Very long-chain phenylpropyl and phenylbutyl esters from Taxus baccata needle cuticular waxes

Article abstract of DOI:10.1016/S0031-9422(02)00286-8

The cuticular wax of Taxus baccata L. needles was found to contain four different classes of long-chain esters that were identified by various chemical transformations with product assignment employing GC-MS. Homologous series of (1) 3-(4′-hydroxyphenyl)-propyl esters of C₂₀-C₃₆ fatty acids, (2) 4-(4′-hydroxyphenyl)-2-butyl esters of C₁₈-C₂₈ fatty acids, (3) 3-(3′,4′-dihydroxyphenyl)-propyl esters of C₂₀-C₃₂ fatty acids, and (4) 4-(3′,4′-dihydroxyphenyl)-2-butyl esters of C₁₈-C₂₈ fatty acids were identified. The four compound classes amounted to 0.1-3.6 μg/cm² of needle surface area, corresponding to 0.2-7.6% of the wax mixture, respectively. While both phenylpropyl ester series had a maximum for the homolog containing tetracosanoic acid, in the phenylbutyl esters homologs containing eicosanoic and docosanoic acids predominated.

Full text of DOI:10.1016/S0031-9422(02)00286-8

Phytochemistry 61 (2002) 579–587
www.elsevier.com/locate/phytochem
Very long-chain phenylpropyl and phenylbutyl esters from
Taxus baccata needle cuticular waxes
¨
Reinhard Jetter*, Adelheid Klinger, Stefanie Schaffer
Julius-von-Sachs-Institut fu¨r Biowissenschaften, Universita¨t Wu¨rzburg, Julius-von-Sachs-Platz 3, D-97082 Wu¨rzburg, Germany
Received in revised form 8 July 2002
Abstract
The cuticular wax of Taxus baccata L. needles was found to contain four different classes of long-chain esters that were identified
0
by various chemical transformations with product assignment employing GC–MS. Homologous series of (1) 3-(4 -hydroxyphenyl)-
0
0
0
propyl esters of C20–C36 fatty acids, (2) 4-(4 -hydroxyphenyl)-2-butyl esters of C18–C28 fatty acids, (3) 3-(3 ,4 -dihydroxyphenyl)-
0
0
propyl esters of C –C fatty acids, and (4) 4-(3 ,4 -dihydroxyphenyl)-2-butyl esters of C –C fatty acids were identified. The four
18
2
0
32
28
2
compound classes amounted to 0.1–3.6 mg/cm of needle surface area, corresponding to 0.2–7.6% of the wax mixture, respectively.
While both phenylpropyl ester series had a maximum for the homolog containing tetracosanoic acid, in the phenylbutyl esters
homologs containing eicosanoic and docosanoic acids predominated.
#
2002 Elsevier Science Ltd. All rights reserved.
Keywords: Taxus baccata; Gymnospermae; Yew; Cuticular wax; Leaves; Phenolics; Fatty acid esters
1
. Introduction
wax components have been identified in cuticular wax
mixtures of only a small number of plant species (Wal-
ton, 1990). The occurrence of phenolics near the plant
surface is of special interest, as these compounds might
serve as protectants because of their light absorbing
properties (Vogelmann, 1993; Krauss et al., 1997). Also,
the geometry and electronic attributes of the aromatic
ring system differ distinctly from those of other wax
constituents, and it has been speculated that aromatic
compounds might disturb the molecular arrangement of
cuticular waxes. Compounds containing both phenolic
and long-chain aliphatic moieties could influence the
ecophysiological properties of the cuticle (Riederer and
Schreiber, 1995).
In the course of ongoing studies on the chemical
composition of cuticular lipids of diverse plant taxa,
gymnosperm needles were analyzed. Cuticular waxes on
Taxus baccata (Taxaceae) needles were found to contain
various unknown constituents that had MS character-
istics similar to phenolic compounds. The structure elu-
cidation of these compounds is a prerequisite for further
investigations into the structure and function of T. bac-
cata cuticles. Therefore, the objective of the present
work was to identify these novel constituents by various
chemical transformations and product structure assign-
ment employing GC–MS.
The cuticle is an extracellular membrane covering all
primary aboveground plant organs. It consists of a
matrix of cutin (a biopolymer of di- and trihydroxy
fatty acids) that is encrusted with ‘intracuticular waxes’
and covered with ‘epicuticular waxes’. Diverse biologi-
cal functions have been attributed to these waxes: pre-
venting transpirational water loss over the plant surface
(
¨
Schonherr, 1976); guarding leaf surfaces from accumu-
lation of air-borne particles and spores (Barthlott and
Neinhuis, 1997); keeping leaf surfaces dry (Holloway,
1
970) and thus preventing the germination of pathogen
spores (Kolattukudy, 1980; Deising et al., 1992); and
directing the behavior of herbivorous insects that probe
the surface for infochemicals (Espelie et al., 1991;
Eigenbrode and Espelie, 1995).
The specific physiological and ecological functions of
plant cuticles can only be understood based on their
characteristic wax composition. On the one hand, satu-
rated, unbranched very-long chain aliphatics have been
reported as predominant wax constituents of diverse
plant taxa (Baker, 1982). On the other hand, aromatic
*
Corresponding author. Fax: +49-931-888-6235.
