peak is consistent with
cis-4-decenoyl-iso-CoA [17]. Fig. 6B shows the MS chromatogram
(total ion current [TIC] = m/z 200–1000) that was collected simul-
taneously. The MS spectrum of the chromatographic peak eluting
at 34.0 min (Fig. 6C) shows a mass signal at m/z 918 (the expected
mass of cis-4-decenoyl-CoA). The full-scan MS/MS spectrum on m/z
918 (m/z 200–1000 at a relative collision energy of 25 [Fig. 6D])
contains fragment ion consistent with CoASH: m/z 408 and 426.
Fig. 7A shows the 3-phenylpropionyl-CoA UV chromatogram with
the 3-phenylpropionyl-CoA peak (retention time = 25.8 min), rep-
resenting 99.2% of the UV peak areas. The MS spectrum (Fig. 7C)
shows a mass signal at m/z 898. The full-scan MS/MS spectrum
Acyl-CoA dehydrogenase activity studies
The dehydrogenation of acyl-CoAs is catalyzed by enzymes with
different chain length specificities. Our standard assay procedure
uses three substrates: butyryl-CoA (predominantly measures
short-chain acyl-CoA dehydrogenase [SCAD]), octanoyl-CoA (pre-
dominantly measures MCAD), and palmitoyl-CoA (measures both
LCAD and VLCAD). The quality control samples for our determina-
tion of acyl-CoA dehydrogenase activities are frozen isolated rat
skeletal muscle mitochondria. We used five different aliquots of
the quality control material to evaluate activities using our current
regular substrates (butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA)
as well as the three acyl-CoAs prepared above (cis-4-decenoyl-CoA,
3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA) (see
Fig. 9). MCAD and LCAD are significantly different in their substrate
specificity toward cis-4-decenoyl-CoA (LCAD oxidizes cis-4-dece-

122
Synthesis and characterization of acyl-CoAs / H.F. Sobhi et al. / Anal. Biochem. 401 (2010) 114–124
Fig. 6. Analytical HPLC–UV–MS–MS/MS of cis-4-decenoyl-CoA. (A) UV chromatogram at 259 nm. (B) TIC–MS chromatogram (m/z 700–1000). (C) MS spectrum of cis-4-
decenoyl-CoA (m/z 918). (D) Full-scan MS/MS fragmentation of m/z 918.
Fig. 7. Analytical HPLC–UV–MS–MS/MS of 3-phenylpropionyl-CoA. (A) UV chromatogram. (B) TIC–MS chromatogram. (C) MS spectrum of 3-phenylpropionyl-CoA (m/z 898).
(D) Full-scan MS/MS fragmentation of m/z 898.
noyl-CoA at a rate of ꢀ30% of that observed with MCAD in bovine
liver [29]). In our study, the reaction rate of cis-4-decenoyl-CoA is
slightly higher than that of octanoyl-CoA. 3-Phenylpropionyl-CoA
(specific substrate for MCAD), unlike octanoyl-CoA, shows zero
activity with SCAD in rat liver [4]. 2,6-Dimethylheptanoyl-CoA (a
specific substrate for LCAD) has zero activity with VLCAD in human
fibroblasts [8]. Because palmitoyl-CoA is a substrate for both LCAD
and VLCAD, and in our study the activity of palmitoyl-CoA is seven
times higher than that of 2,6-dimethylheptanoyl-CoA, 2,6-dimeth-
ylheptanoyl-CoA can be used to differentiate between LCAD and

Synthesis and characterization of acyl-CoAs / H.F. Sobhi et al. / Anal. Biochem. 401 (2010) 114–124
123
Fig. 8. Analytical HPLC–UV–MS–MS/MS of 2,6-dimethylheptanoyl-CoA. (A) UV chromatogram. (B) TIC–MS chromatogram. (C) MS spectrum of m/z 906. (D) Full-scan MS/MS
fragmentation of m/z 906.
