Synthesis of 1-octacosanol and GC-C-IRMS discrimination of samples from different origin

Article abstract of DOI:10.1080/14786410903194498

Lately, long-chain primary alcohols have been investigated in depth on account of their biological activities. In particular, 1-octacosanol (C ₂₈H₅₇OH), the main component of policosanol, the hypolipidaemic fatty alcohol mixture obtained from sugar cane wax, has been the subject of a multitude of pharmacological studies. The aim of this work was to search a convenient synthetic protocol for the preparation of 1-octacosanol in a gram scale. The key step was a Wittig reaction between the octadecyltriphenylphosphonium ylide and the methyl 10-oxodecanoate. Some steps were further improved by power ultrasound and microwave irradiation, either alone or in combination. Our methodology is suitable for a rapid generation of homologues by varying the chain length in the alkyl halide. Due to the high commercial value, a series of 1-octacosanol samples, either isolated from natural sources or from synthesis (different origin and suppliers), were analysed by gas chromatography-combustion-isotopic ratio mass spectrometry (GC-C-IRMS) and according to the carbon isotopic content, classified on the basis of their origin.

Full text of DOI:10.1080/14786410903194498

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Synthesis of 1-octacosanol and GC-C-
IRMS discrimination of samples from
different origin
Giancarlo Cravotto a , Emanuela Calcio Gaudino a , Alessandro
Barge a , Arianna Binello a , Andrea Albertino b & Costanza
Aghemo b
a Dipartimento di Scienza e Tecnologia del Farmaco , Università di
Torino , 10125, Torino, Italy
b Dipartimento di Chimica IFM , Università di Torino , 10125,
Torino, Italy
Published online: 17 Mar 2010.
To cite this article: Giancarlo Cravotto , Emanuela Calcio Gaudino , Alessandro Barge , Arianna
Binello , Andrea Albertino & Costanza Aghemo (2010) Synthesis of 1-octacosanol and GC-C-IRMS
discrimination of samples from different origin, Natural Product Research: Formerly Natural
Product Letters, 24:5, 428-439, DOI: 10.1080/14786410903194498
To link to this article: http://dx.doi.org/10.1080/14786410903194498
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Natural Product Research
Vol. 24, No. 5, 20 March 2010, 428–439
Synthesis of 1-octacosanol and GC-C-IRMS discrimination of
samples from different origin
a
a
a
*
Giancarlo Cravotto , Emanuela Calcio Gaudino , Alessandro Barge ,
Arianna Binelloa, Andrea Albertinob and Costanza Aghemob
a
`
Dipartimento di Scienza e Tecnologia del Farmaco, Universita di Torino, 10125 Torino,
Italy; Dipartimento di Chimica IFM, Universita di Torino, 10125 Torino, Italy
b
`
(Received 27 June 2009; final version received 20 July 2009)
Lately, long-chain primary alcohols have been investigated in depth on
account of their biological activities. In particular, 1-octacosanol
(C28H57OH), the main component of policosanol, the hypolipidaemic
fatty alcohol mixture obtained from sugar cane wax, has been the subject of
a multitude of pharmacological studies. The aim of this work was to search
a convenient synthetic protocol for the preparation of 1-octacosanol in
a
gram scale. The key step was a Wittig reaction between the
octadecyltriphenylphosphonium ylide and the methyl 10-oxodecanoate.
Some steps were further improved by power ultrasound and microwave
irradiation, either alone or in combination. Our methodology is suitable for
a rapid generation of homologues by varying the chain length in the alkyl
halide. Due to the high commercial value, a series of 1-octacosanol
samples, either isolated from natural sources or from synthesis (different
origin and suppliers), were analysed by gas chromatography-combustion-
isotopic ratio mass spectrometry (GC-C-IRMS) and according to the
carbon isotopic content, classified on the basis of their origin.