E-mail address: jetter@botanik.uni-wuerzburg.de (R. Jetter).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00286-8

580
R. Jetter et al. / Phytochemistry 61 (2002) 579–587
2
. Results and discussion
TMSi ether derivatives. Although derivatization
improved both the GC separation of individual com-
pounds and the MS information obtainable from them,
few reference spectra for the acetates or TMSi ethers of
hydroxyphenylpropanol esters were available. The
The surface extracts from needles of Taxus baccata
were separated into 14 distinct fractions by TLC on
silica gel with CHCl –EtOH (99:1). Among these, two
3
prominent bands migrating with Rf 0.21 and 0.16
between primary alcohol and fatty acid standards were
found to contain uncommon wax constituents and were
designated as fractions A and B. According to their GC
retention behavior (Table 1) and their MS character-
istics, the constituents of these two bands belonged to
two pairs of related homologous series A , A and B ,
TMSi ether MS of compounds A showed a base peak
1
at m/z=206 (Fig. 1a) that had previously been inter-
preted as an acid elimination product [TMSiO–Ph–
+
CH -CH=CH ] of this compound type (Olsen et al.,
2
2
1998). These derivatives were additionally characterized
by other prominent MS fragment ions, either consisting
+
of the phenolic [TMSiO–Ph–CH ] , or the phenylpro-
2
1
2
1
+
B , respectively.
2
pyl moiety [TMSiO–Ph–(CH ) –OH] , [Me SiO–Ph–
2 3
2
+
+
In a first experiment, GC–MS data of underivatized
fraction A were acquired. Although pairs of compounds
could only be partially separated, the base ions
m/z=134 and 148 could be assigned to the compound
classes A and A , respectively, according to their GC-
CH –CH=CH ] , or the entire molecule [M] , [M–
2 2
+
Me] . Analogous fragments were found for the acetate
derivatives of the compounds of the A type, including
1
the ions [M]+ and [M-ketene] , representing the entire
+
+
ester structure, the acid elimination products [M-acid]
+
1
2
MS single ion traces. Homologs of class A were found
1
and [M-acid-ketene] , as well as the phenylpropyl
+
to elute shortly after those of A (Table 1). A fragment
2
alcohol [AcO–Ph–(CH ) –OH] and its ketene elimina-
2 3
ion at m/z=134, interpreted as
xyphenylpropenyl ion generated by elimination of the
acid moiety, had previously been described for 3-(4 -
hydroxyphenyl)-propyl esters (Boll et al., 1992; Olsen et
al., 1998). All other MS signals reported for these esters
were also characteristic of the compounds in class A1,
a
hydro-
tion product. Finally, the hydroxybenzyl ion at m/
z=107 confirmed the phenolic structure, while two ser-
ies of fragments m/z=55, 69, 83, etc. and m/z=57, 71,
85, etc. indicated the presence of an alkyl moiety.
0
Taken together, these data confirmed that compounds
0
of the A type represented fatty acid esters of 3-(4 -
1
and it was surmised that the homologs in A represented
1
hydroxyphenyl)-propanol. Seventeen homologs with acid
0
fatty acid esters of a hydroxyphenylpropanol.
This structure assignment was corroborated by GC–
MS analysis of the corresponding acetate ester and
chain lengths of C –C were identified. Only 3-(4 -
2
0
36
hydroxyphenyl)-propyl tetracosanoate had been descri-
bed as a constituent of stem and leaf wax of Piper clarkii
Table 1
GC retention times (min) of phenolic esters in Taxus baccata needle wax under the conditions used and MS data available
Fatty acid
carbon number
Fraction A
0
1
Fraction A
0
2
Fraction B
0
1
Fraction B
2
0
0
0
(4 -hydroxyphenyl)-
propyl esters
(4 -hydroxyphenyl)-
2-butyl esters
(3 ,4 -dihydroxyphenyl)-
propyl esters
(3 ,4 -dihydroxyphenyl)-
2-butyl esters
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
–
26.1 t
–
–
29.0 t
–
–
–
29.8 T A
31.6 t a
29.7 T a u
31.5 T
32.7 T
34.4 t
36.2 T A
32.5 T A
34.2 T A
a
a
a
a
a
a
33.4 T A U
33.3 T a u
35.0 T
35.9 T A
37.5 T A
35.1 T A u
36.8 T A U
38.5 T A u
40.1 T A U
41.7 T A
43.3 T A
44.8 t
37.8 T A
39.5 T A
41.0 T A
42.6 T A
44.3 T a
45.7 T A
47.1 t
48.5 t
50.0 t
51.5 t
–
36.7 T a u
38.4 T
40.0 T
39.2 T A
40.7 T A
42.3 T A
44.1 t
45.6 t
–
43.2 t
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
46.4 T A
47.9 t
49.3 T A
51.3 t
53.1 T A
55.8 t
58.2 T a
–
–
–
T, A, U: full mass spectra of the TMSi ether, the acetate or the underivatized compound, respectively, could be acquired. t, a, u: the TMSi ether, the
acetate or the underivatized compound was identified by inspection of characteristic fragments.
a
Cf. Fig. 1.

R. Jetter et al. / Phytochemistry 61 (2002) 579–587
581
Fig. 1. Mass spectra of phenylalkyl esters from needle wax of Taxus baccata. Spectra of (a–d) the TMSi ethers or (e and f) the acetate esters are
shown for one representative homolog from each of the classes of esters.

582
R. Jetter et al. / Phytochemistry 61 (2002) 579–587
+
+
+
(Piperaceae) (Boll et al., 1992). The same ester, together
with homologs containing C , C , C and C34 fatty
(CH ) –OH] ,
2
[M–acid] ,
+
[M–acid–HOTMSi] ,
+
3
[(TMSiO) –Ph–CH ] and [TMSiO–Ph–CH ] (Fig. 1c).
2
2
5
26
32
2
2
acids, had previously been reported as constituents of
needles of Taxus canadensis, but the possible localiza-
tion of these compounds in the cuticular waxes had not
been investigated (Olsen et al., 1998).
