Chemical syntheses and properties of hydroxycinnamoyl coenzyme A derivatives

Full text of DOI:10.1515/znc-1975-5-609

Chemical Syntheses and Properties of Hydroxycinnamoyl-
Coenzyme A Derivatives
J. Stoekigt and M. H. Zenk
Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, Bochum
(Z. Naturforsch. 30 c, 352 —358 [1975] ; received February 3, 1975)
Coenzyme A Thiol Esters, Cinnamic Acids
Acyl-CoA derivatives of several hydroxylated cinnamic acids have been synthesized in 30 to 50%
yield via a. acyl phenyl thiol esters, b. acyl N-hydroxysuccinimide esters, and c. glucocinnamoyl
derivatives. Properties of the intermediates have been determined. The cinnamyol-CoA thiol esters
were characterized by their chromatographic behaviour and UV spectra. The molar extinction
coefficients of these important intermediates in plant phenylpropane metabolism have been un-
equivocally determined. Recently published values 13 for the molar extinction coefficients of these
derivatives are incorrect; the methodological reason for this error has been established.
Cinnamoyl-coenzyme A thiol esters have long been
discussed as central intermediates in phenylpropane
metabolism in plants. The biosynthesis of flavonoid
compounds is assumed to proceed via the conden-
sation of cinnamoyl-CoA thiol esters either with
essary to develop chemical methods of synthesis.
Previous attempts to synthesize hydroxycinnamoyl-
CoA derivatives by chemical means have failed 12,
due to the reactivity of the phenolic hydroxyl group
and the danger of substitution of SH-compounds to
the double bond of the acryloyl side chain. Very
acetyl-CoA
1
or malonyl-CoA2. Activated cinnamic
acids have also been postulated as intermediates in recently Johns13 published a method for the syn-
the formation of lignin 3, benzoic acids 4, and other
phenolics of higher plants 5.
Recently proof of the participation of cinnamoyl-
CoA thiol esters was found in the cell-free formation
thesis of cinnamoyl-CoA derivatives involving the
direct synthesis of cinnamoyl phenyl thiol esters and
subsequent thiol ester exchange 1 4 , 1 5 onto CoA-SH.
Using these derivatives, Johns13 counterchecked
of naringenin6, the biosynthesis of cinnamyl alco- some of the properties of the enzymatically synthe-
hols 7-9, and the formation of chlorogenic acid 10. sized cinnamoyl-CoA derivatives prepared by Gross
The established role of cinnamoyl-CoA thiol esters
in the biosynthesis of plant phenolics made it neces-
and Zenk
3
and criticized several aspects of their
work. We now present three independent chemical
sary to develop methods for the chemical synthesis syntheses for the preparation of hydroxycinnamoyl-
of these important intermediates. Previously only
enzymatic methods for the preparation of cinnamoyl-
CoA derivatives were available 3>n . These techniques
give small amounts of the desired products but allow
the synthesis of radioactive intermediates with high
specific activity. The necessity of having larger
amounts of hydroxycinnamoyl-CoA derivatives at
hand, especially for the application of optical assays
in reactions involving a loss of the characteristic
CoA derivatives with high metabolic activity and
present data which prove that the analytical methods
used by Johns 13 gave, in most cases, inconclusive
results.
M aterial and M ethods
Biochemicals of the highest purity available were
obtained from Boehringer, Mannheim. Radioactive
cinnamic acids were synthesized by standard Knoe-
thiol ester absorption in the long UV-region
3
and
as substrates for substitution reactions, made it nec- vennagel procedures using appropriate aldehydes
and [2-1 4 C]malonic acid (NEN, Boston). p-Cou-
maric, caffeic and ferulic acid had specific activities
Abbreviations:
CoA, Coenzyme A; DCC, Dicyclohexyl
of 1.90, 1.95, and 1.85 //Ci per /<mol, respetive-
ly. Mass spectra were recorded on VARIAN
carbodiimide.
a
Requests for reprints should be sent to Dr. J. Stoekigt,
Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bo-
chum, D-4630 Bochum, W.-Germany.
MAT 111. C,H,N-analyses were performed by Fa.
Pascher, Bonn.