Keywords: octacosanol; synthesis; GC-C-IRMS; ultrasound; microwaves
1. Introduction
Since the 1980s, higher aliphatic primary alcohols have been a subject of intensive
research. Early work was mainly focused on the biological activities of 1-octacosanol
(C28H57OH) and 1-triacontanol (C30H61OH), isolated from germ oils and beeswax
(Houtz, Ries, & Tolbert, 1985; Norris, Denys, & Fallat, 1986; Saint-John &
McNaughton, 1986; Sho, Chinen, & Fukuda, 1984). It was reported that the
addition of sugarcane wax to the diet lowered the levels of plasma lipids in the rat,
although the authors did not attribute the effect to long-chain alcohols (Sho et al.,
1984). Later on, when the effects of 1-octacosanol on lipid metabolism were
investigated in mice submitted to exercise training, a reduction of hepatic lipid
(mainly triglycerides) was observed (Shimura, Hasegawa, Takano, & Suzuki, 1987).
*Corresponding author. Email: giancarlo.cravotto@unito.it
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410903194498
http://www.informaworld.com

Natural Product Research
429
It was only from the 1990s that a spate of results appeared, documenting the
cholesterol-lowering, anti-platelet and antioxidant effects of a substance named
policosanol (Hernandez et al., 1992; Mas, 2000). Policosanol was described by
´ ´
Cuban researchers as a mixture of eight higher aliphatic primary alcohols obtained
from the wax of sugarcane (Saccharum officinarum L.). It contained: 1-tetracosanol
(C24), 1-hexacosanol (C26), 1-heptacosanol (C27), 1-octacosanol (C28) – the most
abundant (60–70%), 1-nonacosanol (C29), 1-triacontanol (C30), 1-dotriacontanol
(C32) and 1-tetratriacontanol (C34). The relative abundance of these alcohols and
their total content defines the identity of policosanol. These mixtures are easily
analysed by gas chromatography (GC) and GC coupled with mass spectrometry
(GC-MS) (Sierra, Gonzalez, & Magraner, 2002). Although more work is needed for
´
a definitive conclusion to be reached, the overwhelming majority of clinical
investigations corroborate the dose-dependent efficacy of original policosanol as
an anti-lipidaemic agent (Viola, Oliaro, Binello, & Cravotto, 2008). Pure octacosanol
has been investigated as a possible treatment for Parkinsonism and amyotrophic
lateral sclerosis (Norris et al., 1986; Snider, 1984). It has also been used by athletes to
enhance their performance (Beltz & Doering, 1993; Voy, 1986; Woolley, 1991). Most
of the studies related to the pharmacokinetics and metabolism of octacosanol in
human and animal subjects were performed using radioactively labelled octacosanol
(Kabir & Kimura, 1993, 1994, 1995; Menendez et al. 1996). A direct absorption of
´
the fatty alcohol was shown in the mucosa cells without complete oxidation to the
corresponding fatty acid metabolite. More recently, a clinical study conducted by
Keller, Gimmler and Jahreis (2008) demonstrated that octacosanol decreases in the
concentration of faecal cholesterol end products that should be derived from a
systemic effect on cholesterol metabolism, even though the serum cholesterol levels
were not influenced. Octacosanol is commercially available and it can be obtained by
isolation from natural sources, i.e. by molecular distillation under a high vacuum
(Chen et al., 2007) or by synthesis (Cravotto, 2005). The present article describes a
new efficient method of synthesis of 1-octacosanol and a way to easily discriminate
samples coming from different natural sources, place of production, manufacturing
protocol or starting materials for the synthesis by means of isotopic ratio mass
spectrometer (IRMS).
2. Results and discussion
The present article describes an efficient synthetic procedure to obtain 1-octacosanol
in gram scale. Some critical steps were improved by irradiation with power
ultrasound and/or microwaves, alone or combined. In particular, the formation of
the diol by epoxide opening, as described in a previous paper (Palmisano et al.,
2007), was rapidly achieved by simultaneous irradiation (Scheme 1). Undecylenic
acid and stearyl bromide are commercially available cheap starting materials that
make the synthesis very attractive for industrial application (Cravotto, 2005). All the
steps promoted by US or MW irradiation may also be performed under conventional
conditions, though with slightly lower yields and longer reaction times. The key step
was a Wittig reaction between an octadecyltriphenyl phosphonium ylide and
a methyl 10-oxodecanoate, the two building blocks of the long-chain monofunctio-
nalised target molecule. In agreement with a flow-chart study of our synthetic

