One-pot synthesis of β-keto esters and preparation of 3-ketopalmitoyl-CoA

Article abstract of DOI:10.1055/s-0031-1291149

β-Keto esters were synthesized from acyl chlorides and sodium ethyl acetoacetate in EtOH using a simple one-pot, one-step' method. The deacetylation of α-acetyl β-keto ester to β-keto ester was performed simply, by heating the reaction mixture at reflux for 12 hours, without the addition of additional reagents (e.g., NHl, NH MeOH, or NaOH). One of the β-keto esters prepared using this method was ethyl 3-oxohexadecanoate, a key intermediate in the synthesis of 3-ketopalmitoyl coenzyme A (3-oxohexadecanoyl coenzyme A), a potential substrate of the biologically important enzyme 17β-hydroxysteroid dehydrogenase type 12. Georg Thieme Verlag Stuttgart ? New York.

Full text of DOI:10.1055/s-0031-1291149

LETTER
▌1609
letter
One-Pot Synthesis of β-Keto Esters and Preparation of 3-Ketopalmitoyl-CoA
One-Pot Synthesis of β-Keto Esters
Urban Košak, Andreja Kovač, Stanislav Gobec*
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia
Fax +386(1)4258031; E-mail: gobecs@ffa.uni-lj.si
Received: 29.02.2012; Accepted after revision: 10.04.2012
7a–e (Scheme 1). These were further deacetylated simply
Abstract: β-Keto esters were synthesized from acyl chlorides and
by heating the reaction mixture at reflux for 12 hours. Af-
sodium ethyl acetoacetate in EtOH using a simple ‘one-pot, one-
ter filtration of the precipitated NaCl, solvent evaporation,
and acid workup, the β-keto esters 2a–e were purified by
step’ method. The deacetylation of α-acetyl β-keto ester to β-keto
ester was performed simply, by heating the reaction mixture at re-
flux for 12 hours, without the addition of additional reagents (e.g., flash column chromatography. A series of β-keto esters
NH4Cl, NH3, MeOH, or NaOH). One of the β-keto esters prepared
using this method was ethyl 3-oxohexadecanoate, a key intermedi-
ate in the synthesis of 3-ketopalmitoyl coenzyme A (3-oxohexa-
Overall yields for these three-step preparations of purified
decanoyl coenzyme A), a potential substrate of the biologically
important enzyme 17β-hydroxysteroid dehydrogenase type 12.
2a–e were thus obtained in a one-pot procedure from acyl
chlorides without isolation of any intermediates (Table 1).
β-keto esters were between 30% and almost 50%, which
is comparable to yields in analogous methods.4
Key words: β-keto esters, one-pot reaction, deacetylation, 3-keto-
palmitoyl coenzyme A, 17β-hydroxysteroid dehydrogenase type 12
+
Na
+
–
–
Na
O
O
O
O
O
EtOH
+
– EtOAc
OEt
R
R
1a–e
Cl
OEt
17β-Hydroxysteroid dehydrogenase type 12 (17β-
HSD12) is a member of the 17β-HSD that catalyze the re-
duction of 17-ketosteroids or the oxidation of 17β-hy-
droxysteroids.1 17β-HSD12 has an important role in
estrone formation, by selectively and efficiently catalyz-
ing the transformation of the weak estrogen estron into the
potent estrogen estradiol.2 This enzyme also has ketoacyl-
coenzyme A reductase activity and it is involved in lipid
metabolism through participation in fatty-acid elonga-
tion.3 3-Ketopalmitoyl coenzyme A is hypothesized to be
a substrate of this enzyme.3 To investigate this hypothesis,
milligram quantities of 3-ketopalmitoyl coenzyme A were
required, and so a total synthesis is needed to be devel-
oped.