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353
J. Stöckigt and M. H. Zenk • Syntheses of CoA Thiol Esters
Cinnamoyl thiophenyl ester was also synthesized
via the acid chloride and thiophenol17. Analysis of
the cinnamoyl phenyl thiol ester m.p. 92 —93 °C
(found C: 74.73%; H: 4.95%; S: 13.35%; calcd.
for C1 5 H12OS C: 74.97%; H: 5.03%; S: 13.34%).
0
- c - s
base
H2Ct
-CO2 ^ - Ö - C H ^ H - C - S
r'^2^cho
'C -O H
ii
FT
0
0
II
b. Synthesis of N-hydroxysuccinimide esters of cin-
namic acids
CH=CH -C-OH
HO-N
3
]
0
The cinnamic acid in question (15 mmol) was
dissolved under heating in absolute ethyl acetate,
N-hydroxysuccinimide (15 mmol) and, after cooling
to 30 °C, dicyclohexyl carbodiimide (17 mmol) was
added. After 24 hours the dicyclohexylurea was
filtered off and the filtrate extracted with 1 M sodium
bicarbonate. After drying the ethyl acetate phase
over sodium sulfate, the solvent was evaporated and
the esters purified by thinlayer chromatography
(silicagel GF; solvent: CHCl3/MeOH = 2 0 :1 ). The
esters were recrystallized from benzene. Some ana-
lytical data and yields of the pure products are given
in Table II.
Scheme I. Synthesis of activated cinnamoyl esters.
R = R '= R " = H cinnamoyl derivatives
R = R "=H ; R '=O H
R = H; R '= R " —OH
R = H; R '=O H ; R" = OCH3
R = R" = OCH3; R' = OH
p-coumaroyl derivatives
caffeoyl derivatives
feruloyl derivatives
sinapoyl derivatives
Synthesis of intermediates
a. Synthesis of cinnamoylthiophenyl esters
To the corresponding substituted benzaldehyde
(1.4 mmol) was added crystalline malonyl thio-
phenol16 (1.7 mmol) and the mixture dissolved in
0.4 ml of absolutely dry pyridine/piperidine (1 0 :1 ).
After
time the course of the reaction can be followed by
observing C 0 set free during the reaction, the solu-
tion was diluted with water and acidified with 2N
hydrochloric acid. After 12 hours the mixture was
exhaustively extracted with ethyl acetate. The dried
extract was chromatographed on silicagel GF using
6
hours at room temperature, during which
c. Glucocinnamic acids
/5-D-Glucocinnamic acids were synthesized accord-
ing to published methods 18. Melting points agreed
with published values.
2
Thiol ester exchange reaction and transesterification
CHCI /ethyl acetate/benzene/ethyl methyl ketone/
3
For the synthesis of hydroxycinnamoyl-CoA from
light petrol (40 —60 °C) in a ratio of 7 :1 :2 : 3: 3. cinnamoyl thiophenyl esters or the cinnamoyl N-
The thiol ester was eluted using ethyl acetate/metha-
nol and after evaporation of the solvent the esters procedure was applied. Nitrogen was bubbled
hydroxysuccinimide esters the following general
crystallized, except for sinapoyl phenyl thiol ester.
Some properties and yields of these esters are given
through an aqueous solution ( 2 ml) of CoA
(1 0 //mol) at a low rate, solid NaHC0 (100 //mol)
3
in Table I. UV-spectra were recorded in methanol, was added and subsequently the thiophenyl
Imax values for the maximal absorption of the thiol
ester linkage are presented.
(33 //mol) or N-hydroxysuccinimide ester (50 //mol)
added. Acetone was added till the mixture formed a
Table I. Melting points, optical values, mass spectral characteristics, Rf values and yields for hydroxylated series of
phenyl thiol esters. Chromatography on silica gel. I: in chloroform/methanol 20:1; II: in chloroform/ethyl acetate/benzene/
ethyl methyl ketone/light petrol. 7 :1:2 :3:3.
m.p.
