Identification and synthesis of new sex-specific components of olive fruit fly (Bactrocera oleae) female rectal gland, through original Negishi reactions on supported catalysts

Article abstract of DOI:10.1016/j.tet.2018.07.003

In the present study, eleven new sex-specific components extracted from female rectal gland of olive fruit flies were synthesized and identified. The quantitative determination of those components by GC and GC/EI-MS, at different moments of the insect life span, highlighted the growing trend of their secretion. While for the synthesis of saturated esters, conventional transesterification methods could be adopted, for the synthesis of unsaturated components, a Negishi cross-coupling between organozinc halides and (Z)-1-bromo-1-alkenes was developed. To the extent of our knowledge, this reaction represents the first example of supported-catalyst promoted Negishi coupling, between an alkylzinc reagent and an alkenyl halide.

Full text of DOI:10.1016/j.tet.2018.07.003

Accepted Manuscript
Identification and synthesis of new sex-specific components of olive fruit fly
(Bactrocera oleae) female rectal gland, through original Negishi reactions on
supported catalysts
Graziano Fusini, Davide Barsanti, Gaetano Angelici, Gianluca Casotti, Angelo
Canale, Giovanni Benelli, Andrea Lucchi, Adriano Carpita
PII:
S0040-4020(18)30794-4
10.1016/j.tet.2018.07.003
TET 29663
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 24 April 2018
Revised Date: 28 June 2018
Accepted Date: 2 July 2018
Please cite this article as: Fusini G, Barsanti D, Angelici G, Casotti G, Canale A, Benelli G, Lucchi A,
Carpita A, Identification and synthesis of new sex-specific components of olive fruit fly (Bactrocera
oleae) female rectal gland, through original Negishi reactions on supported catalysts, Tetrahedron
(2018), doi: 10.1016/j.tet.2018.07.003.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
Identification and synthesis of new sex
specific components of olive fruit fly
(Bactrocera oleae) female rectal gland,
through original Negishi reactions on
supported catalysts

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Identification and synthesis of new sex-specific components of olive fruit fly
(
Bactrocera oleae) female rectal gland, through original Negishi reactions on
supported catalysts
a
a
a, 1
a
b
Graziano Fusini , Davide Barsanti , Gaetano Angelici ∗ , Gianluca Casotti , Angelo Canale , Giovanni
b
b
a, 2
Benelli , Andrea Lucchi and Adriano Carpita ∗
a
Department of Chemistry and Industrial Chemistry,
University of Pisa, via G. Moruzzi 13, 56124 Pisa, Italy
b
Department of Agriculture, Food and Environment,
University of Pisa, via del Borghetto 80, 56124 Pisa, Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
In the present study, eleven new sex-specific components extracted from female rectal gland of
olive fruit flies were synthesized and identified. The quantitative determination of those
components by GC and GC/EI-MS, at different moments of the insect life span, highlighted the
growing trend of their secretion. While for the synthesis of saturated esters, conventional
transesterification methods could be adopted, for the synthesis of unsaturated components, a
Negishi cross-coupling between organozinc halides and (Z)-1-bromo-1-alkenes was developed.
To the extent of our knowledge, this reaction represents the first example of supported-catalyst
promoted Negishi coupling, between an alkylzinc reagent and an alkenyl halide.
Available online
Keywords:
Olive Fruit Fly
Negishi coupling
Organozinc compounds
Supported catalysis
Pheromones synthesis
2
009 Elsevier Ltd. All rights reserved.
—
——
∗
Corresponding authors. Tel.: +39-050-2219281; fax: +39-050-2220673; e-mail: gaetano.angelici@unipi.it
Tel. +39 050 2219275; fax: +39 050 2219260; e-mail: adriano.carpita@unipi.it

2
Tetrahedron
1
. Introduction
the most classic Wittig olefination. For example, Murata et al. in
ACCEPTED MANUSCRIPT
2
the efforts to obtain [C6- H ]-dioleoylphosphatidylcholine,
2
1
.1. Olive fruit fly (Bactrocera oleae) control
synthesised ethyl (Z)-tridec-4-enoate, through a Wittig reaction
18
with 50% yield. Many other authors described similar results
with a major formation of the Z-alkene. Notably, Darwish
reported the preparation of conformationally pure deuterated (Z)-
Since Plato’s sacred olive tree stood up over the most famous
academic institutions, the olive fruit fly, Bactrocera oleae (Rossi)
Diptera: Tephritidae), is known as the major pest of olive crops
(
1
9
1
methyl oleate via the Wittig reaction, with 60% yield. Another
strategy is the well-known and generally efficient, stereoselective
catalytic hydrogenation of the corresponding alkyne. The alkyne,
however, must be previously synthesised, through the formation
of Lithium acetylides, like shown by Miyoshi in the synthesis of
worldwide. This invasive pest, widely known in the
Mediterranean region, threated California olive crops as well.
