Very-long-chain hydroxyaldehydes from the cuticular wax of Taxus baccata needles

Article abstract of DOI:10.1016/j.phytochem.2007.05.029

In the cuticular wax of Taxus baccata needles, homologous series of very-long-chain 1,5-alkanediols and 5-hydroxyaldehydes were identified by various chemical transformations with product assignment using GC-MS. The 1,5-alkanediols had chain lengths ranging from C₂₈ to C₃₈, with strong predominance of even carbon numbers and a maximum at C₃₂ (29%). The series of 5-hydroxyaldehydes comprised chain lengths C₂₄ and C₂₆-C₃₆, and showed a pronounced prevalence of even-numbered homologues. 5-Hydroxyoctacosanal was the most abundant compound of the series (42%). The 5-hydroxyaldehydes together amounted to 0.4 μg/cm², corresponding to 1.2% of total wax of the needles. A polyketide-like biosynthetic pathway is proposed based on the (similar) chain length distributions and functional group patterns for both compound classes.

Full text of DOI:10.1016/j.phytochem.2007.05.029

PHYTOCHEMISTRY
Phytochemistry 68 (2007) 2563–2569
www.elsevier.com/locate/phytochem
Very-long-chain hydroxyaldehydes from the cuticular wax
of Taxus baccata needles
*
Miao Wen, Reinhard Jetter
Department of Botany, University of British Columbia, 6270 University Blvd., Vancouver, Canada V6T 1Z4
Department of Chemistry, University of British Columbia, 6174 University Blvd., Vancouver, Canada V6T 1Z3
Received 6 February 2007; received in revised form 4 May 2007
Available online 24 July 2007
Abstract
In the cuticular wax of Taxus baccata needles, homologous series of very-long-chain 1,5-alkanediols and 5-hydroxyaldehydes were
identified by various chemical transformations with product assignment using GC–MS. The 1,5-alkanediols had chain lengths ranging
from C28 to C38, with strong predominance of even carbon numbers and a maximum at C32 (29%). The series of 5-hydroxyaldehydes
comprised chain lengths C24 and C26–C36, and showed a pronounced prevalence of even-numbered homologues. 5-Hydroxyoctacosanal
was the most abundant compound of the series (42%). The 5-hydroxyaldehydes together amounted to 0.4 lg/cm2, corresponding to 1.2%
of total wax of the needles. A polyketide-like biosynthetic pathway is proposed based on the (similar) chain length distributions and
functional group patterns for both compound classes.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Taxus baccata; Taxodiaceae; Yew; Alkanediols; Aldehydes; Chain length; GC–MS
1. Introduction
biosynthesized by elongation of acyl-CoA precursors, and
modification either by reduction to alcohols or by decarbo-
nylation to alkanes (Kunst and Samuels, 2003). Some plant
waxes contain compounds with two functional groups in
primary and/or secondary positions. Most wax compo-
nents with secondary/secondary functional groups have
hydrocarbon backbones with odd numbers of carbons
(Holloway and Brown, 1977; Franich et al., 1979), while
the compound classes with primary/secondary functional
groups have predominantly even-numbered chains (Jetter
and Riederer, 1999a,b; Vermeer et al., 2003).
The surfaces of aboveground, non-woody, plant organs
are covered with a cuticle that consists of cutin and waxes
(Jetter et al., 2006). The primary physiological function of
the cuticle is to limit non-stomatal water loss, and it is also
of ecological importance as it represents the outermost
layer of the plant organs. The cuticular waxes render plant
surfaces water repellent, thereby keeping them dry, guard-
ing them from accumulation of particles, and preventing
germination of pathogen spores.