sodium
O
ethyl acetoacetate
7a–e
(not isolated)
+
–
acid
workup
Na
O
O
O
O
R
OEt
R
OEt
2a–e
8a–e
(not isolated)
Scheme 1 Reaction pathway
β-Keto ester 2b was further used for coupling with
CoASH. As coupling of β-ketopalmitoic acid and differ-
ent thioles with no protection on the β-keto functionality
proved to be unsuccessful, we first introduced the 1,3-di-
oxolane protecting group onto the β-keto group. β-Keto
ester 2b was treated with ethylene glycol in the presence
of a catalytic amount of PTSA·H2O. The ethyl ester 3 was
prepared with 60% yield. Compound 3 was then hydro-
lyzed using 1 M aqueous LiOH solution in a THF–H2O
mixture to provide, after acidic treatment, the carboxylic
acid 4 with 92% yield. This was followed by the prepara-
tion of the activated esters with the use of known activat-
ing agents, such as N-hydroxysuccinimide5 or N-hydroxy-
phthalimide5a and dicyclohexylcarbodiimide. These acti-
vated derivatives reacted poorly with methyl thioglyco-
late, a model thiol reagent that was used to probe the
reaction before the use of the more expensive CoASH.
Compound 4 was then finally converted into a mixed an-
hydride using ethyl chloroformate in the presence of N-
methylmorpholine in tetrahydrofuran (THF) at –5 °C and
reacted in situ with an aqueous solution of the trilithium
salt of CoASH. The pH of the reaction was 8–9 and tetra-
n-butylammonium chloride was used as a phase-transfer
catalyst to provide the protected thioester 5. This was fol-
lowed with one of the most demanding steps, the deprot-
To date, a number of syntheses for β-keto esters have been
reported.4 Acylation of acetoacetic esters at the C2 carbon
followed by deacetylation of α-acetyl β-keto esters to β-
keto esters is a well-known method. Yuasa and Tsuruta4a
discussed and solved a number of problems of previously
reported methods. In their procedure, deacetylation of α-
acetyl β-keto ester to β-keto ester is still performed by
adding an additional reagent (MeOH) to the reaction mix-
ture. To avoid this additional step, we developed a general
‘one-pot, one-step’ process for the synthesis of β-keto es-
ters from acyl chlorides and sodium ethyl acetoacetate in
EtOH, and applied this to the synthesis of 3-ketopalmitoyl
coenzyme A. The main advantage of this procedure is that
it is very simple, and it gives reasonably good yields.
Aliphatic or aromatic acyl chlorides 1a–e (Table 1, entries
1–5) were reacted with a solution of sodium ethyl aceto-
acetate in EtOH at 0 °C, to form the α-acetyl β-keto esters
SYNLETT 2012, 23, 1609–1612
Advanced online publication: 13.06.2012
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
DOI: 10.1055/s-0031-1291149; Art ID: ST-2012-B0183-L
© Georg Thieme Verlag Stuttgart · New York

1610
U. Košak et al.
LETTER
O
O
O
ection of the 1,3-dioxolane protecting group on the β-keto
functionality. We found that the best way to deprotect in-
termediate 5 was by using a mixture of 4 M aqueous HCl
and a small amount of THF at 40 °C. After alkaline work-
up, the thioester 6 was purified by automated reverse-
phase flash column chromatography (Scheme 2). The
yield over the last two steps was 36%, calculated to the
trilithium salt of CoASH. Other methods for the removal
of 1,3-dioxolane were also tried; for example, using a cat-
alytic amount of PTSA in water, a method described by
Kurosawa et al.,6 and a mixture of 2 M HCl and THF;
however, they were not efficient in this case.
O
i
ii
OEt
Cl
12
12
1b
2b
O
O
O
iv
iii
O
O
O
3
O
OEt
OH
O
12
12
4
O
O
v
SCoA
SCoA
12
12
5
6
NH2
N
N
N
Table 1 One-Pot Synthesis of β-Keto Esters
OH
H
N
H
N
O
O
N
O P O P O
OH OH
MeCOCH2COOEt (1.1 equiv),
CoASH = HS
O
O
O
O
NaOEt (1.1 equiv),
O
O
O
P
OH
EtOH, 0 °C to reflux, 12 h
R
Cl
R
OEt
O
OH
1a–e
2a–e
OH
Entrya Acyl chloride
Product
Yield (%)b
Scheme 2 Reagents and conditions: i. MeCOCH2COOEt, NaOEt,
EtOH, 0 °C to reflux, 12 h, 33%; ii. HOCH2CH2OH, PTSA·H2O
(cat.), PhMe, reflux, 16 h, 60%; iii. (a) 1 M LiOH aq, THF–H2O, 0 °C
to r.t., 24 h, (b) 2 M HCl aq, 0 °C, 92%; iv. (a) ClCOOEt, NMM, THF,
–5 °C, 90 min, under argon; (b) Li3CoASH, 1 M LiOH aq, H2O,
TBAC (cat.), 0 °C to r.t., pH 8–9, 45 min, under argon; (c) 1 M HCl
aq (to pH 6–7), 0 °C; v. (a) 4 M HCl aq, THF, 40 °C, 90 min; (b) 1 M
LiOH aq (to pH 6–7), 0 °C, 36% (for steps iv and v, calcd to
Li3CoASH).