430
G. Cravotto et al.
OH
O
NaOH, H2O
MCPBA
CH2Cl2
H2C CH(CH2)8COOH
H2C CH(CH2)8COOH
H2C CH(CH2)8COOH
US/MW
55°C, 15 min
1
2
OH
NaIO4
2
MeOH, TMOF
OHC(CH2)8COOH
OHC(CH2)8COOCH3
Dioxane/H2O
MW 40W, 5 min
3
4
80 min, US
TPP
BuLi
CH3(CH2)16CH2Br
CH3(CH2)16CH2PPh3 Br
CH3(CH2)16CH PPh3
Toluene rfx
THF
6
5
THF
6
OHC(CH2)8COOCH3
CH3(CH2)16CH CH(CH2)8COOCH3
4
7
(cis, trans)
LiAlH4
H2 30 bar, Pd/C
70°C, THF
7
CH3(CH2)16CH
CH(CH2)8CH2OH
CH3(CH2)26CH2OH
THF, rt
9
8
Scheme 1. Synthesis of 1-octacosanol.
process, it emerged that procedure and costs were compatible with an industrial
scale up.
Direct gas chromatography-combustion-isotopic ratio mass spectrometry
(GC-C-IRMS) analysis, without any derivatisation of the samples, afforded ꢀ13C
data reported in Table 1 with standard deviations. 13C data of octacosanol samples
were ranging in a wide interval, between ꢀ16 and ꢀ36%. Each value resulted as an
average of four runs (SD 5 2%). All ꢀ 13C data versus PDB are plotted with error
bars in Figure 1.
Octacosanol samples prepared by synthesis in two different laboratories (Batch 1
and 2) using starting materials from different suppliers showed 13C deviations close
to each other, but with significantly different mean values: ꢀ29.7% for Batch 1 and
ꢀ32.5% for Batch 2.
The ꢀ13C values of the industrial samples vary (ꢀ29% for Sigma-Aldrich and
higher for Giellepi, up to ꢀ17%), proving a different origin of the starting material.
In fact, octacosanol samples provided by Giellepi showed that 13C figures are not
significantly different from natural Cuban samples (Dalmer sa.), conclusive evidence
for the same natural source (i.e. sugarcane wax) of the samples. 1-Octacosanol can be
isolated from the wax of sugarcane (Saccharum officinarum, L.) after hydrolysis and
purification. Samples have a content of 1-octacosanol ranging from 70 to 95%.
Under our GC experimental conditions, the C28 alcohol was easily separated from
other fatty alcohols present in low amounts (mainly C26 and C30). Surprisingly,
Indian octacosanol samples of natural origin (claimed to be from sugar cane wax)
showed a considerably different content of 13C compared to the Cuban ones. This
fact is supporting evidence for a rather different pedo-climatic environment in which
the plants grew. Standard deviations are lower than 0.5% for industrial, natural
samples and the product coming from Batch 1; octacosanol samples from Batch 2
showed less precise figures, with standard deviation values around 1%.

Natural Product Research
431
Table 1. ꢀ 13C values with standard deviations for octacosanol samples
from industrial, natural and synthetic sources.