The specific functions of plant cuticles can only be
understood on the basis of their characteristic wax compo-
sition and biosynthetic origin. Cuticular waxes typically
consist of complex mixtures of very-long-chain fatty acids,
aldehydes, primary alcohols, esters and alkanes. It is gener-
ally accepted that these (monofunctional) compounds are
There are two fundamentally different pathways for the
introduction of the secondary functional groups in wax
components: (1) direct hydroxylation has been proposed
with functional groups on/near the central carbon, e.g.,
nonacosane-14,15-diol in Brassica oleracea (Holloway
and Brown, 1977), and octacosane-1,14-diol as well as
hentriacotane-9,16-diol in Pisum sativum (Wen et al.,
2006a); (2) polyketide biosynthetic pathways are leading
to b-diketones (von Wettstein-Knowles, 1995) and to wax
*
Corresponding author. Fax: +1 604 822 6089.
E-mail address: jetter@interchange.ubc.ca (R. Jetter).
0031-9422/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.05.029

2564
M. Wen, R. Jetter / Phytochemistry 68 (2007) 2563–2569
components with 1,3-, 1,5-, 1,7-, 1,9-substitution patterns
(Jetter and Riederer, 1999a,b; Vermeer et al., 2003).
To further substantiate these pathways, potential inter-
mediates and corresponding products have to be identified.
Therefore, the objective of the present work was to eluci-
date the structure of candidate wax components that
appeared to have two functional groups. A number of pre-
viously unidentified compounds in the cuticle of Taxus bac-
cata (yew) were targeted here. In order to identify them, the
cuticular wax mixture was extracted from yew needles and
separated by TLC. The fractions containing unknown
structures were transformed into various derivatives and
analyzed by GC–MS.
After derivatization with bis-(N,N-trimethylsilyl)-triflu-
oroacetamide (BSTFA), the MS of the most prominent
compound in B showed fragments m/z 73, 75 and 103
characteristic for an OTMSi group (Fig. 1a). While this
indicated the presence of one hydroxyl group, the lack
of a diol signal m/z 147 made it unlikely that a second
hydroxyl group was present. Instead, the characteristic
base peak m/z 119, which had previously been reported
as a characteristic fragment for non-vicinal ketols (Jetter
and Riederer, 2000; Shanker et al., 2007), indicated the
presence of one or more carbonyl groups in the molecule.
The fragment m/z 173 could be interpreted as an a ion
[C5H8O2TMSi]+ and thus pointed to the 1,5-geometry
between a carbonyl and a hydroxyl group. The com-
pound was further characterized by another a ion m/z
425 [C24H48OTMSi]+, a fragment m/z 481 [MÀCH3]+
and the molecular ion m/z 496. In the spectra of all the
other compounds in B only the latter three signals dif-
fered by 14 mass units (Table 1), indicating that they
were homologous ketols. It can be concluded that these
homologues differed in the number of CH2 groups in a
hydrocarbon chain attached on one side of the 1,5-
bifunctionality, rather than between both functional
groups.
The number and position of carbonyl groups in the
compounds was assessed by reduction of the homologous
mixture with LAH, followed by BSTFA derivatization
(Fig. 1b). The fragments m/z 147 and 149 showed that
the reduced derivatives were diols, and that they con-
tained one primary and one secondary hydroxyl group,
respectively (Jetter and Riederer, 1999a). All the charac-
teristic signals for 1,5-alkanediols (see above, Jetter and
Riederer, 1999b) were detected in the MS of all 12 homo-
logues, including the fragments m/z 85, m/z 247
[C5H9(OTMSi)2]+ and its daughter ion m/z 157
[C5H8OTMSi]+. The major compound in the mixture of
reduction products showed fragments m/z 425 and 555
[MÀCH3]+, thus confirming its structure as octacosane-
1,5-diol, and the structure of the other compounds as
homologous 1,5-alkanediols (Table 1). Taken together,
this experiment confirmed the 1,5-ketol geometry of com-
pounds in B and excluded the presence of any further
functional groups.
2. Results and discussion
TLC separation of the wax mixture from needles of T.
baccata yielded 12 fractions with all the reported wax con-
stituents and two bands of previously unidentified com-
pounds, designated as compound classes A and B.