1
1a R = Me(CH2)4
1b R = Me(CH2)12
1c R = Ph
2a R = Me(CH2)4
2b R = Me(CH2)12
2c R = Ph
30
33
47
35
32
2
3
4c
5
1d R = 4-O2NC6H4
1e R = 4-ClC6H4
2d R = 4-O2NC6H4
2e R = 4-ClC6H4
a Conditions: acyl chlorides (35.91 mmol), ethyl acetoacetate (5.0 ml,
39.50 mmol, 1.1 equiv), Na (0.908 g, 39.50 mmol, 1.1 equiv), EtOH
(55 mL).
column used for method A and method B was a Zorbax Eclipse Plus
C18 analytical 150 × 4.6 mm column, at 5 micron (Agilent). The
column used for method C was a Luna 5u C18(2) 100A 250 × 4.6
mm column, at 5 micron (Phenomenex). HPLC Guard Cartridge
systems were used on both columns, as a Security Guard Cartridge
C18 CODS, octadecyl 4 mm × 3.0 mm ID (Phenomenex). The
HPLC columns were thermostatted at 25 °C.
b Isolated yield.
c Solid acyl chloride was dissolved in THF (10 mL).
To conclude, we successfully synthesized a complex mol-
ecule, 3-ketopalmitoyl-CoA, which is a potential sub-
strate for the biologically relevant 17β-HSD12 enzyme.
Additionally, a previously unknown ‘one-pot, one-step’
method for the synthesis of β-keto esters from acyl chlo-
rides was developed.
Method A: 20 μL of sample solution was injected and eluted at a
flow rate of 0.7 mL/min, using a linear gradient of mobile phase A
(MeCN) and mobile phase B (aq phosphate buffer: 20 mM, pH
4.95). Gradient for method A: from 3% to 70% mobile phase A in
20 min, then 6 min at 70% mobile phase A, and back to 3% mobile
phase A in 6 min.
Method B: 20 μL of sample solution was injected and eluted at a
flow rate of 0.7 mL/min, using a linear gradient of mobile phase A
(MeCN) and mobile phase B [aq phosphate buffer (20 mM, pH
4.95) with 3% MeCN]. Gradient for method B: from 0% to 67%
mobile phase A in 20 min, then 6 min at 67% mobile phase A, and
back to 0% mobile phase A in 6 min. Method C: 20 μL of sample
solution was injected and eluted at a flow rate of 1.5 mL/min, using
a linear gradient of mobile phase A (MeCN) and mobile phase B (aq
phosphate buffer: 25 mM, pH 5.0). Gradient for method C: 3 min at
5% mobile phase A, then in 12 min to 70% mobile phase A, then 15
min at 70% mobile phase A, and back to 3% mobile phase A in 10
min. Automated reverse-phase flash column chromatography was
carried out with a Biotage Isolera One System. The cartridge used
was a 30 g Biotage SNAP Cartridge KP-C18-HS.