Sample identifier
Origin of samplea
ꢀ13C vs. PDB
1
2
3
4
5
6
7
8
9
Sigma–Aldrich
Sigma–Aldrich
Sigma–Aldrich
Sigma–Aldrich
Sigma–Aldrich
Sigma–Aldrich
Sigma–Aldrich
Sigma–Aldrich
Giellepi
Giellepi
Giellepi
Giellepi
Giellepi
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
S
S
S
S
S
S
S
S
S
S
S
S
S
S
(ꢀ29.24 ꢁ 0.04)
(ꢀ29.04 ꢁ 0.03)
(ꢀ29.05 ꢁ 0.06)
(ꢀ29.09 ꢁ 0.06)
(ꢀ29.46 ꢁ 0.07)
(ꢀ29.38 ꢁ 0.09)
(ꢀ29.46 ꢁ 0.33)
(ꢀ29.21 ꢁ 0.18)
(ꢀ16.76 ꢁ 0.02)
(ꢀ16.70 ꢁ 0.09)
(ꢀ17.08 ꢁ 0.17)
(ꢀ16.96 ꢁ 0.17)
(ꢀ16.84 ꢁ 0.16)
(ꢀ16.87 ꢁ 0.14)
(ꢀ17.13 ꢁ 0.15)
(ꢀ17.13 ꢁ 0.15)
(ꢀ17.06 ꢁ 0.02)
(ꢀ16.90 ꢁ 0.08)
(ꢀ16.78 ꢁ 0.08)
(ꢀ16.72 ꢁ 0.06)
(ꢀ16.93 ꢁ 0.47)
(ꢀ17.34 ꢁ 0.27)
(ꢀ16.86 ꢁ 0.04)
(ꢀ16.89 ꢁ 0.20)
(ꢀ36.42 ꢁ 0.01)
(ꢀ36.59 ꢁ 0.03)
(ꢀ36.88 ꢁ 0.03)
(ꢀ36.70 ꢁ 0.06)
(ꢀ36.47 ꢁ 0.17)
(ꢀ36.78 ꢁ 0.26)
(ꢀ36.55 ꢁ 0.30)
(ꢀ36.39 ꢁ 0.06)
(ꢀ30.21 ꢁ 0.19)
(ꢀ29.90 ꢁ 0.15)
(ꢀ29.52 ꢁ 0.17)
(ꢀ29.88 ꢁ 0.17)
(ꢀ29.44 ꢁ 0.04)
(ꢀ29.51 ꢁ 0.22)
(ꢀ33.12 ꢁ 0.52)
(ꢀ32.99 ꢁ 0.45)
(ꢀ32.48 ꢁ 0.53)
(ꢀ32.16 ꢁ 0.55)
(ꢀ32.35 ꢁ 0.28)
(ꢀ32.23 ꢁ 0.32)
(ꢀ32.18 ꢁ 0.32)
(ꢀ32.45 ꢁ 0.20)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Giellepi
Giellepi
Giellepi
SCW (Cuba)
SCW (Cuba)
SCW (Cuba)
SCW (Cuba)
SCW (Cuba)
SCW (Cuba)
SCW (Cuba)
SCW (Cuba)
SCW (India)
SCW (India)
SCW (India)
SCW (India)
SCW (India)
SCW (India)
SCW (India)
SCW (India)
Batch 1
Batch 1
Batch 1
Batch 1
Batch 1
Batch 1
Batch 2
Batch 2
Batch 2
Batch 2
Batch 2
Batch 2
Batch 2
Batch 2
a
Note: Industrial (I), natural (N) or synthetic (S) origin.

432
G. Cravotto et al.
Figure 1. ꢀ13C for samples vs. PDB plotted with error bars: Sigma–Aldrich (g), Cuba (?),
India (f), Giellepi (^), Batch 1 (p), Batch 2 ( ).
Owing to the high commercial value of 1-octacosanol and its unique biological
properties, an easy access to this fatty alcohol could stimulate further investigations
for its definitive acceptance as food supplement or drug. Moreover, our
GC-C-IRMS method enables the discrimination of octacosanol samples origin
from different manufacturers and geographical areas.
3. Experimental
3.1. Materials and methods
All solvents used were of analytical reagent grade and were purified according to the
published procedures (Amarengo & Perrin, 1998). Starting materials, reagents and
standards (octadecane) were purchased from Sigma–Aldrich, Fluka and Alpha-
Aesar.
For simultaneous MW/US irradiation, the professional oven (Microsynth
Milestone – Sorisole, BG) was equipped with a US probe featuring a pyrex horn
(frequency 21.4 kHz) (Danacamerini – Torino).
IR spectra were recorded with a Shimadzu FT-IR 8001 spectrophotometer.
NMR spectra were recorded with two Bruker Avance spectrometers: 300 MHz and
600 MHz at 300 K; chemical shifts were calibrated to the residual proton resonances
of the solvent: CDCl3 (ꢀH ¼ 7.26).
Low-resolution mass spectra were recorded on a Finnigan-MAT TSQ70 in
electron impact (EI) and chemical ionisation (CI) with isobutane as reactant gas;
ESI-mass spectra were recorded on a Waters Micromass ZQ equipped with
ESI source.