Compound class A (Rf = 0.05), migrating between second-
ary/secondary alkanediols (Rf = 0.08) and fatty acids
(Rf = 0.03), likely contained either a primary and a sec-
ondary hydroxyl group, or else three or more secondary
hydroxyl and carbonyl groups. The TMSi derivative of
the most prominent compound in A showed a MS frag-
ment m/z 85, which had been interpreted as [C5H9O]+
and found to be characteristic for 1,5-alkanediols (Jetter
and Riederer, 1999b). The TMSi derivative was further
characterized by an a-fragment m/z 247 [C5H9(OTMSi)2]+
together with its daughter ion m/z 157 [C5H8OTMSi]+, a
second a-fragment m/z 425 [C24H48OTMSi]+, a fragment
m/z 555 [MÀCH3]+ and molecular ion m/z 570. Taken
together, these data suggested that the compound was
octacosane-1,5-diol. The GC profile of fraction A showed
10 more peaks, all with matching MS characteristics of
TMSi ethers of primary/secondary diols (Table 1). Based
on the chain length specific a-fragments and molecular
ions, the compounds were identified as a homologous ser-
ies of 1,5-alkanediols ranging from C28 to C38.
Compound class B (Rf = 0.14) migrated between pri-
mary alcohols (Rf = 0.17) and secondary/secondary alkan-
ediols (Rf = 0.08) on TLC, and therefore likely contained
compounds with a (secondary) hydroxyl group and one
(or more) carbonyl group(s). Further GC separation
resulted in 12 distinct peaks with similar mass spectral
characteristics, indicating that compound class B was a
homologous series. Combining all the mass spectral data
from different derivatives of all 12 compounds, it was even-
tually concluded that compound class B represented a
homologous series of 5-hydroxyaldehydes comprising
chain lengths C24 and C26–C36. This was corroborated by
further GC–MS comparisons with a synthetic standard of
5-hydroxyoctacosanal. Since this is also the most abundant
homologue in the wax, the structure elucidation will be
described using it as an example.
To further corroborate the 5-hydroxyaldehyde struc-
ture, compounds in B were reduced with LAH and then
further derivatized with acetic anhydride. The resulting
products (Fig. 1c) had a prominent MS fragment m/z 61,
interpreted as [AcOH2]+ diagnostic for acetates. Further-
more, the fragments m/z 450 [MÀ60]+, m/z 407
[MÀ43À60]+ and m/z 390 [MÀ120]+, involving the loss
of one and two acetyl moieties, were characteristic for bis
acetates of alkanediols (Wen et al., 2006a). The molecular
ion m/z 510 of octacosanediol bis acetate confirmed the
presence of exactly two hydroxyl functions. Different from
the fragmentation patterns of primary/secondary diol ace-
tates from pea wax (Wen et al., 2006a), the daughter frag-
ment Dm/z 42 of the a-ion (m/z 334) had relatively low

Table 1
MS spectral data of 1,5-alkanediol TMSi ethers, 5-hydroxyaldehyde derivatives, and underivatized 5-hydroxyaldehydes
Compounds
Relative intensity (%)
Fragments characteristic for the compound classa
Fragments characteristic for the homologue
TMSi ether of 1,5-alkanediols
Octacosane-1,5-diol
Nonacosane-1,5-diol
73
40