Experimental Section
1H NMR, 13C NMR, and 31P NMR were recorded on a Bruker
Avance III NMR spectrometer at 400, 100, and 162 MHz respec-
tively. The chemical shifts (δ) are reported in parts per million
(ppm) and are referenced to either the internal standard TMS (when
CDCl3 was used) or the deuterated solvent (D2O) used. The cou-
pling constants (J) are reported in Hz, and the splitting patterns are
indicated as: s (singlet), br s (broad singlet), d (doublet), dd (doublet
of doublets), t (triplet), br t (broad triplet), dt (doublet of triplets), q
(quartet), and m (multiplet). IR spectra were recorded on a Perkin-
Elmer FT-IR System Spectrum BX. Microanalyses were performed
on a PerkinElmer C, H, N Analyzer 240 C. The analyses are indi-
cated by the symbols of the elements and they were within ±0.4%
of the theoretical values. Centrifugation was performed on a
Tehtnica Centric 150. Analytical reversed-phase HPLC was per-
formed on an Agilent 1100 LC modular system equipped with an
autosampler, a quarternary pump system, a photodiode array detec-
tor, a thermostatted column compartment, and a ChemStation data
system. The detector was set to 210.8, 254.16, and 280.16 nm. The
Method D: the sample was eluted at a flow rate of 22 mL/min using
a linear gradient of mobile phase A (MeCN) and mobile phase B
(double-distilled water). Gradient for method D: 5 column volumes
of 0% mobile phase A, then in 8 column volumes to 30% mobile
phase A. The detector was set at 254 and 210 nm.
MS were recorded on a VG-Analytical AutoSpec Q Micromass
mass spectrometer. Melting points were determined on a Leica hot
stage microscope and are uncorrected. Evaporation of solvents was
Synlett 2012, 23, 1609–1612
© Georg Thieme Verlag Stuttgart · New York

LETTER
One-Pot Synthesis of β-Keto Esters
1611
performed at reduced pressure. Reagents and solvents were pur-
chased from Acros Organics, Aldrich, Carlo Erba, Euriso-Top,
Fluka, Janssen Chimica, J.T. Baker, Kemika, Merck, Panreac,
Riedel-de Haën, Sigma, and Sigma-Aldrich, and were used without
further purification, unless otherwise stated. The trilithium salt of
coenzyme A was from yeast at ≥93% purity, as purchased from
Sigma. Double-distilled water was obtained using a Millipore Ad-
vantage A10 system. THF was refluxed under argon over sodium
and benzophenone for 2 h, then fractionally distilled under argon
through a helices-packed column prior to use. Ethylene glycol was
dried with NaOH and distilled under vacuum prior to use. Ethyl
chloroformate was washed with H2O and sat. brine solution, then
fractionally distilled under argon through a 20 cm Vigreux column
prior to use. N-Methylmorpholine was refluxed under argon over
sodium for 3 h, then fractionally distilled under argon through a 20
cm Vigreux column prior to use. Tetra-n-butylammonium chloride
was crystallized from acetone by addition of Et2O, then dried in vac-
uo at r.t. in the presence of NaOH, P2O5, and silica gel prior to use.
Flash column chromatography was performed on silica gel 60
(0.040–0.063 mm) for column chromatography (particle size, 230–
400 mesh). Analytical TLC was performed on silica gel Merck 60
Ethyl 3-(4-Nitrophenyl)-3-oxopropanoate (2d)
Purified by flash column chromatography using Et2O–PE (4:21) as
the eluent to produce the β-keto ester 2e as a slightly yellow solid
(35% yield). Rf = 0.45 (n-hexane–EtOAc = 4:1); mp 63–67 °C. 1H
NMR (400 MHz, CDCl3): δ = 1.27 (1.32 H, keto, t, J = 7.15 Hz),
1.36 (1.68 H, enol, t, J = 7.15 Hz), 4.04 (0.88 H, keto, s), 4.23 (0.88
H, keto, q, J = 7.15 Hz), 4.30 (1.12 H, enol, q, J = 7.05 Hz), 5.77
(0.56 H, enol, s), 7.94 (1.12 H, enol, dt, J1 = 4.89 Hz, J2 = 2.20 Hz),
8.12 (0.88 H, keto, dt, J1 = 4.89 Hz, J2 = 2.13 Hz), 8.28 (1.12 H,
enol, dt, J1 = 4.89 Hz, J2 = 2.20 Hz), 8.34 (0.88 H, keto, dt, J1 = 4.89
Hz, J2 = 2.16 Hz), 12.57 (0.56 H, enol, s).
Ethyl 3-(4-Chlorophenyl)-3-oxopropanoate (2e)
Purified by flash column chromatography using Et2O–PE (1:8) as
the eluent to produce the β-keto ester 2f as a slightly pink liquid
1
(32% yield). Rf = 0.48 (n-hexane–EtOAc = 4:1). H NMR (400
MHz, CDCl3): δ = 1.26 (2.31 H, keto, t, J = 7.15 Hz), 1.34 (0.69 H,
enol, t, J = 7.15 Hz), 3.97 (1.54 H, keto, s), 4.22 (1.54 H, keto, q,
J = 7.11 Hz), 4.27 (0.46 H, enol, q, J = 7.19 Hz), 5.64 (0.23 H, enol,
s), 7.38–8.11 (4 H, m), 12.59 (0.23 H, enol, s).