2
The substantial economic impact of the damage losses of fruits
and oil quality is dealt adopting different control strategies,
including3 insecticide-based control (e.g., dimethoate and
20
4
some cardiolipins, or by Kronenberg for the synthesis of natural
fenthion), biological control using natural enemies, sterile
21
5
6
7
Sphingomonas glycolipids. Such step is not compatible with the
presence of ester functions, so in our case, we would have needed
some extra-steps for the protection of the functional group. We
evaluated more appealing, for our purpose, an approach based on
cross-coupling reactions catalysed by transition metals,
especially for the possibility of improving convenience and
greenness of the method, by using heterogeneous catalysts. It is
indeed largely recognized that supported catalysts, even though
generally requiring harsher conditions, can be re-used, presenting
insect techniques, attraction to baits lured with colours and
pheromones. A pesticide-free approach would be of course the
most appealing, for many reasons, including the risk for human
and environmental health, not to mention the insurgence of
pesticide resistance. In our group, in the past years, we focused
mainly on the development of semiochemicals-based control
approaches. This standpoint requires a deep knowledge of the
exact chemical components of sex-specific pheromones, at
different life age and conditions of the insects. The story of the
discovery of olive fruit fly sex-specific pheromones is complex
and sometimes controversial. It is well-known that B. oleae
females attract conspecific males by using racemic 1,7-
dioxaspiro[5,5]undecane (olean),
monitoring the insect with different strategies (lure and kill or
pheromone-based mating disruption), leading to fluctuating
results. The failure of those preliminary tests on the guided
control against B. oleae, could be attributed to the lack of
knowledge on its intraspecific chemical communication. In our
efforts to solve this issue, in the past decade, we identified (Z)-9-
8
9,10
2
2
as well, the advantage to avoid expensive purifications.
Nevertheless, for the synthesis of biologically interesting
molecules, the use of supported-catalysts to promote cross-
coupling reactions, are still limited. In literature, there are several
interesting examples of Csp3-Csp2 Suzuki couplings promoted by
homogeneous catalysts, for the selective synthesis of stereo
11
which was used for
23
12
chemically pure (Z) ω-alkenoates. The same Suzuki showed the
alkylation reaction through boronic esters on bromo-acrylate and
3
-bromo-5,5-dimethylcyclohex-2-en-1-one,
with
60-70%
24
yields. Using an excess of Ag O, the group of Falck improved
the reaction between2 boronic acids and alkyl and aryl
electrophiles additive. Furthermore, adding triphenylarsine,
2
1
3,14
tricosene (muscalure) as male-produced female attractant,
nine carboxylic esters as female-produced components, two of
and
5
15
Bellina et al. could synthesise unsymmetrically disubstituted 3,4-
dialkyl-2(5H)-furanones. Interestingly, Santelli et al. exploiting
a less air-sensitive bidentate complex PdCl(dppb)(C H ) as pre-
catalyst, and Cs CO as base, could achieve Csp3-C coupling,
with moderate catalyst loading (1-2 mol%) and without the use of
expensive Ag O. However, many authors claim that alkyl
boronic acids are difficult to handle, as a result of their instability
in the air. Moreover, catalyst loadings are generally too high (1-
which with intraspecific semiochemical activity. However,
from GC/MS chromatograms of female rectal gland extracted,
eleven components were not unequivocally identified and
quantified, because relating standards were not commercially
available. From the fragmentation analysis of those components
26
3
5
2
3
sp2
27
(see TIC images in Supporting Information) and from the
2
correspondence of their mass spectra in the most common
databases (Wiley Registry of Mass Spectral Data and NIST02
Mass Spectral Library) we speculated the presence of four
generic saturated butyl esters (decanoate, dodecanoate,
tetradecanoate, hexadecanoate), two hexadecenoate methyl
esters, two hexadecenoate ethyl esters, two octadecenoate methyl
esters and one octadecenoate ethyl ester. To confirm our
hypothesis, we synthesized the corresponding molecules,
developing as well, a new methodology for supported-catalyst
promoted Negishi coupling, between an alkyl electron donor and
an alkenyl electron acceptor.