–
85
83
–
103
25
–
30
27
7
9
6
6
3
147
23
–
149
10
–
10
9
8
8
8
9
157
9
3
10
9
8
8
7
8
9
247
5
2
7
8
7
7
7
7
M
M-15
555
569
583
597
611
625
639
653
667
681
695
CnH2nOTMSi
570
584
598
612
626
640
654
668
682
606
710
–
–
0.5
–
0.2
–
–
–
–
–
–
2
2
2
1
0.4
0.6
0.5
0.5
0.6
0.6
0.7
425
439
453
467
481
495
509
523
537
551
565
100
100
100
100
100
100
100
95
Triacontane-1,5-diol
45
32
32
36
25
26
22
24
25
90
87
86
93
95
100
100
100
100
25
20
13
15
12
12
10
12
14
Hentriacontane-1,5-diol
Dotriacontane-1,5-diol
Tritriacontane-1,5-diol
Tetratriacontane-1,5-diol
Pentatruacontane-1,5-diol
Hexatriacontane-1,5-diol
Heptatriacontane-1,5-diol
Octatriacontane-1,5-diol
9
8
9
8
7
7
94
96
95
6
4
9
8
TMSi ether of 5-hydroxyaldehydes
5-Hydroxytetracosanal
5-Hydroxyhexacosanal
5-Hydroxyheptacosanal
5-Hydroxyoctacosanal
5-Hydroxynonacosanal
73
8
9
103
27
25
27
22
21
21
22
19
23
19
20
20
119
100
100
100
100
100
100
100
100
100
100
100
100
173
29
25
24
16
17
17
16
18
18
17
16
19
M
M-15
425
453
467
481
495
509
523
537
551
565
579
593
CnH2nOTMSi
440
468
482
496
510
524
538
552
566
580
594
608
2
2
1
1
1
1
1
1
2
1
1
1
22
369
397
411
425
439
453
467
481
495
509
523
537
19
12
13
9
9
10
9
10
10
10
9
23
20
13
13
13
12
12
14
11
12
13
10
11
12
12
11
11
13
14
12
13
5-Hydroxyteiacontanal
5-Hydroxylhentriacontanal
5-Hydroxydotriacontanal
5-Hydroxytritriacontanal
5-Hydroxytetratriacontanal
5-Hydroxypentatriacontanal
5-Hydroxyhexatriscontanal
9
Acetates of 1,5-alkanediols
Tetracosane-1,5-diol
Hexacosane-1,5-diol
Heptacosane-1,5-diol
Octacosane-1,5-diol
Nonacosane-1,5-diol
Triacontane-1,5-diol
Hentriacontane-1,5-diol
Detriacontane-1,5-diol
Tritriacontane-1,5-diol
Tetratriacontane-1,5-diol
Pentatriacontane-1,5-diol
Hexatriacontane-1,5-diol
61
8
8
9
8
9
9
10
9
9
85
100
100
100
100
100
100
100
100
100
100
100
100
M-60-43
365
393
M-120
348
376
390
404
418
432
446
460
474
488
502
516
19
17
19
18
15
14
16
14
14
13
15
14
30
28
30
29
30
28
29
32
29
35
32
29
407
421
435
449
463
477
491
505
10
9
8
519
533
5-Hydroxyaldehydes
98
111
36
30
M-18
350
378
5-Hydroxytetracosanal
5-Hydroxyhexacosanal
5-Hydroxyheptacosanal
100
100
100
7
7
4
29
392
(continued on next page)

2566
M. Wen, R. Jetter / Phytochemistry 68 (2007) 2563–2569
abundance. Since the base peak m/z 85 was found for both
the bis acetates and for the corresponding bis TMSi ethers,
it is clearly diagnostic for 5-hydroxyalkanediols irrespective
of the hydroxyl derivatives.
The MS of underivatized 5-hydroxyoctacosanal (Fig. 1d)
showed prominent fragments m/z 98 and m/z 111 that can
be interpreted as C6H10O+ resulting from water elimination
from the molecular ion followed by McLafferty rearrange-
ment, and C7H11O+ resulting from water elimination
followed by b-fragmentation, respectively. Due to the pres-
ence of a hydroxyl group, water can be easily eliminated
from the molecular ion, resulting in the fragment m/z 406
[MÀH2O]+ with relatively high abundance. For the other
homologues, this fragment differed by 14 mass units,
whereas the other fragments m/z 98 and m/z 111 stayed con-
stant (Table 1).