Hence, very similar fragmentation patterns were infer-
red for both derivatives, confirming that the con-
stituents of fraction B represented a series of 3-(3 ,4 -
1
0
0
dihydroxyphenyl)-propanol esters. Thirteen homologs
comprising esterified C –C fatty acids were identified.
For compounds of class A , the MS of the under-
2
20
32
ivatized constituents were found to be indistinguishable
from those of class A , except for the hydro-
Only one of these, dihydroxyphenylpropanyl arachi-
date, had previously been described as a constituent of
aerial parts of Symphyopappus compressus (Asteraceae)
(Bohlmann et al., 1981) and Phonus arborescens (Aster-
aceae) (Barrero et al., 1997).
1
xyphenylalkenyl ion at m/z=148. Additionally, the
TMSi derivatives of compounds A showed fragmenta-
2
tion patterns that were analogous to those of class A1
(
Fig. 1b). Ions interpreted to contain the entire molecule
+
Compounds in fraction B2 eluted shortly before
those of B (Table 1) and yielded acetate and TMSi
+
[
ketene] , [M-acid] , [M–Me-acid] were shifted by
M] , [M–Me] or the phenylalkyl moiety [M–acid
+
1
+
+
derivatives with MS characteristics very similar to
those of B (Fig. 1d and f). On one hand, all the small
Ám/z=14 as compared to A types. In contrast, those
1
1
fragments comprising only the benzylic moiety
[
mass signals representing either only the alkyl chain of
the fatty acid or the (di-)hydroxybenzyl moiety of the
alcohol were identical for both compound classes. On
the other hand, those fragments containing the alkyl
side chain of the aromatic alcohol, together with their
secondary products generated by loss of ketene or
HOTMSi units, had masses shifted by Ám/z=+14
between B and B -types. Consequently, compounds in
TMSiO–Ph–CH ]+ were identical for both classes. The
acetates of compounds A also showed all the fragments
2
2
described for the corresponding hydroxyphenylpropyl
esters, but all those ions containing the alkyl side chain
of the aromatic alcohol were shifted by 14 mass units.
Together with the similarities in the chromatographic
behavior of both A series, this information led to the
1
2
assumption that class A compounds comprised esters
2
B could be regarded as homologs of the dihydroxy-
2
of a second phenolic alcohol with an additional methyl
group in the alkyl side chain. Although the possible
hydroxyphenylbutyl alcohol isomers cannot be dis-
tinguished using the current spectroscopic information,
phenylpropanol esters identified in B , and the addi-
1
tional methyl group had to be located either on the
terminal or the central methylene group of the phenyl-
0
0
propyl side chain. An alcohol of this type, 4-(3 ,4 -dihy-
droxyphenyl)-2-butanol, had previously been described
as a constituent of T. baccata needles (Das et al., 1993a,
b). By analogy to the structural assignment for com-
it seems very plausible that A -type compounds con-
2
0
tained esters of 4-(4 -hydroxyphenyl)-2R-butanol (alias
(
-)-rhododendrol), an alcohol previously identified in
the needles of Taxus baccata (Das et al., 1993a, b) and
T. brevifolia (Chu et al., 1994). Accordingly, the two
compound classes A and A were found to represent
pound class A , it can be inferred that compounds in
2
0
0
class B were of the 4-(3 ,4 -dihydroxyphenyl)-2-butanol
2
ester series. Ten homologs comprising esterified C –C
28
1
2
18
fatty acid esters of two homologous phenolic diols, 3-
0 0
fatty acids were identified, all of which represented
novel compounds.
(
4 -hydroxyphenyl)-propanol and 4-(4 -hydroxyphenyl)-
butanol. In the second series, ester homologs containing
fatty acids with chain lengths C –C were identified,
all of which represented novel compounds.
To further verify the structure of the four classes of
phenolic esters, one homolog of each series was synthe-
sized by transesterification of methyl docosanoate with
1
8
28
0
In accordance with these findings and with reference
data, spectra of class B -type compounds could be
interpreted. Their acetate MS showed two series of ions
respective alcohols. Only 3-(4 -hydroxyphenyl)-propanol
being commercially available, the other three alcohols
were generated by reduction of the corresponding
phenylbutanones and phenylpropanoic acid and char-
acterized using their acetate and TMSi ether MS
(Kraus and Spiteller, 1997). The resulting compounds,
after transformation into TMSi derivatives, exactly
matched the GC retention behavior and MS character-
istics of one representative of the compound classes A1,
A , B and B2, respectively. Consequently, both the
1
[
[
M] , [M–ketene] , [M–2xketene] and [M–acid]+,
+
+
+
+
+
M–acid-ketene] , [M–acid-2xketene] (Fig. 1e) that
0
0
had previously been described for 3-(3 ,4 -dihydrox-
yphenyl)-propyl esters (Bohlmann et al., 1981). A cor-
responding series of three fragments, not reported
before, could be rationalized as the alcohol [(AcO) –Ph–
2
+
(
CH ) –OH] and its two ketene elimination products.
2 3
2
1
Finally, the dihydroxybenzyl ion m/z=123 confirmed
the phenolic structure, while two series of alkyl frag-
ments at m/z=55, 69, 83, etc. and m/z=57, 71, 85, etc.
indicated the presence of the fatty acid moiety. The MS
postulated alcohol structures and the fatty acid chain
length assignment for all four homologous series of
phenolic esters in T. baccata wax were unambiguously
confirmed.
of TMSi ethers of compound class B showed very pro-
1
The homolog pattern of the esterified fatty acids was
further investigated after cleavage of the original esters.