1
0 mol%) and there are still no examples in literature of Suzuki
cross-coupling reactions between alkenyl halides and alkyl-boron
species, catalysed by supported catalyst. We estimated more
convenient an approach based on Negishi cross-coupling
reactions, between organo-zinc compounds and alkenyl halides,
like described in the retrosynthetic scheme reported on Scheme 1.
2
8
1
.2. Choice of synthetic strategies
For the synthesis of saturated esters, we chose a convenient
esterification and transesterification method proposed by Kaimal
16
et al., using catalytic amount of molecular iodine and an excess
of the desired alcohol. The method is simple, mild and non-toxic,
generally leading to high yields, regardless of potentially
sensitive functional groups present in the molecule. For the
synthesis of monounsaturated esters, we focused on synthetic
strategies which lead mainly to the formation of (Z)-olefines, as
in nature, fatty acids are mainly present in cis conformation, with
some rare exceptions. Furthermore, it is essential to obtain high
stereoisomeric purities, because even small amount of the
undesired isomers could bias the bioactivity results of pheromone
components. Several methods, for the synthesis of (Z)-
monounsaturated esters are available in literature, starting from
Scheme 1. Retrosynthetic approach of the Negishi cross-coupling
reaction
Even though the preparation of organozinc halides through
classic metal-metal exchange is not compatible with the presence
of ester functions, it is possible to use a direct oxidative addition
17
29
of Zn (0). Knochel et al., for example, prepared organozinc
halides from alkyl-iodides, activating Zinc powder with 1,2-
30
dibromoethane and trichloromethylsilane. Alternately, on alkyl-
bromide, Hou et al. used moleculare iodine and N,N-

3
3
1
a
Product purified by low pressure fractional distillation
Product purified by flash chromatography
dimethylacetamide. Knochel and other author
A
s a
C
dd
C
ed
E
L
P
iC
T
l
E
sa
D
lt, MANUSCRIPT
b
to remove from the metal surface, the already formed organozinc
Generally, Negishi reactions can be performed
easily with low-cost catalysts, like Pd(PPh ) or PdCl (PPh ) in
32,33,34
compound.
2
.2.2. Preparation of organozinc halides
3
4
2
3 2
THF, and as showed by Tamaru et al., it is possible to synthesise
As mentioned in section 1.2, transmetallation is not a
stereo- specifically, both cis and trans olefins, with high purity
convenient method for our organozinc intermediates, due to the
high reactivity of ester functional groups to organometallic
species. A suitable approach is the direct oxidative addition of Zn
(0), which has been described and improved by Knochel in
35
and good yields. Due to its efficiency, Negishi coupling was
used also in the synthesis of rather complex molecules, like (-)-
36
37
rasfonin, and mycestericin A. Most relevant, for our needs,
was the study of Lipshutz et al. on the stereo chemistry of the
32
2006, adding 1 equivalent of LiCl and 2 equivalents of Zinc
powder, previously activated with 10 mol% of 1,2-
dibromoethane and 1 mol% of trimethylsilyl chloride (TMSCl).