In summary, the spectral information indicated the
presence of one hydroxyl and one carbonyl group with
primary/secondary 1,5-geometry, narrowing the candidate
structures of the prevalent homologue to the isomers 5-
hydroxyoctacosanal and 1-hydroxyoctacosan-5-one. The
latter structure seemed highly unlikely because: (1) the
presence of a primary hydroxyl group and a carbonyl
group would give rise to a polarity higher than that
observed in our TLC experiments; (2) the a-fragment of
the mid-chain functional group (m/z 425 for the TMSi
derivatives) was not affected by LAH treatment, showing
that this functionality cannot be reduced and therefore
likely is a hydroxyl group; (3) the MS of the TMSi ether
of 1-hydroxyoctacosan-5-one would very likely give an
a-fragment m/z 351 (instead of m/z 425), which could
not be detected. Since the spectral interpretation strongly
favoured a 5-hydroxyaldehyde structure for compounds
in B, one representative of the homologous series was
synthesized for final proof of structure (Njardarson
et al., 2002). It was found that the GC characteristics
and MS fragmentation pattern of authentic 5-hydroxyoc-
tacosanal were identical to those of one homologue in
compound class B (data not shown). The compounds in
the needle wax of T. baccata were thus identified as a ser-
ies of 5-hydroxyaldehydes comprising chain lengths C24
and C26–C36.
The total needle wax contained approximately 0.4 lg/
cm2 of 5-hydroxyaldehydes. The homologous series was
dominated by even-numbered compounds, containing
42% of 5-hydroxyoctacosanal, 22% of 5-hydroxytriacont-
anal, 19% of 5-hydroxydotriacontanal, and 17% of the
other homologues (Fig. 2a). The 5-hydroxylaldehydes
could be detected in a wax sample isolated from the
abaxial side of the needle, but not in the wax from the
adaxial side. It is not clear whether they were absent from
this side of the needle, or not detectable due to the lower
total wax amounts on the adaxial surface (Wen et al.,
2006b). The 1,5-alkanediols were present in the needle
wax only at trace levels. Interestingly, this homologous
series was also dominated by even-numbered chains
(Fig. 2b).

M. Wen, R. Jetter / Phytochemistry 68 (2007) 2563–2569
2567
100
80
60
40
20
0
a
425
119
O
OTMSi
CH
3
H
22
173
103
100
173
481
496
425
m/z
600
50
150
200
250
300
300
300
300
350
400
450
500
550
100
80
60
40
20
0
b
c
425
85
425
OTMSi
OTMSi
CH
3
147
22
247
157
-TMSiOH
149
129
100
247
157
200
555
m/z
600
50
150
150
150
250
250
250
350
400
450
500
550
100
80
60
40
20
0
85
395
OAc
OAc
61
CH
3
22
395
390
187
407
187
450
450
m/z
600
50
100
200
350
400
500
550
100
80
60
40
20
0
d
O
OH
98
CH
3
H
22
111
406
m/z
600
50
100
200
350
400
450
500
550
Fig. 1. Mass spectra of representative derivatives of 5-hydroxyaldehydes in the needle wax of Taxus baccata. (a) TMSi ether of 5-hydroxyoctacosanal,
(b) bis TMSi ether of octacosane-1,5-diol, (c) bis acetate of octacosane-1,5-diol, and (d) underivatized 5-hydroxyoctacosanal.
50
40
30
20
10
0
50
40
30
20
10
0
a
b
Fig. 2. Chain length distribution (%) of (a) 5-hydroxyaldehydes (n = 5, SE) and (b) 1,5-alkanediols (n = 3, SE) in T. baccata needle wax.