[°C]
Yield
[%]
Phenyl thiol
esters
IR
[cm-
UV
^max
[nm]
MS
m/e
(Intens.%)
Rf
I
II
1440 1330;
Cinnamoyl
p-Coumaroyl
Caffeoyl
9 2 -9 3 * 1680
1019
1670 1435
302
338
352
352
356
240(2) ; 131(100) ;
109(5) ; 103 (33)
256(2) ; 147(100) ;
119(19) ; 109(4)
272(1) ; 163(100) ;
135(10) ; 109(8)
750
690
1325;
686
0.94
0.76
0.40
0.90
0.90
0.99
0.84
0.59
0.85
0.78
30
20
23
22
11
1 1 6 -1 1 9
1 2 6 -1 2 9
7 2 -7 5
oil
1020
1655
1020
750
1440 1350;
737 686
1660 1430 1380;
Feruloyl
276(2) ; 177(100) ;
149(6) ; 109 (5)
1018
1665
1022
740
1430 1340;
748 690
687
Sinapoyl
316(2) :
175(31)
207(100) ;
; 109(3)
* Literature value17: 91 °C; Johns13: 90—91 °C.
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354
J. Stoekigt and M. H. Zenk • Syntheses of CoA Thiol Esters
Table II. Melting points, optical values, mass spectral characteristics, Rf values and yields for hydroxylated series of N-
hydroxysuccinimide esters. Chromatography on silica gel. I: in chloroform/methanol 20:1; II: in chloroform/ethyl ace-
tate/benzene/ethyl methyl ketone/light petrol. 7:1:2:3:3.
N-Hydroxy-
succinimide
esters
m.p.
[°C]
IR
[cm-1 ]
UV
MS
ra/e
(Intens.%)
Yield
[%]
Rf
I
II
Anax
[nm]
Cinnamoyl
p-Coumaroyl
Caffeoyl
1 8 0 -1 8 2
1 8 4 -1 8 8
1 7 6 -1 7 9
1 4 5 -1 4 8
1 1 6 -1 1 8
1760 1740; 1628;
1370 1073; 644
286
332
342
341
344
245(2); 131(100);
103(30)
261(2); 147(100);
119(23)
277(4); 163(100);
135(13)
291(9); 177(100);
145(74)
321(15) ; 207(100) ;
175(46)
0.89
0.42
0.23
0.66
0.66
0.67
0.37
0.17
0.35
0.29
81
36
15
30
52
1760
1710; 1620;
1378 1071; 645
1765 1725; 1630;
1370 1070; 660
1760 1725; 1630;
Feruloyl
1378
1070; 645
Sinapoyl
1750 1730; 1620;
1378 1070; 650
single phase. The solutions were kept at 4 °C for a
total of 24 hours. The organic solvent was eva-
porated by a stream of nitrogen. The aqueous phase
Identification of hydroxycinnamoyl-CoA thiol esters
The reaction products were checked for their iden-
tity as cinnamoyl-CoA thiol esters by several proce-
dures. Ascending chromatography was done at room
temperature on Whatman No. 3 paper in the solvent
systems given in Table III. The hydroxycinnamoyl-
CoA thiol esters gave a characteristic yellow color
upon treatment with nitroprusside reagent under
alkaline conditions 2 0 as previously observed for this
class of compounds 3. Free phenolic OH-groups were
detected by spraying with diazotized sulfanilic acid.
The thiol esters yielded the corresponding hy-
droxamic acids upon treatment with hydroxylamine
at pH 7.0. The most sensitive assay for the purified
derivatives was their characteristic UV-spectrum re-
corded in 0.1 M potassium phosphate buffer at
pH 7.0 in combination with the change in spectral
characteristics after hydrolysis of the thiol ester
linkage 3.
was desalted with Dowex 50 W-X , extracted with
8
ethyl acetate, freeze dried, and the residue purified
by chromatography. Yields, estimated spectroscopi-
cally (UV), ranged from 30 to 50% for the hydroxy-
cinnamoyl-CoA derivatives based on CoA.
Cinnamoyl-CoA derivatives via gluco-cinnamoyl-
CoA’s
Glucose derivatives of hydroxycinnamic acids
were esterified in the presence of CoA (10 to
50 //mol) and dicyclohexyl carbodiimide by the
technique used for the synthesis of feruloyl-AMP 19.
Gluco-hydroxycinnamoyl-CoA derivatives were iso-
lated in 35 —50% yield. These derivatives were in-
cubated at pH 5.0 with /?-glucosidase (Boehringer,
Mannheim, which proved to be absolutely free of
thiol esterase activity) and kept for
2
0
min at 30 °C.