Natural Product Research
433
3.2. GC-C-IRMS analysis
Until now, GC has been commonly used to analyse policosanols and other fatty
alcohol mixtures with and without derivatisation. Most of the derivatisation
methods employed N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) as silyla-
tion reagent. Unfortunately, for the purpose of this work, this procedure adds a
relatively high number of carbon atoms to the molecule, which is a complication for
IRMS analysis. Recently, some researchers developed GC methods for policosanol
analysis that did not involve any derivatisation (Wang et al., 2007). Overall, carbon
isotopic ratios were measured with a Trace GC Ultra (Thermo, Italy) equipped with
a 95% polydimethylsiloxane capillary column (50 m ꢂ 0.2 mm i.d., 0.33 mm coating
thickness). The results were expressed in ꢀ13C %, corresponding to:
13C=12Csample
ꢀ
13 C=12Cstd
1000 ꢂ
,
13C=12Cstd
where the subscript suffix std refers to the international carbonate standard V-PDB
(Coplen, 1994, 1995).
GC-C-IRMS analyses were performed as follows. Octacosanol solutions used for
injection were prepared by dissolving 10 mg of product in chloroform (10 mL) in the
presence of octadecane (10 mg) as internal standard. Samples were injected at 250ꢃC
with a split ratio of 1 : 10. After 2 minꢀ, 1at 150ꢃC (initial temperature of the oven) the
temperature was ramped at 25ꢃC min up to 300ꢃC and maintained for 6 min in an
isotherm. Helium was used as the carrier gas and CO2 as the reference. The effluent
from the column was combusted at 940ꢃC in an alumina tube (0.55 mm inner
diameter, 1.55 mm outer diameter, 320 mm long), which acts as an oxidation reactor.
The tube contains three wires made of CuO, NiO and Pt, respectively (all 240 mm
long and 0.125 mm thick). Nitrogen oxides were reduced to N2 at 640ꢃC in a
reduction furnace made by three twisted copper wires of 0.125 mm diameter.
A hygroscopic NaphionÕ membrane, situated just before the spectrometer,
eliminated excess water, avoiding CO2 protonation and the consequent formation
of HCOþ2 (m/z ¼ 45).
Each sample was analysed three times and the results were averaged. The CO2
reference was calibrated using alkane mixtures supplied by Arndt Schimmelmann
of Indiana University with certified ꢀ13C values against V-PDB. The ꢀ13C of 15 linear
alkanes, ranging from C16 to C30, were calculated by Schimmelmann’s method
according to Coplen et al.’s (2006) method. Figure 2 reports the GC-C-IRMS spectra
of all 1-octacosanol samples.
3.3. Samples
As many as six different sets of samples of octacosanol (70–98%) of various origins
were investigated. Two industrial sets were supplied by Sigma–Aldrich and by
Giellepi Chemicals; two sets were natural wax, extracted from the sugar cane
produced in different geographical areas (Cuba, India) and two sets were obtained by
synthesis from two different batches. With the aim of checking the repeatability of
the synthetic protocol, the synthesis was performed in two different laboratories
(Batch 1 and 2), beginning from the starting material of different suppliers.

434
G. Cravotto et al.
Figure 2. GC-C-IRMS spectra of 1-octacosanol samples from Sigma–Aldrich (a), octacosanol
from Cuba (b), octacosanol from India (c), octacosanol from Giellepi (d), octacosanol from
synthesis Batch 1 (e) and Batch 2 (f ). Peak A represents the internal standard (octadecane),
peak B indicates the polialcohols present in smaller percentages than octacosanol and peak C
represents octacosanol.

Natural Product Research
435
3.4. Synthetic and purification procedures
The synthetic protocol is depicted in Scheme 1; some steps have been dramatically
improved by means of non-conventional techniques, such as power ultrasound
and microwaves alone or combined. Crude products were usually purified by flash
chromatography (direct phase) using the CombiFlash Rf system (Teledyne-Isco) with
hexane/ethyl acetate mixtures as eluent.