2568
M. Wen, R. Jetter / Phytochemistry 68 (2007) 2563–2569
O
OH
OH
OH
C23H47
C23H47
C23H47
OH
H
O
O
OH
OH
O
Elongase2
O
O
KCR
C23H47
SCoA
C23H47
SCoA
C23H47
SCoA
OH
DH
O
KCS
C23H47
SCoA
ECR
O
C23H47
OH
H
O
O
O
O
Elongase3
Elongase1
C23H47
SCoA
C23H47
SCoA
C23H47
SCoA
C23H47
OH
C23H47
Fig. 3. Proposed biosynthetic pathway leading to 5-hydroxyaldehydes and 1,5-alkanediols in the needle wax of Taxus baccata. Only the C28 homologues
are depicted as an example. In normal wax biosynthesis (shown in boxes with black lines), acyl CoA chains are elongated by elongase complex(es)
containing 3-ketoacyl CoA synthase (KCS), 3-ketoacyl CoA reductase (KCR), dehydratase (DH) and enoyl CoA reductase (ECR) (shown in box with
shade). Multiple rounds of elongation lead to C28 acyl CoA, which is further modified into the corresponding free fatty acid, aldehyde and alcohol (circled
with black line). A polyketide-like pathway is proposed for biosynthesis of 5-hydroxyaldehydes and 1,5-alkanediols (shown in box with dashed lines). A
hydroxacyl CoA intermediate is elongated directly and the resulting 5-hydroxyacyl CoA is further modified (shown in dashed cycle). Two or more of the
elongase complexes shown may be identical.
3. Conclusions
of British Columbia. Mature needles were cut from the twigs
using razor blades. Batches of 30–40 needles were used for
total wax analysis, while 5–8 needles were used for brushing
to extract waxes from abaxial and adaxial surfaces separately.
The similar chain length distributions of 5-hydroxyaldehy-
des and 1,5-alkanediols suggest a biosynthetic relationship
between both compound classes. Furthermore, the predomi-
nance of even-numbered homologues in both series makes it
very likely that both compound classes are formed, in analogy
to other common wax components, on pathways involving
acyl reduction rather than decarbonylation. We therefore
conclude that the primary function of both compound classes
originates by reduction of a derivative generated by fatty acid
elongation. In contrast, the constant 1,5-geometry of all
alkanediols and hydroxyaldehydes detected here suggests
that the secondary functional group is introduced during
elongation. In analogy to polyketide biosynthesis, one round
of elongation may start with 3-hydroxyacyl CoA intermedi-
ates instead of the corresponding acyl CoAs (Fig. 3). The
resulting 5-hydroxyacyl CoAs could be modified into
5-hydroxyacids, 5-hydroxyaldehydes, or 1,5-alkanediols. 5-
Hydroxy fatty acids were not detected in yew needle wax,
but their d-lactones have been identified in leaf cuticular
wax of Cerinthe minor (Jetter and Riederer, 1999b).
4.2. Wax extraction
For total wax extraction, cut needles were immediately
immersed twice for 30 s in CHCl3 at room temperature.
To selectively extract waxes from the abaxial and adaxial
surfaces of needles, either of the two surfaces was brushed
gently with fabric glass that had been pre-extracted (in a
Soxhlet apparatus) and soaked with CHCl3 (Wen et al.,
2006b). The resulting solutions were filtered, dried, and
stored at 4 °C until they were analyzed.
4.3. Qualitative and quantitative analysis of 5-
hydroxyaldehydes
Compound classes in total waxes were separated by
TLC (sandwich technique (Tantisewie et al., 1969), silica
gel, mobile phase CHCl3) and visualized by staining with
primuline and UV light. Bands were removed from the
plates, eluted with CHCl3, filtered, concentrated in a
stream of N2 and stored at 4 °C. Two fractions contained
unknown compounds and were subjected to detailed qual-
itative analyses. Relative compositions (wt%) of homo-
logues were quantified based on the abundance of
characteristic MS fragments. To this end, constituents were
4. Experimental
4.1. Plant material
Twigs were harvested in spring from plants of Taxus bac-
cata L. growing continuously on the campus of the University

M. Wen, R. Jetter / Phytochemistry 68 (2007) 2563–2569
2569
studied with capillary GC (5890N, Agilent, Avondale, PA,
USA; column 30m HP-1, 0.32 mm i.d., df = 0.1 lm) with
He carrier gas inlet pressure programmed for constant flow
of 1.4 ml/min and mass spectrometric detector (5973N,
Agilent). GC was carried out with temperature pro-
grammed injection at 50 °C, oven 2 min at 50 °C, raised
by 40 °C/min to 200 °C, held for 2 min at 200 °C, raised
by 3 °C/min to 320 °C and held for 30 min at 320 °C.