+
minent ions interpreted as [M] , [(TMSiO) –Ph–
2

R. Jetter et al. / Phytochemistry 61 (2002) 579–587
583
2
Treatment with LiAlH yielded series of unbranched
4
0.1–3.6 mg/cm , representing 0.2–7.6% of the total wax
mixture (Table 2). In all four classes, those homologs
containing fatty acids with even carbon numbers were
dominant. It is noteworthy that both phenylpropyl ester
series had a maximum for the homolog containing tet-
racosanoic acid, while in the phenylbutyl ester homo-
logs eicosanoic and docosanoic acids predominated
(Table 2). This finding could either imply that two dif-
ferent pools of fatty acid precursors are used for the
esterification of the phenylpropyl and the phenylbutyl
alcohols or that two acyl transferases differing both in
alcohol and in fatty acid chain length specificities are
involved.
Other very long-chain derivatives of phenolics, most
of them containing a propenyl side chain, had been
identified previously from various plant species. In
many cases, a phenyl propenoic acid had been found
esterified with typical wax alcohols, e. g. p-coumarates
in Rosa rugosa (Hashidoko et al., 1992). The localiza-
tion of these highly lipophilic compounds within the
plant had rarely been investigated, except for the long-
chain caffeates and ferulates of poplar bud exudates
(Greenaway and Whatley, 1991), and p-coumaryl alco-
hol esters in the stem bark of Buddleja globosa
(Houghton, 1989). Interestingly, all these phenolic esters
combine the aliphatic and aromatic moieties known as
C –C and C –C alcohols for fractions A and B,
20
2
0
30
36
respectively. A corresponding series of fatty acid methyl
esters, with identical homolog patterns, was released by
BF /MeOH treatment. Hence, the original ester struc-
3
ture and the exclusive presence of unbranched, satu-
rated very long-chain fatty acids were confirmed. Beside
the acid moieties of the original esters, the alcohols
could also be detected, by both reductive cleavage and
transesterification, which yielded two pairs of alcohols
from fractions A and B, respectively. All four alcohol
products were further characterized by GC co-injection
experiments as well as MS comparisons with synthetic
standards. Both their TMSi ethers and their acetates
proved to be identical with corresponding derivatives of
0
0
0
synthetic 3-(4 -hydroxyphenyl)-propanol, 3-(3 ,4 -dihy-
0
droxyphenyl)-propanol, 4-(4 -hydroxyphenyl)-2-butanol
0
0
and 4-(3 ,4 -dihydroxyphenyl)-2-butanol, respectively.
The alcohol moieties of the T. baccata wax esters were
hence unambiguously identified, ruling out all isomeric
alternatives and confirming the ester structures inferred
above.
All four homologous series of phenolic esters identi-
fied in the respective TLC fractions of T. baccata needle
wax could also be detected in the whole wax mixture.
These compounds amounted to surface coverages of
Table 2
2
Composition of the phenolic ester fractions from Taxus baccata needle wax. Leaf coverages (mg/cm ) of the compound classes were assessed in the
original wax mixture by adding respective GC–FID peak areas. Relative amounts (%) of the individual homologs were quantified by GC-MS using
characteristic fragments (m/z) of respective TLC fractions.
Fatty acid
carbon number
Fraction A
0
1
Fraction A
0
2
Fraction B
0
1
Fraction B
2
0
0
0
(4 -hydroxyphenyl)-
propyl esters
(4 -hydroxyphenyl)-
2-butyl esters
(3 ,4 -dihydroxyphenyl)-
propyl esters
(3 ,4 -dihydroxyphenyl)-
2-butyl esters
Characteristic fragment
2
06
220
294
308
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
–
–
tra
–
–
tr
–
–
2.9
0.4
20.8
2.6
51.0
2.7
13.4
0.5
2.6
tr
32.7
1.6
39.8
1.4
22.1
0.6
1.7
–
9.1
0.6
14.7
1.5
49.3
2.8
17.2
0.8
3.1
tr
36.5
1.6
35.2
1.4
23.3
0.5
1.4
tr
tr
0.1
–
–
0.9
tr
–
0.6
tr
–
–
–
1.0
tr
–
0.3
–
–
–
–
0.9
tr
–
–
–
–
–
–
0.2
–
–
–
Total coverage
1.5
0.1
3.6
3.5
a
tr: 0.01–0.1% detectable.

584
R. Jetter et al. / Phytochemistry 61 (2002) 579–587
ꢀ
building blocks of the polymer suberin. The geometry of
mixed ester molecules could indicate that covalent links
exist between the aliphatic and aromatic domains of
suberin.
70 C) was performed and the reaction products were
isolated by addition of H O and extraction with Et O;
(4) alternatively, esters were reductively cleaved by
2
2
addition to excess of LiAlH in refluxing THF over 48
4
h. The mixt. of LiAl-alcoholate complexes was hydro-
lyzed with 10% H SO , with the alcohols being
obtained by extraction of the soln with Et O. The cor-
2
4
3
. Experimental
2
responding compounds of interest were purified by TLC
(SiO , CHCl -EtOH 99:1) and were transformed into
the corresponding TMSi ethers.