We used the Knochel method on (Z)-1-bromohex-1-ene (5),
testing different conditions, to obtain 5-methoxycarbonyl-1-
pentylzinc bromide (7), as shown in Table 3. The use of LiCl was
indeed necessary for the reaction, which otherwise would not
happen. Anyway, considering the long reaction time of 44 h, to
obtain a complete conversion, we increased the amount of 1,2-
dibromoethane up to 20 mol% and TMSCl up to 5 mol%, added
in two portions as described in the experimental section,
shortening the reaction time to 24 h. Noteworthy, in all the 3
conditions tested in Table 2, we observed traces of methyl 6-
chlorohexanoate as byproduct. With the optimized conditions,
38
cross coupling with (Z)-alkenyl halides, which strongly depends
on the catalyst ligand. It was indeed reported that excellent
results, in terms of selectivity and yields, are reached using
PdCl (PPh ) /TMEDA, with only 1 mol% catalyst loading. Even
2
3 2
though organozinc halides are air-sensitive, Negishi reaction, at
least for alkyl-alkenyl coupling, looks to have better yields with
lower catalyst loading, respect to Suzuki reaction. Until now, also
in the case of Negishi coupling, there are no example of
supported catalysis, between an alkyl electron donor and an
alkenyl electron acceptor and this contribution represent a first
example.
2
. Results and Discussion
(
10-methoxy-10-oxodecyl) zinc(II) bromide (8) was also
2
.1. Synthesis of saturated isobutyl esters
synthesized with similar results, as described in the experimental
section. The THF solutions of 7 and 8 were titrated by iodometry,
using a literature method,40 generally resulting with a title
comprehended between 0.56 M and 0.73 M.
To exclude the possibility of confusing the isomeric nature of
butyl esters, n-butyl, sec-butyl, iso-butyl and tert-butyl esters
were synthesized. Once confirmed, by their different retention
time on GC analysis, that only the iso-butyl ester isomers were
present in the female rectal gland extracted, only those were
completely isolated and characterized. The isobutyl
transesterification reactions were easily performed through the
16
already described molecular iodine method, using commercially
available starting materials and obtaining iBu decanoate (1), iBu
dodecanoate (2), iBu tetradecanoate (3), iBu hexadecanoate (4) in
good yields, as shown in the supporting info.
Table 2. Optimization of organozinc halides formation
2
2
.2. Synthesis of alkenyl esters
.2.1 Preparation of (Z)-alkenyl halides
The intermediates (Z)-alkenyl halides, which have to be used
in the following Negishi coupling, were prepared through well-
1,2-dibromethane
(mol %)
TMSCl
(mol %)
LiCl
(equiv.)
Time
(h)
GC a
Entry
39
Conv.
known synthetic procedures. The approach is based on the
initial regio- and stereo-controlled syn-hydroboration of the
corresponding alkyne with catecholborane. Bromine is then
added onepot on the obtained boronic ester, to give addition to
the double bond with an anti-mechanism. Finally, the alkenyl
bromine is obtained by anti-elimination reaction with sodium-
hydroxide as a base. This method allowed us to synthesize (Z)-1-
bromohex-1-ene (5) and (Z)-1-bromodec-1-ene (6) with good
yields, depending on the purification method used, as shown in
Table 1. Crucial for our synthesis was the high stereoisomeric
purity of the compounds, that once obtained were directly used
for the following steps, without further characterizations.
1
2
10
1
0
29
0%
45%,82%,
100%
2
0,
10
1
1
2
9,44
3
20
5
1
24
97%
a
Conversion evaluated as ratio between the product area and the s.m. area
2
.2.3. Negishi cross-coupling reaction on homogeneous catalysis
Coupling reaction was, in the first place, tested for the
synthesis of methyl (Z)-dodec-7-enoate (9), on homogeneous
catalysis, using PdCl (PPh ) /TMEDA, as this couple was
reported in literature to give excellent selectivity and yields, in
similar Negishi reactions. The results were indeed satisfactory,
as shown in entry 1 of Table 3, with high yield, short reaction
time and preserving the Z/E ratio (99:1). In the attempt to
decrease the catalyst loading, we found 0.05 mol% of
PdCl (PPh ) , to be a good compromise to obtain a good yield in
2
3 2
38
Table 1. Synthesis of alkenyl halides
2
3 2
2
hours reaction time.
c
Alkyne
Product
Z/E ratio
Yields
a
1
-hexyne
-decyne
(Z)-1-bromohex-1-ene (5)
(Z)-1-bromodec-1-ene (6)
99:1
51%
b
1
99:1
85%

4
Tetrahedron
le SC5.