Ethyl 2-(2-Tridecyl-1,3-dioxolan-2-yl)acetate (3)
F254 aluminium sheets (0.20 mm), using visualization with ultravio-
Ester 2b (3.361 g, 11.26 mmol, 1.0 equiv) was dissolved in toluene
(50 mL). Ethylene glycol (1.884 mL, 33.78 mmol, 3.0 equiv) and
PTSA·H2O (214.21 mg, 1.126 mmol, 0.1 equiv) were added, and
the mixture was refluxed for 16 h. During the reaction, the toluene–
H2O azeotrope was removed using a Dean–Stark apparatus. The re-
action mixture was washed with sat. aq NaHCO3 solution (50 mL)
followed by sat. brine solution (50 mL), dried over anhyd Na2SO4,
and evaporated. The residue was purified by flash column chroma-
tography using Et2O–PE (1:5) as the eluent to produce 2.303 g of 3
as a colorless oil (60% yield). Rf = 0.63 (n-hexane–EtOAc = 1:3).
IR (NaCl): 3650, 2925, 2854, 1739, 1466, 1369, 1304, 1219, 1040,
949, 842, 722 cm–1. 1H NMR (400 MHz, CDCl3): δ = 0.90 (3 H, t,
J = 6.89 Hz), 1.05–1.52 (25 H, m), 1.78–1.84 (2 H, m), 2.66 (2 H,
s), 3.94–4.05 (4 H, m), 4.17 (2 H, q, J = 7.10 Hz). 13C NMR (100
MHz, CDCl3): δ = 14.13, 14.19, 22.70, 23.54, 29.37, 29.58, 29.66,
29.70, 29.72, 31.93, 37.79, 42.59, 60.50, 65.09, 109.49, 169.62.
HRMS (ESI+): m/z calcd for C20H39O4: 343.2848; found: 343.2842.
Anal. Calcd for C20H38O4: C, 70.13; H, 11.18. Found: C, 70.35; H,
11.41.
let light and/or visualization reagents.
General Procedure for the Synthesis of β-Keto Esters 2a–f
A solution of sodium (1.1 equiv) in commercial absolute EtOH was
cooled to 0 °C and ethyl acetoacetate (1.1 equiv) was added drop-
wise. Acyl chloride 1a–e (1.0 equiv) was added dropwise (solid
acyl chloride 1c was dissolved in a minimal amount of THF and
then added dropwise). The resulting suspension was refluxed for 12
h, then diluted with 96% EtOH, filtered through a pad of Celite, and
evaporated. Toluene and H2O were added to the residue, and the
mixture was adjusted to pH 1–2 with 2 M aq HCl. The organic phase
was washed with sat. brine solution, dried over anhyd Na2SO4, and
evaporated. The residue was purified by flash column chromatogra-
phy to produce the β-keto esters 2a–e.
Ethyl 3-Oxooctanoate (2a)
Purified by flash column chromatography using Et2O–PE (1:10) as
the eluent, to produce β-keto ester 2a as a colorless liquid (30%
1
yield). Rf = 0.52 (n-hexane–EtOAc = 4:1). H NMR (400 MHz,
CDCl3): δ = 0.89 (3 H, t, J = 7.00 Hz), 1.23–1.35 (7 H, m), 1.56–
1.64 (2 H, m), 2.54 (2 H, t, J = 7.40 Hz), 3.44 (1.84 H, keto, s), 4.19
(0.16 H, enol, q, J = 7.15 Hz), 4.20 (1.84 H, keto, q, J = 7.15 Hz),
4.98 (0.08 H, enol, s), 12.12 (0.08 H, enol, s).