Gluco-hydroxycinnamoyl-CoA derivatives were thus
transformed into hydroxycinnamoyl-CoA derivatives
in essentially quantitative yield. Isolation and puri-
Hydroxamate te st21
For the quantitative determination of hydroxycin-
fication of the CoA thiol esters was done by chro- namoyl thiol esters the hydroxamate procedure by
matography (Table III). Johns 13 was followed exactly. For the purified phe-
Table III. Rf values of hydroxycinnamic acids, their CoA thiol esters and hydroxamic acids on paper. I. in n-butanol/
glac. acetic acid/water 5:2:3; II: in i-butyric acid/ammonia/water 66:1:33; III: in ethanol/0.1 N sodium acetate pH 4.5
1:1; IV: in n-butanol/glac. acetic acid/water 20:1:4.
Parent acid
CoA thiol ester
Hydroxamic acid
IV
Acid
I
II
III
I
II
III
Cinnamic
p-Coumaric
Caffeic
Ferulic
Sinapic
0.94
0.85
0.72
0.77
0.77
0.92
0.86
0.77
0.83
0.85
0.85
0.85
0.52
0.79
0.66
0.75
0.75
0.66
0.68
0.70
0.83
0.69
0.45
0.60
0.42
0.59
0.42
0.42
0.40
0.66
0.43
0.58
0.37
0.46
0.34
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J. Stöckigt and M. H. Zenk • Syntheses of CoA Thiol Esters
355
nyl thiol esters we used, 0.5 ml of 0.2 M hydroxyl-
amine (pH 8.0) which corresponds to 100 /<mol
NH2OH per test (Johns, pers. commun.). Cinnamoyl
hydroxamate was synthesized from cinnamoyl ethyl
ester, using a general method 22. The hydroxamate
of the desired thiol esters. To circumvent these diffi-
culties, three independent syntheses have been de-
veloped. To avoid the use of free thiol, and there-
fore the possibility of an addition of SH-groups to
the double bond of the cinnamic acids, malonyl thio-
phenol was condensed onto a properly substituted
benzaldehyde in a Knoevennagel reaction according
to Scheme I, A. This reaction gave a satisfactory
yield of the desired thiol ester intermediate (Table I).
Johns 13 stated that Michael addition of SH-groups
to the double bond of cinnamic acids is not a serious
problem. This observation was made independently
by us during the course of this work. However,
direct reaction of hydroxycinnamic acids with thio-
phenol in the presence of DCC according to the pro-
cedure of Johns 13 yielded after thinlayer chromato-
graphy, at least five different products in about
equal amounts. One of these products was the de-
sired thiol ester. Johns13 did obtain these phenyl
thiol esters of hydroxycinnamic acids by his
method, but apparently not in crystalline form since
no mention was made in his paper of the analytical
properties of these intermediates. Thus, we consider
that the condensation of malonyl thiophenol onto
benzaldehydes, to yield cinnamoyl phenyl thiol esters
is superior to the direct method 13.
was recrystallized from H 0/M e0H . The pure com-
2
pound had a m.p. of 117.5 —119 °C. Analysis of
the product gave C: 65.94%; H: 5.41%; N: 8.54%
(calcd. for C
8.58%).
9
H9NOo C: 66.25%; H: 5.56%; N:
Purification of hydroxycinnamoyl-CoA derivatives
For small scale purification of hydroxycinnamoyl-
CoA derivatives paper chromatography using the
solvent systems given in Table III, proved very use-
ful. Since column chromatography of these deriva-
tives on Sephadex G- 1
0
11 did not work satisfactorily
in our hands, larger amounts of synthetic cinnamoyl
derivatives were chromatographed at 4 °C on DEAE
cellulose columns (2 .5 x 8 cm), using an HCOOH/
HCOONa gradient23. The effluent of the column
was monitored automatically and simultaneously at
254 nm and 339 nm. The CoA thiol ester containing
fractions showed peaks at both wavelengths. The
pooled fractions containing the CoA thiol ester were
isolated as in I.e. 2 3 and kept in the dry state at
—
2
0
°C; under these conditions deterioration of the
compound was minimal. Yields of the CoA thiol
ester purified by this method were about 75% of the
applied compound.
Chemical protection of the free phenolic groups
of cinnamic acids during the preparation of CoA
thiol esters is difficult since the chemical removal of
the protecting group, after the thiol ester formation
had been achieved, would in most cases lead to a
destruction of the labile ester linkage 12. Protection
of the free phenolic group by glucosidation was
attempted by the technique previously described for
the synthesis of feruloyl-AMP 19. One of the pre-
requisites for a successful employment of this re-
action is that the /?-glucosidase be absolutely free
of thiol esterase which was fortunately the case for
our commercial sample.