3.4.1. Synthesis of 9-(oxiran-2-yl)nonanoic acid (1)
In a 100 mL round-bottomed flask placed in an ice bath, undecenoic acid (1.8 g,
9.8 mmol) and dichloromethane (30 mL) were added. To the stirred solution (0ꢃC),
m-chloroperbenzoic acid (MCPBA, 2.7 g, 15.68 mmol) was added portionwise. The
reaction mixture was stirred for 4 h at room temperature and monitored by TLC
(hexane : ethyl acetate 7 : 3 þ 0.5% trifluoroacetic acid as eluent). The mixture was
then diluted with dichloromethane (10 mL), washed with water (15 mL, three times)
and brine (40 mL), then dried over sodium sulphate. The crude product was purified
by flash chromatography using a hexane : ethyl acetate (9 : 1) mixture as eluent.
Compound 1 was obtained as a colourless oil (1.9 g, 97% yield). FT-IR (KBr):
ꢁ (cmꢀ1) 3502, 2914, 1705, 1469, 1433, 1323, 912, 844, 721; 1H-NMR (CDCl3,
300 MHz) ꢀ 2.92 (1H, m, H-10), 2.65 (1H, m, HA-11), 2.48 (1H, m, HB-11), 2.37
(2H, t, J ¼ 7.5 Hz, H-2), 1.65 (2H, m, H-3), 1.57 (2H, m, H-9), 1.42 (10H, m,
H-4, 5, 6, 7, 8). CIMS 201 [M þ 1]þ [C11H20O3 þ H].
3.4.2. Synthesis of 10,11-dihydroxyundecanoic acid (2)
In a 250 mL two-necked conic pyrex flask equipped with an optic-fibre thermometer,
compound 1 (1.9 g, 9.5 mmol) and 0.2 N NaOH (90 mL) were added, and
simultaneously irradiated with ultrasound and microwaves (US/MW) (Cravotto &
Cintas, 2007; Palmisano et al., 2007) for 15 min at 55ꢃC (power: 20 and 50 W
respectively). Reaction outcome was checked by TLC with hexane : ethyl acetate
7 : 3 þ 0.5% trifluoroacetic acid as eluent. The reacted mixture was cooled to room
temperature and brought to pH 5 with 2 N HCl and a whitish amorphous solid was
precipitated and collected on a paper filter, then the filtrate was extracted with
dichloromethane to recover the residual product. 1.64 g of pure 2 were obtained
(79% yield). FT-IR (KBr): ꢁ (cmꢀ1) 3584, 2926, 2361, 2341, 1738, 1713, 1265, 1076,
723. 1H-NMR (CDCl3, 300 MHz) ꢀ 4.36 (2H, br, OH), 3.70 (1H, m, H-10), 3.63
(1H, m, HA-11), 3.49 (1H, m, HB-11), 2.36 (2H, t, J ¼ 7.2 Hz, H-2), 1.66 (2H, m, H-9),
1.53 (2H, m, H-3), 1.45 (2H, m, H-8), 1.27 (8H, m, H-4, 5, 6, 7). CIMS 219 [M þ 1]þ
[C11H22O4 þ H].
3.4.3. Synthesis of 10-oxodecanoic acid (3)
To a solution of 2 (1.5 g, 6.9 mmol) in dioxane : water 3 : 1 (60 mL), sodium periodate
(3 g, 13.8 mmol) was added and the mixture was sonicated in a cavitating tube
(19.2 kHz, 80 W) (Cravotto et al., 2008) at room temperature for 80 min. The
reaction was monitored by TLC with hexane : ethyl acetate 7 : 3 þ 0.5% trifluor-
oacetic acid as eluent. The residue was filtered off on a Buckner flask with a sintered
glass. The filtrate was concentrated at 50% of the volume under vacuum, poured