2-(2-Tridecyl-1,3-dioxolan-2-yl)acetic acid (4)
A solution of 1 M aq LiOH (67.24 mL, 67.24 mmol, 10 equiv) was
added dropwise to a solution of ester 3 (2.303 g, 6.724 mmol, 1.0
equiv) in a mixture of THF (40 mL) and H2O (15 mL) at 0 °C. The
reaction mixture was allowed to warm to r.t. and then stirred for 24
h. The reaction mixture was cooled to 0 °C and adjusted to pH 2
with 2 M aq HCl, and extracted with EtOAc (3 × 75 mL). The com-
bined organic phases were washed with sat. brine solution (75 mL),
dried over anhyd Na2SO4, and evaporated. The residue was crystal-
lized from EtOAc by adding n-hexane to produce 1.974 g of 4 as
white crystals (92% yield). Rf = 0.54 (EtOAc–n-hexane–AcOH =
1:2:0.25); mp 65–68 °C. IR (KBr): 2917, 2848, 1715, 1466, 1430,
Ethyl 3-Oxohexadecanoate (2b)
Purified by flash column chromatography using Et2O–PE (1:15) as
the eluent to produce the β-keto ester 2b as a slightly golden oil that
solidified into a white solid after cooling (33% yield). Rf = 0.56 (n-
hexane–EtOAc = 4:1); mp 27–30 °C. IR (KBr): 3652, 2925, 2853,
2362, 1744, 1718, 1468, 1318, 1233, 1032, 945, 721, 591 cm–1. 1H
NMR (400 MHz, CDCl3): δ = 0.90 (3 H, t, J = 6.8 Hz), 1.06–1.37
(23 H, m), 1.53–1.63 (2 H, m), 2.54 (2 H, t, J = 7.35 Hz), 3.44 (1.92
H, keto, s), 4.20 (0.08 H, enol, q, J = 7.16 Hz), 4.21 (1.92 H, keto,
q, J = 7.16 Hz), 4.99 (0.04 H, enol, s), 12.11 (0.04 H, enol, s).
HRMS (ESI+): m/z calcd for C18H35O3: 299.2586; found: 299.2596.
Anal. Calcd for C18H34O3: C, 72.44; H, 11,48. Found: C, 72.76; H,
11.87.
1326, 1224, 1140, 1049, 940, 751, 724, 646, 562 cm–1. H NMR
1
(400 MHz, CDCl3): δ = 0.90 (3 H, t, J = 6.90 Hz), 1.12–1.42 (22 H,
m), 1.78-1.83 (2 H, m), 2.72 (2 H, s), 4.00–4.08 (2 H, m), 10.91 (1
H, br s). HRMS (ESI–): m/z calcd for C18H33O4: 313.2379; found:
313.2387. Anal. Calcd for C18H34O4: C, 68.75; H, 10.90. Found: C,
69.08; H, 11.29.
Ethyl 3-Oxo-3-phenylpropanoate (2c)
Purified by flash column chromatography using Et2O–PE (1:7) as
the eluent to produce the β-keto ester 2d as a slightly pink liquid
(47% yield). Rf = 0.47 (n-hexane–EtOAc = 5:1). IR (NaCl): 3647,
2984, 1741, 1688, 1625, 1450, 1268, 1198, 1037, 943, 757, 690,
593 cm–1.1H NMR (400 MHz, CDCl3): δ = 1.26 (2.52 H, keto, t, J =
7.10 Hz), 1.34 (0.48 H, enol, t, J = 7.10 Hz), 4.0 (1.68 H, keto, s),
4.22 (1.68 H, keto, q, J = 7.11 Hz), 4.27 (0.32 H, enol, q, J = 7.03
Hz), 5.67 (0.16 H, enol, s), 7.40-7.97 (5 H, m), 12.59 (0.16 H, enol,
s).
2-(2-Tridecyl-1,3-dioxolan-2-yl)acetic Acid Coenzyme A
Thioester (5)
To a 25 mL round-bottomed flask equipped with a magnetic stirring
bar was added 3 (400.0 mg, 1.272 mmol, 1.0 equiv) under an argon
atmosphere. THF (10 mL) was added with a glass syringe, and the
resulting solution was cooled to –5 °C. N-Methylmorpholine (140.0
μL, 1.272 mmol, 1.0 equiv) was added dropwise with a glass sy-
ringe, and the solution was stirred for 15 min before ethyl chlorofor-
mate (121.6 μL, 1.272 mmol, 1.0 equiv) was added dropwise with a
© Georg Thieme Verlag Stuttgart · New York
Synlett 2012, 23, 1609–1612