Results and Discussion
Acids containing one or more free phenolic
groups in the aromatic ring system and a double
bond in the side chain present difficult problems in
the chemical esterification with coenzyme A or with
other thiols. Problems arise using either carboxyl
activation reactions or coupling agents. Since attack
at the free phenolic hydroxyl is likely to occur12
and Michael addition of the thiol to the double bond
of the cinnamic acid is a possibility 20. In both cases
these side reactions would interfere with formation
C o A - S H 't R - < ( jV C H = C H -C -S -C o A
B.
1. DCC
H S -C o A 2^
C G h M K f » -C H = C H -C -0 H
♦
_
_
h 0 ^
(
H 0 -(^ > -C H :C H -C -S-CoA
R = H or OCH3
Scheme II. Synthesis of cinnamoyl CoA thiol esters.
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356
J. Stöckigt and M. H. Zenk • Syntheses of CoA Thiol Esters
/?-D-Gluco-p-coumaric acid and /?-D-glucoferulic
moyl-CoA derivatives from each other will only
rarely occur in praxi.
acid were coupled directly with CoA using DCC.
The resulting thiol ester glucoside was isolated by
chromatography and the glucose moiety subsequently
split off by treatment with /?-glucosidase as shown in
Scheme II, C. The hydroxycinnamoyl-CoA deriva-
tives thus obtained were identical in all respects to
those synthesized by alternative chemical or enzy-
Further identification of the synthetic CoA deriva-
tives was done as described in material and methods.
The “delayed” nitroprussid reaction and the for-
mation of hydroxamates and their chromatographic
properties (Table III) proved especially useful to
supplement the optical characteristics of the esters.
matic means. Thus glucose can be used as protect- Since the UV-spectra of both caffeoyl-CoA and sina-
ing group in the synthesis of very labile biochemical
intermediates of phenols.
poyl-CoA have not been published previously these
curves are given in Fig. . These synthetic cinna-
1
moyl-CoA thiol esters are metabolically active in two
different enzyme assays 9’ 10.
Since N-hydroxysuccinimide esters of aliphatic
acids were shown to present useful activated inter-
mediates for the formation of aliphatic acyl-CoA
thiol esters 24, we attempted the direct synthesis of
hydroxycinnamoyl N-hydroxysuccinimide esters us-
ing DCC without protection of the phenolic group of
the cinnamic acids in question. It was hoped that the
reactive hydroxysuccinimide would, under these
conditions, attack the carboxyl group of the acids
more readily than the phenolic group. This was in-
deed the case in spite of the fact that quite a number
of other derivatives were also formed. The desired
esters were, however, readily isolated by preparative
chromatography in satisfactory yields (Table I I ) .
Thiol ester exchange of the phenyl thiol ester, as
well as transesterification of the hydroxysuccinimide
esters under the same conditions as given in the
material and methods section, gave the correspond-
ing CoA thiol esters in about 40% yield. Unreacted
esters were removed by ethyl acetate extraction and
the CoA esters purified by either paper or column
chromatography. Table III shows the /^-values of
parent acids as compared with their CoA thiol esters
in three different solvent systems which had been
used previously for the purification of enzymatically
synthesized cinnamoyl-CoA esters 3. The previously
n m --------------
Fig. 1. UV-spectrum of caffeoyl-CoA (a) and sinapyl-CoA
(b) both recorded in 0.1 phosphate buffer pH 7.0.