3
.1. Plant waxes and sample preparation
Plants were grown in the Botanical Garden of the
2
3
University of Wu
¨
rzburg. Four batches of twigs carrying
3.3. Quantitative analyses
ca. 500 current-year needles (fresh weight 25 g) were
harvested in early summer and immediately immersed
twice for 30 s in CHCl at room temp, yielding ca. 12
For quantitative analysis of the entire wax mixture,
compounds containing hydroxyl groups were trans-
formed to TMSi derivatives (as above). Surface cov-
erages of individual compounds were then determined
3
mg of crude extract. For absolute quantification of
phenylalkyl esters within the whole wax mixture, a
defined amount of tetracosane was added as internal
standard. The resulting solns of cuticular waxes were
dried, filtered and the solvent removed under reduced
pressure. Extracted needle surface areas were calculated
by multiplying average needle numbers per twig, the
number of twigs and the average (projected) needle sur-
face area. The latter was assessed by multiplication of
average needle lengths and widths.
by GC-FID (as above, with H carrier gas inlet pres-
2
ꢁ1
sures 41 min at 50 kPa, 10 kPa min to 150 kPa, 30
min at 150 kPa). Needle surface coverages of individual
constituents were calculated as mean values of four
independent analyses; values for the separate analyses
0
differed between 9% and 24% for 3-(4 -hydroxyphenyl)-
0
0
propyl esters and 3-(3 ,4 -dihydroxyphenyl)-propyl
esters, respectively. Values for compound classes were
calculated by summing those of all identified homologs.
Homolog patterns were assessed using the TMSi ether
derivatives of compounds in fractions A and B. Single
ion chromatograms were reconstructed for four distin-
guishable compound classes A , A , B and B using
3
.2. Qualitative analyses
Substance classes were separated by TLC (sandwich
technique, silica gel, mobile phase CHCl –EtOH (99:1),
3
1
2
1
2
+
and localized by staining with primuline and UV-light).
Bands were immediately removed from the plates,
eluted with CHCl , and filtered. Finally, the solvent was
corresponding fragments [TMSiO–Ph–CH –CH=CH ] ,
2 2
+
[TMSiO–Ph–CH –CH=CH–CH ] ,
[(TMSiO) –Ph–
2
2
+
3
CH –CH=CH ] and [(TMSiO) –Ph–CH –CH=CH–
2 2 2 2
3
+
removed in a stream of N and samples were stored in
2
CH ] , respectively, generated by acid elimination from
3
ꢀ
the dark at 4 . Two bands, designated as fractions A (Rf
0
the esters. Relative abundances of individual homologs
were calculated by integration of these single ion traces.
.21) and B (R 0.16), contained unknown compounds
f
and were subjected to detailed qualitative analyses. To
this end, either the underivatized constituents or their
derivatization products were analyzed using GC (30 m
3.4. Synthesis of selected phenylalkyl esters
ꢀ
DB-1 WCOT i.d. 320 mm, on-column-injection at 50 C,
oven 2 min at 50 C, 40 C min to 200 C, 2 min at
Very-long-chain fatty acid methyl esters were transes-
terified with phenylalkyl alcohols by modification of a
procedure for selective monoacetylation of diols (Bre-
ton, 1997). Unless stated otherwise, chemicals were
purchased from Sigma-Aldrich, Deisenhofen, FRG.
ꢀ
ꢀ
ꢁ1
ꢀ
ꢀ ꢀ ꢀ ꢀ
ꢁ1
2
carrier gas inlet pressures 1 min at 5 kPa, 4 kPa min
to 18 kPa, 0,6 kPa min to 40 kPa, 37 min at 40 kPa)
with MS detection (70 eV, m/z 50–650).
00 C, 3 C min to 320 C, 30 min at 320 C and He
ꢁ1
ꢁ1
0
Commercial 3-(4 -hydroxyphenyl)-propanol was used
0
0
Constituents of fractions A and B were derivatized in
four alternative reactions: (1) compounds containing
free hydroxyl groups were transformed into TMSi
ethers by reaction with bis-(N,N-trimethylsilyl)-tri-
without further purification. 3-(3 ,4 -Dihydroxyphenyl)-
0
propanol and 4-(4 -hydroxyphenyl)-2-butanol were
generated from the corresponding acid and ketone,
0
respectively, using LiAlH as described above. 4-(3 -
4
ꢀ
0
fluoroacetamide in pyridine for 30 min at 70 C; (2)
hydroxyl groups were acetylated by adding pyridine and
Methoxy-4 -hydroxyphenyl)-2-butanone (0.5 mmol,
Pfaltz and Bauer, Waterbury, CT, USA) was demethy-
ꢀ
ꢀ
Ac O to the dried fraction, heating the mixture to 70 C
2
lated using 1 M BBr (5.0 mmol) in THF (ꢁ78 C, 1h),
3
for 5 min, keeping it at RT overnight and then removing
the solvent in a stream of N ; (3) for qualitative analysis
the reaction mixture was allowed to warm to room temp
and extracted with Et O/water. The organic phase was
2
2
of the alcohols and fatty acids in esters of fractions A
and B, transesterification with BF /MeOH (60 min at
dried over Na SO , concentrated under N , and purified
4
2
2
by TLC (SiO , CHCl –EtOH 99:1). The resulting 4-
2 3
3

R. Jetter et al. / Phytochemistry 61 (2002) 579–587
585
0
0
+
(3 ,4 -dihydroxyphenyl)-2-butanone was isolated and
reduced to the corresponding alcohol with LiAlH as
[(TMSiO) –Ph–CH ]
2
267 (50), [TMSiO–Ph–CH –
2
2
CH=CH2]+ 205 (5), 193 (5), [TMSiO–Ph–CH2] 179
+
4
described above. GC–MS of the TMSi and acetate
derivatives served to identify the intermediate and final
reaction products. In four parallel reactions, the pheny-
lalkyl alcohols (ca. 100 mmol) and methyl docosanoate
(100), 166 (6), 149 (2), 147 (1), 85 (0.1), 83 (0.1), 73 (61),
0
+
0
71 (3), 69 (2), 57 (7), 55 (2). 3-(3 ,4 -Dihydroxyphenyl)-
propyl tetracosanoate [M]+ not detectable, [M-Me]
+
647 (1), [(TMSiO) –Ph–(CH ) –OH] 312 (15), 295
2
2 3
+
(
to heterogenous silica gel-supported NaHSO catalyst.