Fig. 9. Acyl-CoA dehydrogenase activity in rat skeletal muscle mitochondria. The graph shows the results generated from five different aliquots of isolated rat skeletal muscle
mitochondria. Error bars represent standard deviations.
VLCAD activities (Fig. 9). With these good quality control results,
we have added the new substrates to our assays for acyl-CoA dehy-
drogenase in studies involving human and experimental samples.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ab.2010.02.026.
Acknowledgments
References
The authors thank Mariana Rosca for preparing the rat skeletal
muscle mitochondria, Hiral Patel and Colin Naples for performing
the acyl-CoA dehydrogenase activity studies, and Janos Kerner for
engaging in helpful discussions. This work was supported by the
Center for Mitochondrial Diseases.
[1] R.J. Pollitt, Inherited disorders of straight chain fatty acid oxidation, Arch. Dis.
Child. 62 (1987) 6–7.
[2] R. Ensenauer, J.L. Winters, P.A. Parton, D.F. Kronn, J.W. Kim, D. Matern, P.
Rinaldo, S.H. Hahan, Genotypic differences of MCAD deficiency in the Asian
population: novel genotype and clinical symptoms preceding newborn
screening notification, Genet. Med. 7 (2005) 339–343.

124
Synthesis and characterization of acyl-CoAs / H.F. Sobhi et al. / Anal. Biochem. 401 (2010) 114–124
[16] M.S. Chauhan, K. Dakshinamurti, Synthesis of S-erucyl coenzyme A, Prep.
[3] P. Rinaldo, J.J. O’Shea, P.M. Coates, D.E. Hale, C.A. Stanley, K. Tanaka, Medium-
chain acyl-CoA dehydrogenase deficiency: diagnosis by stable-isotope dilution
measurement of urinary n-hexanoylglycine and 3-phenylpropionylglycine, N.
Engl. J. Med. 319 (1988) 1308–1313.
Biochem. 10 (1980) 37–41.
[17] P.E. Minkler, V.E. Anderson, N.C. Maiti, J. Kerner, C.L. Hoppel, Isolation and
identification of two isomeric forms of malonyl-coenzyme A in commercial
malonyl-coenzyme A, Anal. Biochem. 328 (2004) 203–209.
[4] P. Rinaldo, J. Oshea, R. Welch, K. Tanaka, The enzymatic basis for the
dehydrogenation of 3-phenylpropionic acid: In vitro reaction of 3-
phenylpropionyl-CoA with various acyl-CoA dehydrogenases, Pediatr. Res. 27
(1990) 501–506.
[18] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1959) 70–77.
[19] S.J. Jin, K.Y. Tserng, Metabolic origins of urinary unsaturated dicarboxylic acids,
Biochemistry 29 (1990) 8540–8547.
[5] M. Duran, L. Brulnvis, D. Ketting, J.B.C. De Klerk, S.K. Wadman, cis-4-Decenoic
[20] A. Poulos, E. Barone, Partial synthesis of [1–14C]Phytanic acid, Lipids 15 (1980)
19–21.
acid in plasma:
a characteristic metabolite in medium-chain acyl-CoA
dehydrogenase deficiency, Clin. Chem. 34 (1988) 548–551.
[21] I. Richter, H.K. Mangold, Synthesis of muscalure and homologous
hydrocarbons, Chem. Phys. Lipids 11 (1973) 210–214.
[22] K.Y. Tserng, S.J. Jin, D.S. Kerr, C.L. Hoppel, Abnormal urinary excretion of
unsaturated dicarboxylic acids in patients with medium-chain acyl-CoA
dehydrogenase deficiency, J. Lipid Res. 31 (1990) 763–771.
[6] M. Kimura, H.R. Yoon, P. Wasant, Y. Takahashi, S. Yamaguchi, A sensitive and
simplified method to analyze free fatty acids in mitochondrial beta oxidation
disorders using gas chromatography/mass spectrometry and dried blood spots,
Clin. Chim. Acta 316 (2002) 117–121.