436
G. Cravotto et al.
into a separatory funnel and extracted with dichloromethane (3 ꢂ 20 mL), washed
with water (2 ꢂ 60 mL) and dried over sodium sulphate. The solvent was removed
under vacuum and the crude product was purified by flash chromatography using
hexane : ethyl acetate 9 : 1 as eluent, affording 3 as a yellowish solid (1.08 g, 84%
yield). FT-IR (KBr): ꢁ (cmꢀ1) 3321, 2928, 2675, 1716, 1464, 1242, 1047, 943, 725. 1H-
NMR (CDCl3, 300 MHz) ꢀ 9.74 (1H, t, J ¼ 1.8 HZ, –CHO), 2.40 (2H, td, Jt ¼ 8.1 HZ,
Jd ¼ 1.8 HZ, H-9), 2.31 (2H, t, J ¼ 7.5 HZ, H-2), 1.56 (2H, m, H-8), 1.42 (2H, m, H-7),
1.29 (8H, m, H-3, 4, 5, 6). CIMS 187 [M þ 1]þ [C10H18O3 þ H].
3.4.4. Synthesis of methyl 10-oxodecanoate (4)
To a solution of 3 (1.08 g, 5.8 mmol, 1 eq) in methanol (15 mL), trimethylorthofor-
mate (0.6 g, 5.7 mmol, 1 eq) and methansulfonic acid (10 mg) were added and the
solution was irradiated 15 min with MW (50ꢃC, 30 W). The reaction was checked by
TLC with hexane : ethyl acetate 7 : 3 þ 0.5% trifluoroacetic acid as eluent. The
solvent was removed under vacuum, the product was dissolved in dichloromethane
(40 mL) and the organic phase was washed with 5% HCl (2 ꢂ 30 mL), 5% sodium
bicarbonate and brine. The methyl ester containing traces of methyl acetal (less than
2%) was purified by flash chromatography using hexane : ethyl acetate 19 : 1 as
eluent, which afforded 4 as a yellow oil: (1.03 g, yield 89%); FT-IR (KBr): ꢁ (cmꢀ1
)
1
2928, 2675, 1745, 1464, 1242, 1047, 943, 725. H-NMR (CDCl3, 300 MHz) ꢀ 9.74
(1H, t, J ¼ 1.8 Hz, –CHO), 3.60 (3H, s, –OCH3), 2.40 (2H, td, Jt ¼ 8.1 Hz, Jd ¼ 1.8 Hz,
H-9), 2.31 (2H, t, J ¼ 7.5 Hz, H-2), 1.56 (2H, m, H-8), 1.42 (2H, m, H-7), 1.29
(8H, m, H-3, 4, 5, 6). CIMS 201 [M þ 1]þ [C11H20O3 þ H].
3.4.5. Synthesis of octadecyltriphenylphosphonium bromide (5)
To a 100 mL round-bottomed flask containing stearyl bromide (1 g, 2.9 mmol, 1 eq)
dissolved in 25 mL of anhydrous toluene, triphenylphosphine (0.78 g, 2.9 mmol, 1 eq)
was added. The solution was heated under reflux overnight in N2 atmosphere.
After cooling at 0ꢃC and adding ethyl ether (30 mL), the phosphonium salt was
precipitated and it was collected on a paper filter. Compound 5 was obtained as
a whitish product (1.4 g, 81% yield), which was immediately used for the Wittig
reaction.
1H-NMR (CDCl3, 300 MHz) ꢀ 7.85 (15H, m, –ArH), 1.65 (4H, m, H-1, 2), 1.24
(30H, m, H-3-17), 0.87 (3H, t, J ¼ 6.4 Hz, H-18).
3.4.6. Synthesis of (E,Z)-methyl octacos-10-enoate (7)
Phosphonium salt 5 (1.19 g, 2.0 mmol) was dissolved under N2 atmosphere in THF
(20 mL) and the mixture was cooled to 0ꢃC. Butyllithium (1.6 M, 1.2 eq) was added
and the red solution was stirred at room temperature for 15 min. The mixture
of the phosphorus ylide (6) was again cooled to 0ꢃC and a solution in THF (5 mL)
of the aldehyde 4 (0.4 g, 2.0 mmol) was added dropwise and stirred for 2 h at
room temperature. The reaction was checked by TLC using hexane:ethyl acetate 7 : 3
as eluent. The reacted mixture was cooled in ice bath and a mixture of diethyl ether
(20 mL) and 5% HCl (10 mL) was added. The organic layer was washed with
5% HCl (20 mL), water (30 mL), 5% sodium bicarbonate and brine, then dried over

Natural Product Research
437
sodium sulphate, filtered on a paper filter and evaporated under vacuum. The
yellow-orange oil was purified by flash chromatography using hexane : ethyl acetate
19 : 1 as eluent, affording compound 7 in good yield as a yellow oil (0.72 g, 82%).