►
M
The chemical preparation of hydroxycinnamoyl-
CoA thiol esters by the above methods is suitable for
large quantities of the CoA esters in question. For
the preparation of radioactively labelled CoA esters,
especially of high specific activity, the enzymatic
methods3>11 are preferred. Of great importance is
the reliability of the quantitative hydroxamate color
test for cinnamoyl-CoA derivatives. The reason is
twofold. First, several groups still u se26, or used
this test in the past27, for the assay of hydroxy-
cinnamate: CoA ligase. Amounts of hydroxamates
formed were calculated1 1 1 2 8 from the extinction
coefficient for cinnamoyl hydroxamate as determined
by Gross and Zenk3. Secondly, Johns 1 3 computed
the £max values of all the cinnamoyl-CoA derivatives
which he synthesized by comparing the extinction in
the long wavelength UV region with the hydroxamate
assay and used an extinction coefficient of cinna-
published Rf-values
3
agreed reasonably well with
those reported here. We cannot explain why Johns 13
had difficulties in using two of the three reported
chromatographic solvent systems3, especially since
the butanol:glacial acetic acid:H 20 (5 :2 :3 ) sys-
tem gives extraordinary sharp spots and separation
and has been used by others for the separation of a
great many acyl-CoA derivatives even on thinlayer
plates 25. The main purpose of the chromatographic
purification reported above is the separation of par-
ent acids from CoA derivatives which is achieved in
every case. Separation of different hydroxycinna-
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J. Stoekigt and M. H. Zenk • Syntheses of CoA Thiol Esters
357
moyl hydroxamate-FeCl
3
complex as standard. He
40% of the thiol ester present was transformed into
determined this by using an average value of his
series of phenyl thiol esters prepared by hydroxyl-
aminolysis.
the hydroxamate within
1
0
min as revealed by the
colored iron complex, showing that the CoA deriva-
tive is somewhat more reactive under these condi-
tions. Different rates of hydroxylaminolysis for dif-
ferent cinnamoyl CoA esters had been observed be-
fore3. Assuming a 10-fold half-life time for the com-
plete conversion of p-coumaroyl-CoA to p-coumaroyl
While Gross and Zenk
coefficient for the cinnamoyl hydroxamate-iron
complex of 1.54 x 10 cm2/mol [this value is not a
missprint and not X 10 too high as Johns 13 sug-
3
reported an extinction
6
3
hydroxamate (1m NH
reaction would have taken 40 min to go to comple-
tion. Previously published data had already shown
2
OH; pH 7.5; 20 °C)
3
this
gested but represents the molar extinction coefficient
as widely used in biochemical literature], Johns 13
3
determined this coefficient as being 1.05 X 10 cm2/
6
that hydroxylaminolysis may be rate limiting.
mol from his experiments. This value he also
claimed to be more in line with that found for the
No mention was made by Johns 13 of the fact that
acetyl-hydroxamic-iron complex [0.975 X 106] . This the o-dihydroxy groups of the caffeoyl moiety in the
serious discrepancy led us to redetermine our pre- parent acid, as well as in caffeoyl phenyl thiol ester
viously published value. Analytically pure cinnamoyl and caffeoyl-CoA, gives a pronounced color effect at
hydroxamic acid was synthesized and samples dis-
solved in MeOH, at a concentration of //mol/ml
(25m l total). Samples of 0.5m l were removed and
550 nm with the iron chloride reagent used in the
absence of hydroxylamine. Accordingly, the calcu-
lated value 1 3 for caffeoyl-CoA is bond to be incor-
2
assayed as described by Johns 13. An average value rect for this reason alone.
from 10 independent determinations was 1.68 X 10
6
The fact that there was no quantitative conversion
of the thiol esters to the hydroxamates, and that the
rate of hydroxylaminolysis of the phenyl and CoA
thiol esters was different, demonstrates beyond any
doubt that Johns 13 method of calibrating the hy-
droxamate test for the determination of the £max
values of acyl CoA thiol esters ist not accurate. Since
the extinction coefficient of the cinnamoyl hy-
droxamate-iron complex used in the above method
was too low, the reported £max values of the CoA
derivatives are expected to be too high. This is in-
deed the case. We have redetermined the £max
cm2/mol at 540 nm. This corresponds to an extinc-
tion of 0.559 for 1 /«nol cinnamoyl hydroxamate in
3 ml solvent. This value is even higher than that
previously published
3
from this laboratory and
proves that the extinction coefficient determined by
Johns 1 3 is incorrect by more than one third.