5 mmol) were dissolved in 1.5 ml of hexane and added
(70), [(TMSiO) –Ph–CH –CH=CH ]
+
294 (70),
[Me SiO–Ph–CH –CH=CH ] 279 (7), [(TMSiO) –
2
2
2
4
2
2
2
2
ꢀ
Ph–CH2]+ 267 (26), [TMSiO–Ph–CH –CH=CH ]
+
The mixture was heated to 70 C for 1 h, left at RT
overnight, filtered and concentrated under a stream
of N2.
2 2
205 (42), 193 (5), [TMSiO–Ph–CH2]+ 179 (100), 166 (5),
149 (2), 147 (2), 85 (2), 83 (4), 73 (70), 71 (6), 69 (2), 57
0 0
(17), 55 (4). 4-(3 ,4 -Dihydroxyphenyl)-2-butyl doc-
osanoate [M] 648 (21), [M-Me] 633 (0.1), [(TMSiO)2–
+
+
3
.5. Mass spectral data of selected compounds
+
Ph–(CH ) –CHOH–CH ]
2
326 (1), 309 (38),
2
3
+
GC–EIMS (70 eV) m/z (rel. int.). Underivatized phe-
0
[(TMSiO) –Ph–CH –CH=CH-CH ]
2
308
293
(75),
(18),
2
3
+
nylalkyl esters: 3-(4 -hydroxyphenyl)-propyl docosanoate
+
[Me SiO–Ph–CH –CH=CH–CH ]
2 2 3
M]+ 474 (0.1), [HO–Ph–(CH ) –OH] 152 (1), [HO–
[(TMSiO) –Ph–CH ]
2
+
267 (100), [TMSiO–Ph–CH –
2
[
2
3
2
Ph–CH –CH=CH ] 134 (100), [HO–Ph–CH2]+ 107
+
CH=CH–CH3]+ 219 (13), 193 (5), [TMSiO–Ph–CH2]+
179 (61), 149 (1), 147 (1), 85 (1), 83 (1), 73 (37), 71 (2),
2
2
0
(
Hydroxyphenyl)-propyl tetracosanoate [M] 502 (0.1),
[
CH=CH2] 134 (100), [HO–Ph–CH2] 107 (9), 85
(
Hydroxyphenyl)-2-butyl docosanoate [M] 488 (0.1),
[
CH –CH=CH–CH ] 148 (100), [HO–Ph–CH2] 107
14), 85 (1), 83 (2), 71 (1), 69 (0.4), 57 (0.5), 55 (2). 3-(4 -
+
0
0
69 (1), 57 (7), 55 (3). 4-(3 ,4 -Dihydroxyphenyl)-2-butyl
tetracosanoate [M]+ not detectable, [M–Me]+ not
detectable, [(TMSiO) –Ph–(CH ) –CHOH–CH ] 326
2
+
HO–Ph–(CH ) –OH]
+
152 (2),
[HO–Ph–CH2–
+
2
3
+
2 2
3
0
+
0.1), 83 (0.2), 71 (1), 69 (0.4), 57 (2), 55 (1). 4-(4 -
+
(1), 309 (35), [(TMSiO) –Ph–CH –CH=CH–CH ]
2 2 3
+
308 (65), [Me SiO–Ph–CH –CH=CH–CH ] 293 (17),
2
2
3
+
+
HO–Ph–(CH ) –CHOH–CH ]
+
166 (1), [HO–Ph–
+
[(TMSiO) –Ph–CH ]
267 (100), [TMSiO–Ph–CH –
2
2
2
3
2
2
CH=CH–CH3]+ 219 (12), 193 (6), [TMSiO–Ph–CH2]+
179 (54), 149 (1), 147 (2), 85 (2), 83 (1), 73 (30), 71 (4),
69 (2), 57 (7), 55 (3).
2
3
0
+
(
8). 4-(4 -Hydroxyphenyl)-2-butyl tetracosanoate [M]
+
5
16 (0.1), [HO–Ph–(CH ) –CHOH–CH ]
+
166 (1),
HO–Ph–CH –CH=CH–CH ] 148 (100), [HO–Ph–
2
2
3
0
[
CH2] 107 (5).