1612
U. Košak et al.
LETTER
glass syringe. The resulting suspension was stirred for 90 min at
–5 °C, then the stirring was stopped, and the precipitate was allowed
to settle to the bottom of the flask. Then 1.25 mL of the supernatant
was transferred with a glass syringe into a 25 mL round-bottomed
flask equipped with a magnetic stirring bar under an atmosphere of
argon. THF (ca. 9 mL) was added with a double-tipped needle, fol-
lowed by TBAC (3.54 mg). Solution A (see below) was then added
dropwise with a double-tipped needle, and the resulting solution
was stirred for 45 min. At this time, the nitroprusside test7 of the re-
action mixture was negative for the presence of thiols. During the
reaction, this was maintained at pH 8–9 by adding 1 M aq LiOH
(prepared under argon by dissolving solid LiOH in double-distilled
water agitated with a stream of argon) with a glass syringe. The re-
action mixture was then cooled to 0 °C and opened to the air. The
solution was adjusted to pH 6–7 with 1 M aq HCl. The solution was
transferred to a 50 mL round-bottomed flask, and THF was evapo-
rated with the temperature of the water bath of the rotary evaporator
kept at 30 °C. The residue was frozen and lyophilized, which pro-
duced 151.5 mg of a white solid. This product was used in the next
step without further purification. Rf = 0.49 (n-BuOH–AcOH–H2O =
5:2:3). HRMS (ESI–): m/z calcd for C39H67N7O19P3S: 1062.3425;
found: 1062.3452. HPLC purity, 85% at 254.16 nm (method A,
tR = 20.63 min; method C, tR = 16.76 min).
3402, 2925, 2854, 1652, 1244, 1083, 946, 518 cm–1. 1H NMR (400
MHz, D2O): δ = 0.61 (3 H, s), 0.69–0.77 (6 H, m), 0.84–1.14 (20 H,
m), 1.39 (2 H, br t), 2.32 (2 H, t, J = 6.90 Hz), 2.45 (2 H, t, J = 7.28
Hz), 2.59 (1.24 H, keto, s), 2.93 (2 H, t, J = 6.21 Hz), 3.24 (2 H, t,
J = 6.40 Hz), 3.33 (2 H, t, J = 6.90), 3.42 (1 H, dd, J1 = 9.73 Hz, J2
= 4.83 Hz), 3.56 (1 H, dd, J1 = 9.54 Hz, J2 = 5.14 Hz), 3.91 (1 H, s),
4.12 (2 H, br s), 4.27 (0.38 H, enol, br s), 4.46 (0.62 H, keto, br t),
4.51 (0.38 H, enol, t, J = 4.58 Hz), 4.86-–4.92 (0.38 H, enol, m),
6.02 (0.62 H, keto, d, J = 6.27 Hz), 6.09 (0.38 H, enol, d, J = 5.27
Hz), 8.69–8.15 (1 H, m), 8.38–8.44 (1 H, m). 31P NMR (162 MHz,
D2O): δ = –11.25 (d, J = 19.51 Hz), –10.77 (d, J = 19.51 Hz), 1.93–
2.10 (m, enol), 2.36–2.62 (m, keto). HRMS (ESI–): m/z calcd for
C37H63N7O18P3S: 1018.3163; found: 1018.3150. HPLC purity, 95%
at 254.16 nm (method B, tR = 20.55 min; method C, tR = 16.57 min).
Acknowledgment
We thank the Ministry of Higher Education, Science and Technolo-
gy of the Republic Slovenia for financial support, and Dr. Chris
Berrie for critical reading of the manuscript.
References
Preparation of solution A: Double-distilled H2O (5 mL) was stirred
with a stream of argon and was added with a glass syringe into the
container with the trilithium salt of coenzyme A (100 mg, 0.1273
mmol). The solution was transferred with a glass syringe into a 10
mL round-bottomed flask equipped with a magnetic stirring bar un-
der an argon gas atmosphere. The solution was cooled to 0 °C and
adjusted to pH 8–9 by adding 1 M aq LiOH (prepared under argon
by dissolving solid LiOH in double-distilled H2O agitated with a
stream of argon) with a glass syringe.
