470
N. Johns • Synthesis of CoA Thiol Esters
Moisture was excluded throughout the operation.
After a further hour at room temperature, the
chloroform solution was washed twice with 5%
sodium bicarbonate solution and twice with water
(each xlOml), dried over sodium sulphate and
the solvent removed in vacuo. The product general-
ly crystallised completely when excess thiophenol
had been removed by leaving overnight at 0.1 mm
mercury and room temperature. Except in the case
of benzyloxycinnamoyl derivatives, all phenyl thiol
esters were free of the parent acids by this stage,
and were essentially pure (thin layer chromato-
graphy (TLC) silica; benzene plus 10 or 20% ethyl
acetate). Crude yields ranged from 85 —95%. Re-
crystallisation was from methanol or benzene/
hexane, and recovered pure yields were variable,
lying in the range 60 —80%. Methanol gave the best
results but gave lower yields, apparently causing
some methanolysis of the product. Prolonged con-
tact with hot methanol (more than 10 min at re-
flux) or cold methanol (more than 12 hours)
caused noticeable loss of product with formation of
the methyl ester. Analytical data and melting points
observed for the products are recorded in Table I.
a.
Stirred in ice for 3 hours while a stream of
nitrogen was bubbled through, or b. sealed under
nitrogen and shaken in the cold room for 3 hours.
At the end of this time the solution was acidified
with 98% formic acid (50 /d) and extracted three
times with its volume of ether. Yields, estimated
spectroscopically (UV), ranged from approx. 80%
in the case of the non-hydroxylated acid derivatives
(from prepurified phenyl thiol esters) to approx.
30% for most of the hydroxylated derivatives. A
very low yield of o-coumaroyl CoA made deter-
mination of the £max for this compound impracti-
cable.
Hydroxamate test10
Testing of acyl CoA solutions was accomplished
by treating 50, 100, and 150//I aliquots of the
solution with 0.2 M neutralised hydroxylamine (pH
8.0, 50 ju\), making the solution up to 200/d with
water as necessary, and allowing the mixture to
react at 30 °C for 10 min. After treatment with
100 //I of a solution containing ferric chloride
(2.5 g) and trichloroacetic acid (10 g) in I n hy-
drochloric acid (10 ml), followed by centrifugation,
absorption was measured in microcuvettes in a
colourimeter at 546 nm against a suitable blank.
A scan of several wavelengths around this value
gave the shape of the absorption peak.
Table I. Melting points and analytical data for non-
hydroxylated series of phenyl thiol esters.
Phenyl thiol ester
Benzoyl
m.p.
[°C]
Analysis [%]
Calcd Found
Purified phenyl thiol esters were weighed direct-
ly (approx. 1 mg) into centrifuge tubes, dissolved
in methanol (0.5 ml) and treated with 0.2 M hy-
droxylamine (pH 8.0) for 10 min at 30 °C. Water
(1 ml) was then added, followed by the above-
mentioned acid ferric chloride reagent (1ml). The
mixture was shaken vigorously, centrifuged and
transferred to a 3 ml cuvette in which the region
700 —400 nm was scanned against a blank.
72.58
4.60
15.28
55- 56 *
C 72.87
C
H
S
H
S
4.70
14.47
Cinnamoyl
90- 91 ** c 74.47 c 75.50
H
S
5.03
13.34
H
S
5.54
13.24
4-Methoxycinnamoyl
92- 93
116-117
80- 81
c 71.09 c 70.75
H 5.22
S 11.86
H
S
5.15
10.77
Microscale preparation of CoA thiol esters of
phenolic acids
3,4-Dimethoxy-
cinnamoyl
c 67.98
H 5.37
S 10.67
68.19
5.45
10.77
c
H
S
The method was used for p-coumaric acid and
for ferulic acid. a. A solution of the acid in question
(10/<mol) was dissolved in dry dimethylformamide
(50/^1), thiophenol (1/d, approx. 10 //mol) was
added and the mixture was cooled in ice. DCC
(6.0 mg) in dimethylformamide (50/d) was added
at a rate of 5 ju\solution every 5 min. After each
addition the mixture was shaken well and then re-
turned to the ice bath. After the last addition the
mixture was treated with water (50 /d), shaken,
and allowed to stand for 15 min. It was then par-
titioned between water (10 ml) and ether (3 X 10
ml). The ether layers were washed with dilute acid
3,4,5-Trimethoxy-
cinnamoyl
c 65.45
c
H
65.21
5.64
S 10.01
H
S
5.45
9.68
4-Benzyloxycinnamoyl
97- 99
121-123
—
—
3,4-Dibenzyloxy-
cinnamoyl
—
—
* Literature value 55 -56 °C 13.
** Literature value 92 -93 °C 14.
Thiol ester exchange 7>9
CoA (10//mol) was dissolved in 0.1m sodium
bicarbonate buffer (2 ml) and the solution was
cooled in ice. A solution of the thiol ester (10 /umo\ and with water as described above, dried over
or excess) in methanol (1 ml) was added and the sodium sulphate, evaporated to dryness and used
solution was either:
immediately for thiol ester exchange.
Unauthenticated
Download Date | 12/16/15 3:29 AM
Johns