Acetates of phenylalkyl esters: 3-(4 -hydroxyphenyl)-
2
3
+
propyl docosanoate [M]+ 544 (0.1), [M-ketene] 502
+
0
+
TMSi ethers of phenylalkyl esters: 3-(4 -hydroxy-
(0.1), [AcO–Ph–(CH ) –OH] 194 (1), [AcO–Ph–CH –
3
2
2
phenyl)-propyl docosanoate [M]+ 546 (1), [M–Me]+ 531
CH=CH2]+ 176 (28), [HO–Ph–(CH ) –OH] 152 (2),
[HO–Ph–CH –CH=CH ] 134 (100), [HO–Ph–CH2]
2 2
+
2 3
+
+
+
(
CH –CH=CH ]
CH=CH2]+ 191 (15), [TMSiO–Ph–CH2] 179 (13), 85
(
Hydroxyphenyl)-propyl tetracosanoate [M] 574 (1),
[
[
Ph–CH –CH=CH ]
1
(
(
CH3]
2
0.1), [TMSiO–Ph–(CH ) –OH] 224 (4), [TMSiO–Ph–
3
2
+
206 (100), [Me SiO–Ph–CH –
2
107 (7), 85 (0.2), 83 (0.1), 71 (0.5), 69 (0.1), 57 (1), 55
0
2
2
2
+
+
(0.3). 3-(4 -Hydroxyphenyl)-propyl tetracosanoate [M]
572 (0.1), [M-ketene]+ 530 (0.1), [AcO–Ph–(CH ) –
0
0.5), 83 (0.5), 73 (4), 71 (1), 69 (1), 57 (2), 55 (1). 3-(4 -
+
2
3
OH]+ 194 (1), [AcO–Ph–CH –CH=CH ] 176 (37),
+
2
2
M-Me]+ 559 (0.2), [TMSiO–Ph–(CH ) –OH] 224 (4),
+
[HO–Ph–(CH ) –OH]
2 3
+
152
(1), [HO–Ph–CH2–
CH=CH2]+ 134 (100), [HO–Ph–CH2] 107 (7), 85
2
3
+
+
TMSiO–Ph–CH –CH=CH ]
2
206 (100), [Me SiO–
2
191 (16), [TMSiO–Ph–CH2]
79 (15), 85 (1), 83 (1), 73 (4), 71 (1), 69 (1), 57 (2), 55
2
+
+
0
(0.4), 83 (1), 71 (1), 69 (0.4), 57 (1), 55 (1). 4-(4 -
+
2
2
Hydroxyphenyl)-2-butyl docosanoate [M] 558 (0.1),
[M-ketene]+ 516 (0.2), [AcO–Ph–(CH ) –CHOH–
0
+
1). 4-(4 -Hydroxyphenyl)-2-butyl docosanoate [M] 560
+
2
2
CH3]+ 208 (0.3), [AcO–Ph–CH –CH=CH–CH ] 190
+
0.2), [M-Me] 545 (0.1), [TMSiO–Ph–(CH ) –CHOH–
2
2
2 3
+
+
+
238 (1), [TMSiO–Ph–CH –CH=CH–CH ]
3
20 (100), [Me SiO–Ph–CH –CH=CH–CH ]
3
12), [TMSiO–Ph–CH2]+ 179 (20), 85 (0.2), 83 (0.3), 73
(13), [HO–Ph–(CH ) –CHOH–CH ] 166 (1), [HO–Ph–
2 2 3
2
+
CH –CH=CH–CH ] 148 (100), [HO–Ph–CH2]+ 107
+
205
2
2
2 3
(
(
(5), 85 (0.2), 83 (0.1), 71 (0.3), 69 (0.2), 57 (0.3), 55 (0.2).
0
0
+
2), 57 (0.3), 55 (0.2). 4-(4 -Hydroxyphenyl)-2-butyl tet-
+
4-(4 -Hydroxyphenyl)-2-butyl tetracosanoate [M] 586
+
+
racosanoate [M] 588 (1), [M-Me] 573 (0.2), [TMSiO–
+
(0.1), [M–ketene] 544 (0.1), [AcO–Ph–(CH ) –CHOH-
2
2
CH3]+ 208 (0.3), [AcO–Ph–CH –CH=CH–CH ] 190
+
Ph–(CH ) –CHOH–CH ] 238 (2), [TMSiO–Ph–CH2–
2
2
3
2 3
+
+
CH=CH–CH3]
CH=CH–CH3] 205 (8), [TMSiO–Ph–CH2] 179 (14),
220 (100), [Me SiO–Ph–CH –
+
(13), [HO–Ph–(CH ) –CHOH–CH ] 166 (1), [HO–Ph–
2 2 3
CH –CH=CH–CH ] 148 (100), [HO–Ph–CH2]+ 107
2
2
+
+
2 3
0
0
8
Dihydroxyphenyl)-propyl docosanoate [M] 634 (83),
5 (0.2), 83 (0.2), 73 (1), 57 (0.2), 55 (0.1). 3-(3 ,4 -
+
(4), 85 (0.4), 83 (1), 71 (1), 69 (0.4), 57 (1), 55 (1). 3-
0
0
+
(3 ,4 -Dihydroxyphenyl)-propyl docosanoate [M] 574
M-Me]+ 619 (0.4), [(TMSiO) –Ph–(CH ) –OH] 312
+
(0.1), [M–ketene]+ 532 (0.3), [M–2xketene] 490 (13),
[(AcO) –Ph–(CH ) –OH] 252 (3), [(AcO) –Ph–CH –
2
CH=CH2]+ 234 (16), [(AcO)(HO)–Ph–(CH ) –OH]
+
[
2 2 3
+
+
(
(
15), 295 (48), [(TMSiO) –Ph–CH –CH=CH ] 294
2
2
2
2
2 3
2
+
+
63),
[Me SiO–Ph–CH –CH=CH ]
2
279
(8),
2
2
2 3

586
R. Jetter et al. / Phytochemistry 61 (2002) 579–587
+
phenyl)-2-butanol [M]+ 250 (0.3), [M-ketene]+ 208 (3),
2
10 (11), [(AcO)(HO)–Ph–CH –CH=CH ] 192 (40),
2
2
+
[M–HOAc]+ 190 (18), [HO–Ph–CH –CH=CH–CH ]
+
[
CH=CH2] 150 (100), [(HO) –Ph–CH ] 123 (8), 85
(HO) –Ph–(CH ) –OH]
+
168 (2), [(HO) –Ph–CH –
+
2
2 3
2
2
2 3
+