The reason for this lower extinction value found
by Johns 13 was most likely due to incomplete hy-
droxylaminolysis of the purified phenyl thiol esters,
since the amount of hydroxylamine used (0.1 M final
concentration) at pH 8.0 was relatively low. Kinetic
experiments were therefore performed to study the values
conversion of the phenyl thiol ester of cinnamic acid known specific activity. By comparing the extinction
(lm g ) and p-coumaroyl-CoA (0.5 /wnol) to the
corresponding hydroxamates under Johns13 con- tivity of the labelled CoA derivative a calculation of
ditions. It was immediately seen that under these the £max values was possible. The emax value was
conditions hydroxamate formation of both thiol counterchecked by using the enzymatic method of
esters is incomplete. At the time interval ( 1 min)
determining the molar extinction coefficient of cin-
used by Johns 1 3 only about 30% of the thiophenyl namic acid, using hydroxycinnamoyl-CoA ligase19
ester was converted to the hydroxamate and after rather than acyl-CoA synthetase (EC
.2.1.2.) from
25 min 50% was lysed. A complete conversion of beef liver mitochondria. In order to make this re-
mg cinnamoyl thiophenyl ester to cinnamoyl hy- action irreversible, pyrophosphatase (1 U) was in-
droxamate under the given conditions 13, and within
cluded in the assay. Table IV shows the extinction
min, was achieved only using salt free hydroxyl- coefficient values for the hydroxycinnamoyl-CoA
3
using 2-1 4 C-labelled cinnamic acids of
at ^max in the long UV region with the specific ac-
0
3
6
1
1
0
amine. Only under these conditions was the theo- thiol esters, their ^max and Amin values in the long
retical extinction coefficient of the hydroxamate-iron wavelength UV region. These data are average
complex reached. Using 1 M hydroxylamine (final values of between
8
to
1
0
individual determinations
for each compound. No £max value for sinapoyl-CoA
can be given, since a countercheck by the enzymatic
concentration) at pH 8.0 the conversion was only
85% after 10 min. In the case of p-coumaroyl-CoA,
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J. Stoekigt and M. H. Zenk • Syntheses of CoA Thiol Esters
358
ment that his £max values superceded the previously
published ones is therefore not tenable. Besides, if
method was not possible due to the inability of
hydroxycinnamoyl-CoA ligase to activate sinapic
acid 19.
3
optical properties of CoA esters which are synthe-
sized in two different laboratories are compared
(Table IV, ref. 13), these determinations should be
made at the same pH value (Johns 13: pH 2.5 —3.0;
Gross and Zenk: pH 7.0). At any event, the differ-
ence in pH values used was not the reason for the
difference in £max values. Furthermore missprints of
quotations, i. e. Amax values of p-coumaroyl-CoA as
Table IV. UV-Absorption data for hydroxycinnamyol-CoA
derivatives (measured in 0 .1 m phosphate buffer pH 7.0).
CoA
deriva-
tives
^min
[nm]
£max x 106
at ^max II
[cm2/mol]
^max II
[nm]
^•max I
[nm]
285
287
287
289
21
18
19
—
p-Coumaroyl
Caffeoyl
Feruloyl
261
257
257
252
333
346
346
352
351 nm 1 3 instead of 333 nm
3
should be avoided.
In addition Johns1 3 has taken it as an encouraging
sign of the reliability of his £max values for CoA
thiol esters that they were closely similar to those
of the equivalent phenyl thiol esters. We have rede-
termined the £max value of cinnamoyl phenyl thiol
Sinapoyl
As can be seen from the data given in Table IV,
these values agree reasonably well with the values
previously published from our laboratory 3. One ex-
ception is caffeoyl-CoA, where both the Amax and
£max values of our previous determination is incor-
rect; this error had been noticed before 10. While the
Amax values of the different hydroxycinnamoyl-CoA
derivatives of Table IV agree with the ones reported
by Johns 13, his measured £max values are too high
as theoretically expected because of his erroneous
calibration procedure. For example his published
£max value for p-coumaroyl-CoA is about V 3 too high
(35 X 106) about the same order of magnitude by
which his extinction coefficient of the cinnamoyl
hydroxamate-iron complex is too low. Johns 1 3 state-
ester for which he reported an £max of 14.4 X 10
cm /mol. As an average value from independent
determinations of the analytical pure compound we
found 25.2 x 10 cm /mol.
6
2
6
6
2
The excellent technical assistance of Miss B. Ries
and Mr. L. Ändert is gratefully appreciated. Finan-
cial support was provided by the “Deutsche For-
schungsgemeinschaft”, our thanks are due to Prof.
J. McClure for his help in the english version of the
manuscript and to Dr. G. G. Gross and Mr. W.
Kreiten for adapting the column chromatography
method 23 to cinnamoyl-CoA derivatives.
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