(1) Sukurai, N.; Miki, Y.; Suzuki, T.; Watanabe, K.; Narita, T.;
Ando, K.; Yung, T. M. C.; Aoki, D.; Sasano, H.; Handa,
H. J. Steroid Biochem. Mol. Biol. 2006, 99, 174.
(2) Luu-The, V.; Tremblay, P.; Labrie, F. Mol. Endocrinol.
2006, 20, 437.
(3) Moon, Y. A.; Horton, J. D. J. Biol. Chem. 2003, 278, 7335.
(4) (a) Yuasa, Y.; Tsuruta, H.; Yuasa, Y. Org. Process Res. Dev.
1998, 2, 412. (b) Milton, A.; Axelrod, A. E.; Hofmann, K.
J. Am. Chem. Soc. 1950, 72, 1231. (c) Benetti, S.;
Romagnoli, R.; De Risi, C.; Spalluto, G.; Zanirato, V. Chem.
Rev. 1995, 95, 1065. (d) Sato, N.; Saito, N. J. Heterocycl.
Chem. 1988, 25, 1737. (e) Lehmann, F.; Holm, M.; Laufer,
S. J. Comb. Chem. 2008, 10, 364. (f) Riegel, B.; Lilienfeld,
W. M. J. Am. Chem. Soc. 1945, 67, 1273. (g) Paine, J. B.;
Kirshner, W. B.; Moskowitz, D. W.; Dolphin, D. J.
Org.Chem. 1976, 41, 3857. (h) Meyer, W. L.; Brannon, M.
J.; Burgos, C. G.; Goodwin, T. E.; Howard, R. W. J. Org.
Chem. 1985, 50, 438. (i) Anderson, G. W.; Halverstadt, I. F.;
Miller, W. H.; Roblin, R. O. J. Am. Chem. Soc. 1945, 67,
2197. (j) Holmquist, C. R.; Roskamp, E. J. J. Org. Chem.
1989, 54, 3258.
(5) (a) Lucet-Levannier, K.; Lellouch, J. P.; Mioskowski, C.
J. Am. Chem. Soc. 1995, 117, 7546. (b) Al-Arif, A.; Blecher,
M. J. Lipid Res. 1969, 10, 344. (c) Baldwin, J. E.; Widdison,
W. C. J. Am. Chem. Soc. 1992, 114, 2245.
(6) Kurosawa, T.; Fujiwara, M.; Nakano, H.; Sato, M.;
Yoshimura, T.; Murai, T. Steroids 2001, 66, 499.
(7) The nitropruside test was carried out by adding a sample of
the reaction mixture to a mixture of an aqueous solution of
sodium nitroprusside and dilute aqueous ammonia solution.
3-Oxohexadecanoic Acid Coenzyme A Thioester (6)
The raw thioester 5 (100 mg) was suspended in 4 M aq HCl (8 mL)
and heated to 40 °C. Enough THF (ca. 2 mL) was added dropwise
to produce a clear solution from the suspension. The reaction was
monitored by HPLC using method B. After stirring for 90 min at
40 °C, the THF was removed by agitating the reaction mixture with
a stream of nitrogen gas. The resulting suspension was transferred
into a 14 mL centrifuge tube and cooled to 0 °C. Double-distilled
H2O (3.5 mL) was added, and the suspension was left at 0 °C for 10
min. The suspension was centrifuged (3 × 1.5 min at 5,000 rpm),
and the supernatant was removed. HPLC analysis (method B) of the
supernatant showed no presence of thioester 6. Double-distilled
H2O (1 mL) was added to the residue, and the resulting suspension
was cooled to 0 °C. This was adjusted to pH 6–7 with 1 M aq LiOH.
The resulting solution was allowed to warm to r.t. and then purified
by automated reverse-phase flash column chromatography using
method D. The fractions containing thioester 6 were pooled, and the
MeCN was evaporated with the temperature of the water bath of the
rotary evaporator kept at 30 °C. The residue was frozen and lyoph-
ilized to produce 47.8 mg of 6 as a white solid (36% yield for the
last two steps, calculated to the trilithium salt of coenzyme A). Rf =
0.53 (n-BuOH–AcOH–H2O = 5:2:3); mp 185–190 °C. IR (KBr):
Synlett 2012, 23, 1609–1612
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