148 (100), 133 (93), 121 (6) [HO–Ph–CH ] 107 (35),
2
2
2
(
(
(
0.2), 83 (0.1), 71 (0.5), 69 (0.6), 57 (0.6), 55 (0.1). 3-
0 0
[Ph–CH2]+ 91 (7), 77 (8), 65 (0.4), 55 (2). 3-(3 ,4 -Dihy-
0 0
+
droxyphenyl)-propanol [M] 294 (3), [M–ketene]+ 252
+
3 ,4 -Dihydroxyphenyl)-propyl tetracosanoate [M] 602
0.1), [M–ketene]+ 560 (0.2), [M–2xketene] 518 (16),
+
(19), [M–HOAc]+ 234 (5), [(AcO)(HO)–Ph–(CH ) –
2
3
+
OH]+ 210 (97), [(AcO)(HO)–Ph–CH –CH=CH ] 192
+
[
CH=CH2] 234 (24), [(AcO)(HO)–Ph–(CH ) –OH]
(AcO) –Ph–(CH ) –OH] 252 (3), [(AcO) –Ph–CH –
+
2
2 3
2
2
2 2
+
+
(4), [(HO) –Ph–CH –CH=CH ] 150 (100), 149 (11),
2 2 2
2 3
+
+
2
10 (14), [(AcO)(HO)–Ph–CH –CH=CH ] 192 (48),
2
137 (6), [(HO)–Ph–CH –CH=CH ] 132 (14), 131 (7),
2 2
2
+
+
+
[
CH=CH2] 150 (100), [(HO) –Ph–CH ] 123 (9), 85
(HO) –Ph–(CH ) –OH]
+
168 (4), [(HO) –Ph–CH –
+
[(HO) –Ph–CH ] 123 (23), 122 (10), [HO–Ph–CH ]
2 2 2
2
2 3
2
2
+
0
0
107 (3), [Ph–CH2] 91 (9), 77 (9), 65 (3), 55 (3).4–(3 ,4 -
Dihydroxyphenyl)-2-butanol [M]+ 308 (0.4), [M–
ketene]+ 266 (5), [M–HOAc] 248 (18), [(AcO)(HO)–
2
2
0 0
(
droxyphenyl)-2-butyl docosanoate [M] 588 (0.1), [M–
1), 83 (1), 71 (1), 69 (1), 57 (2), 55 (1). 4-(3 ,4 -Dihy-
+
+
ketene]+ 546 (0.1), [M–2xketene] 504 (1), [(AcO) –Ph–
+
Ph–(CH ) –CHOH–CH ] 224 (61), [(AcO)(HO)–Ph–
2 2
+
2
3
+
+
(
CH=CH–CH ] 248 (36), [(AcO)(HO)–Ph–(CH2)2–
CH ) –CHOH–CH ]
+
266 (0.5), [(AcO) –Ph–CH –
CH –CH=CH–CH ]
2
206 (16), [(HO) –Ph–CH –
2 2
2
2
3
2
2
3
+
CH=CH–CH3] 164 (100), 149 (26), [(HO)–Ph–CH2–
+
3
CHOH–CH3]+
CH=CH–CH ] 206 (80), [(HO) –Ph–(CH ) –CHOH–
224
(2),
[(AcO)(HO)–Ph–CH2–
CH=CH–CH ] 146 (5), 137 (6), 131 (9), [(HO) –Ph–
2
3
+
CH2] 123 (29), 122 (31), [HO–Ph–CH2]+ 107 (4), [Ph–
+
3
2
2 2
CH3]+ 182 (1), [(HO) –Ph–CH –CH=CH–CH ] 164
+
CH2] 91 (9), 77 (6), 65 (4), 55 (3).
+
2
2
3
+
(
6
100), [(HO) –Ph–CH ] 123 (18), 85 (1), 83 (1), 71 (1),
2 2
9 (1), 57 (2), 55 (2). 4-(3 ,4 -Dihydroxyphenyl)-2-butyl
tetracosanoate [M] 616 (0.1), [M–ketene]+ 574 (0.1),
0 0
+
+
[
CH3] 266 (1), [(AcO) –Ph–CH –CH=CH–CH ] 248
M–2xketene] 532 (1), [(AcO) –Ph–(CH ) –CHOH–
+
Acknowledgements
2
2 2
+
2
2
3
+
(
47), [(AcO)(HO)–Ph–(CH ) –CHOH–CH ] 224 (2),
3
The authors are indebted to Professor Dr. M. Rie-
derer for instrumental support and for fruitful discus-
sions. Technical assistance by the staffof the botanical
2
2
+
[
[
(AcO)(HO)–Ph–CH –CH=CH–CH ]
+
206
(HO) –Ph–(CH ) –CHOH–CH ] 182 (1), [(HO) –Ph–
(89),
2
3
2
2 2
3
2
+
+
CH –CH=CH–CH ] 164 (100), [(HO) –Ph–CH ]
2
¨
garden of the University of Wurzburg and financial
3
2
2
1
23 (17), 85 (1), 83 (0.1), 71 (1), 69 (1), 57 (2), 55 (1).
TMSi ethers of ester cleavage products: 3-(4 -
support by the Deutsche Forschungsgemeinschaft (SFB
567 ‘Mechanisms of interspecific interactions of organ-
isms’) are gratefully acknowledged.
0
hydroxyphenyl)-propanol [M]+ 296 (7), [M–Me] 281
+
+
(
11),
[TMSiO–Ph–CH –CH=CH ]
2
206
(100),
2
+
[Me SiO–Ph–CH –CH=CH ] 191 (70), [TMSiO–Ph–
2 2 2
CH2] 179 (15), 133 (6), 103 (1), 89 (11), 73 (21). 4-(4 -
Hydroxyphenyl)-2-butanol [M] 310 (3), [M–Me] 295
+
0
+
+
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+
(
4), [TMSiO–Ph–CH –CH=CH–CH ]
220 (100),
205 (77),
TMSiO–Ph–CH ] 179 (12), 147 (3), 103 (13), 89 (2),
2
3
+
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