[7] W. Le, A.S. Abbas, H. Sprecher, J. Vockley, H. Schulz, Long-chain acyl-CoA
[23] T. Wieland, L. Rueff, Synthese von b-oxybutyryl-und S-acetacetyl-coenzyme A,
Angew. Chem. 65 (1953) 186–187.
dehydrogenase is
a key enzyme in the mitochondrial b-oxidation of
unsaturated fatty acids, Biochim. Biophys. Acta 1485 (2000) 121–128.
[8] R.J. Wanders, S. Denis, J.P. Ruiter, L.I. Jlst, G. Dacremont, 2,6-
[24] P.E. Minkler, J. Kerner, S.T. Ingalls, C.L. Hoppel, Novel isolation procedure for
short-, medium-, and long-chain acyl-coenzyme A esters from tissue, Anal.
Biochem. 376 (2008) 275–276.
Dimethylheptanoyl-CoA is
a specific substrate for long-chain acyl-CoA
dehydrogenase (LCAD): evidence for a major role of LCAD in branched-chain
fatty acid oxidation, Biochim. Biophys. Acta 1393 (1998) 35–40.
[9] E.J. Simon, D. Shemin, The preparation of S-succinyl coenzyme A, J. Am. Chem.
Soc. 75 (1953) 2520.
[10] P. Goldman, P.R. Vagelos, The specificity of triglyceride synthesis from
diglygerides in chicken adipose tissue, J. Biol. Chem. 236 (1961) 2620–2623.
[11] S. Werner, S-Palmitoyl coenzyme A, Biochem. Prep. 7 (1960) 80–83.
[12] A. Al-Arif, M. Blecher, Synthesis of fatty acyl CoA and other thiol esters using N-
hydroxysuccinimide esters of fatty acids, J. Lipid Res. 10 (1969) 344–345.
[13] M. Blecher, Synthesis of long chain fatty acyl-CoA thioesters using N-
hydroxysuccinimide esters, Methods Enzymol. 72 (1981) 404–408.
[14] D. Mangroo, G.E. Gerber, Synthesis of acyl-CoA thioesters, Biochem. Cell Biol.
68 (1990) 308–312.
[25] C.L. Hoppel, J.P. DiMarco, B. Tandler, Riboflavin and rat hepatic cell structure
and function: Mitochondrial oxidative metabolism in deficiency states, J. Biol.
Chem. 254 (1979) 4164–4170.
[26] T. Saito, K. Hayamizu, M. Yanagisawa, O. Yamamoto, Spectral Database for
Organic Compounds (SDBS), National Institute of Advanced Industrial Science
and Technology (AIST), Japan.
[27] J.A. Zirrolli, P. Wheelan, R.C. Murphy, Analysis of long-chain acyl coenzyme A
thioesters by negative ion fast-atom bombardment mass spectrometry and
tandem mass spectrometry, J. Am. Soc. Mass Spectrom. 5 (1994) 416–424.
[28] T. Mauriala, K.H. Herzig, M. Heinonen, J. Idziak, S. Auriola, Determination of
long-chain fatty acid acyl-coenzyme
A
compounds using liquid
chromatography–electrospray ionization tandem mass spectrometry, J.
Chromatogr. B 808 (2004) 263–268.
[15] V.B. Rajgarhia, N.D. Priestly, W.R. Strohl, Efficient synthesis of radiolabeled
propionyl-coenzyme A and acetyl-coenzyme A, Anal. Biochem. 224 (1995)
159–162.
[29] V. Dommes, W.H. Kunau, Purification and properties of acyl coenzyme A
dehydrogenases from bovine liver: Formation of 2-trans, 4-cis-decadienoyl
coenzyme A, J. Biol. Chem. 259 (1984) 1789–1797.