The coverage (lg/cm2) of the most abundant homologue,
5-hydroxyoctacosanal, was quantified by GC-FID after
adding a defined amount of n-tetracosane into the total
wax extracts as an internal standard. Inlet pressure was
programmed for constant flow of 2.0 ml/min with H2 as
carrier gas. GC temperature program was the same as for
MS. The coverage of all the 5-hydroxyaldehydes was then
calculated based on the relative abundance of 5-hydroxyoc-
tacosanal in the homologous series.
and then oxidized by trimethyl N-oxide (Sigma–Aldrich,
MO, USA) in refluxing THF for 4 h. The resulting syn-
thetic 5-hydroxyoctacosanal was characterized by GC–
MS as described above: TMSi ether 119 (100), 73 (25),
103 (33), 173 (18), 481 (45), 425 (22), 496 (4); free alcohol
98 (100), 111( 25), 406 (7).
Acknowledgements
The authors gratefully acknowledge technical help by
Dale Chen and financial support from the Special Research
Opportunity program of the Natural Sciences and Engi-
neering Research Council (Canada), the Canadian Foun-
dation for Innovation and the Canada Research Chair
Program.
References
4.4. Derivatization reactions
Franich, R.A., Gowar, A.P., Volkman, J.K., 1979. Secondary diols of
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cuticluar waxes of Cerinthe minor. Phytochemistry 50, 1359–1364.
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The constituents of the fractions containing unknown
compounds were derivatized in three alternative reactions:
(1) compounds containing free hydroxyl groups were trans-
formed into TMSi ethers by reaction with bis-(N,N-trim-
ethylsilyl)-trifluoroacetamide (BSTFA) in pyridine for
30 min at 70 °C; (2) hydroxyl groups were acetylated by
adding pyridine and Ac2O to the dried fraction, heating
the mixture at 70 °C for 5 min, and then keeping it at RT
overnight. The products were isolated by addition of
H2O and extraction with CHCl3; (3) the unknown constit-
uents were subjected to reduction by excess of LiAlH4 in
refluxing tetrahydrofuran overnight, hydrolysis with 10%
H2SO4, and extraction of the solution with CHCl3.
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hitherto unexplored macrocyclization strategies in the epothilone
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4.5. Synthesis of reference compound 5-hydroxyoctacosanal
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1,3-alkanediols and 3-hydroxylaldehydes in leaf cuticular waxes of
Ricinus communis L. Phytochemistry 62, 433–438.
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secondary alcohols and alkanediols in the leaf cuticular waxes of Pisum
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1-Tetracosanol (Sigma–Aldrich, Steinheim, Switzerland)
was oxidized into tetracosanal by pyridinium chlorochro-
mate at RT for 1 h in anhydrous CH2Cl2. Column purified
tetracosanal reacted with 3-butenylmagnesium bromide
(0.5 M solution in THF, Sigma–Aldrich, MO, USA) while
refluxing for 1 h. The reaction was quenched by addition of
water, and then extracted with CHCl3. 1-Octacosen-4-ol
was purified by column chromatography. 5-Hydroxyocta-
cosanal was then synthesized using cross metathesis fol-
lowed by oxidation as described by Njardarson et al.
(2002). Briefly, 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxa-
borolane (3 equiv., Sigma–Aldrich, MO, USA) and
Grubbs catalyst (0.1 equiv., Sigma–Aldrich, MO, USA)
were added to 1-octacosen-4-ol (1 equiv.) solution in
CH2Cl2, and the mixture was kept refluxing for 8 h. The
desired cross metathesis product was purified on TLC
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chemical composition of epicuticular wax crystals on needles of Taxus
baccata L. Phytochemistry 67, 1808–1817.