1
FT-IR (KBr): ꢁ (cmꢀ1) ¼ 2926, 1740, 1466, 1180, 1037, 742, 721, 669. H-NMR
(CDCl3, 300 MHz) ꢀ 5.37 (2H, m, H-10, 11), 3.50 (3H, s, –COOCH3), 2.50
(2H, m, H-2), 2.01 (4H, m, H-9, 12), 1.30 (42H, m, H-3-8, 13-27), 0.89 (3H, t,
J ¼ 6.4 Hz, H-28). CIMS 437 [M þ 1]þ [C29H56O2 þ H].
3.4.7. Synthesis of (E,Z)-octacos-10-en-1-ol (8)
In a 50 mL two-necks round-bottomed flask, equipped with a nitrogen inlet, LiAlH4
(164 mg, 4.3 mmol, 3 eq) was dissolved in THF (8 mL) and was cooled to 0ꢃC.
A solution of 7 (636 mg, 1.44 mmol) in THF (10 mL) was added dropwise. After
the addition was completed, the mixture was kept at room temperature and stirred
for 2 h and monitored by TLC using hexane : ethyl acetate 7 : 3 as eluent. After the
reaction was completed, 5% HCl (30 mL) was added and extracted with diethyl ether
(2 ꢂ 20 mL). The organic phase was extracted twice with brine, dried over sodium
sulphate, filtered with a paper filter and evaporated under vacuum. The crude
product was purified by flash chromatography using hexane : ethyl acetate 19 : 1
mixture as eluent. Compound 8 was obtained as a white solid (529 mg, 90% yield).
FT-IR (KBr): ꢁ (cmꢀ1) ¼ 3500, 2926, 1471, 1292, 964, 891, 729, 648. 1H-NMR
(CDCl3, 300 MHz) ꢀ 5.37 (2H, m, H-10, 11), 3.63 (2H, t, J ¼ 6.6 Hz, H-1), 2.01
(4H, m, H-9, 12), 1.57 (2H, m, H-2), 1.47 (1H, br, OH), 1.28 (42H, m, H-3-8, 13-27),
0.89 (3H, t, J ¼ 6.3 Hz, H-28). CIMS 409 [M þ 1]þ [C28H56O þ H].
3.4.8. Synthesis of octacosan-1-ol (9)
To a solution of octacos-11-en-1-ol (400 mg, 0.98 mmol) in THF (40 mL), 10% of Pd
on charcoal (50 mg) was added and poured into a Parr reactor. The reduction was
carried out at 80ꢃC and 35 bar H2 under stirring for 4 h. Similar results were achieved
when the reaction was performed in a special US-reactor (21.2 kHz) that works
under pressure (Danacamerini – Torino); the reaction was carried out at 40ꢃC and
6 bar H2 for 3 h. In both cases, after filtration on a celite pad and evaporation under
vacuum, a crude product was purified by flash chromatography using hexane : ethyl
acetate 19 : 1 as eluent. Compound 9 was obtained as a white powder (358 mg, 89%
1
yield). FT-IR (KBr): ꢁ (cmꢀ1) ¼ 3500, 2923, 1122, 889, 731, 719, 669. H-NMR
(CDCl3, 300 MHz) ꢀ 3.54 (2H, t, J ¼ 6.6 Hz, H-1), 1.57 (2H, m, H-2) 1.46 (5H, m,
H-3, 4, OH), 1.29 (46H, m, H-5-27), 0.88 (3H, t, J ¼ 6.3 Hz, H-28). CIMS 411
[M þ 1]þ [C28H58O þ H].
Acknowledgements
Financial support from the University of Torino is gratefully acknowledged. We are indebted
to Prof. Marco Vincenti and Dr Alberto Salomone for the IRMS analyses at the CAD
(Orbassano, Turin).

438
G. Cravotto et al.
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