ACCEPTED MA
T
N
ab
U
R
O imization of Negishi cross-coupling on
IPpt
T
O
PdCl2(PPh3)2
THF, TMEDA
O
Br
(CH2)3CH3
O
O
Supported Pd
THF, TMEDA
ZnBr
+
Br
(CH2)3CH3
O
O
(CH2)3CH3
5
5
ZnBr
+
O
O
(CH2)3CH3
7
5
9
5
5
7
5
9
Table 3. Optimization of Negishi coupling
Supported catalysts
a
PdCl
2
(PPh
3
)
2
Time
(h)
b
c
d
Entry
GC Conv.
GC Yield
Yield
(mol%)
En-
try.
Pd
(mol%)
Time
(h)
GC b
GC c
d
a
Catalyst
3 2
PdCl (PPh ) /
Yield
Conv.
Yield
1
2
3
4
5
6
1
2
2
100%
100%
100%
100%
79%
-
83%
2
1
2
2
2
100%
100%
83%
80%
81%
76%
76%
-
-
0.5
0.1
79%
78%
76%
-
-
PS
TM
FibreCat
007
(PPh
PS
2
-
2
3
4
5
6
2
-
-
1
0.05
0.025
0.01
2
71%
PdCl
2
3
3
)
2
/
2
14
2
57%
100%
22%
0.5
0.1
0.1
0.1
20
20
-
-
PdCl
2
(PPh
PS
) /
2
67%
68%
-
14
100%
25%
-
TM
FibreCat
007
a
Reaction conditions: 1.2 equiv. of 7, THF (3.1 mL/mmol of 5), 1.2 equiv. of
14
26
100%
10%
1
TMEDA, reflux, argon atmosphere
TM
Pd En Cat
b
Conversion evaluated as ratio between the product area and the s.m. area
GC yield calculated using naphthalene as internal standard
Product purified by flash chromatography
4
0
c
a
d
Reaction conditions: 1.2 equiv. of 7, THF (3.1 mL/mmol of 5), 1.2 equiv. of
TMEDA, reflux, argon atmosphere
Conversion evaluated as ratio between the product area and the s.m. area
GC yield calculated using naphthalene as internal standard
b
Using this optimized condition, also methyl (Z)-hexadec-7-
enoate (10) and methyl (Z)-hexadec-11-enoate (11) were
synthesized in good yields and complete stereoselectivity, as
shown in Table 4.
c
d
Product purified by flash chromatography
With our best conditions, we synthesized 10 and 11 as well,
using the cheapest PdCl (PPh ) /PS, respect to FibreCat 1007,
TM
Table 4. Synthesis of alkenyl esters
2
3 2
as shown in Table 6, with consistent results.
O
3 2
PdCl (PPh )
O
2
Br
R
ZnBr
+
Table 6. Synthesis of alkenyl esters on supported catalyst
O
O
R
THF, TMEDA
n
n
7
8
: n = 5
: n = 9
6: R = CH
5: R = CH
3
3
(CH
(CH
2
2
)
)
7
-
10: n = 5, R = CH
11: n = 9, R = CH
3
3
(CH
2 7
) -
O
PdCl (PPh ) /PS
O
2
3 2
3
-
(CH
2
)
3
-
Br
R
ZnBr
+
a
Organozinc
halide
Alkenyl
halide
Time GC
c
O
THF, TMEDA
O
R
Entry
Product
b
Yield
n
n
(h)
Conv.
7
8
: n = 5
: n = 9
6: R = CH
5
: R = CH
3
3
(CH
(CH
2
2
)
)
7
-
10: n = 5, R = CH
11: n = 9, R = CH
3
3
2 7
(CH ) -
1
2
10
11
7
8
6
5
2
100%
100%
78%
69%
3
-
2 3
(CH ) -
2
a
Pd
(mol%)
Time
(h)
GC b
c
Entry
Product
Yield
a
Conv.
100%
2 3 2
Reaction conditions: 0.05 mol% of PdCl (PPh ) , 1.2 equiv. of 7 or 8, THF
1
2
10
11
0.1
14
16
73%
-
(
b
3.1 mL/mmol of 5 or 6), 1.2 equiv. of TMEDA, reflux, argon
Conversion evaluated as ratio between the product area and the s.m. area
Product purified by flash chromatography
0.1
0.3
52%
c
3
11
14
100%
67%
a
Reaction conditions: 1.2 equiv. of 7 or 8, THF (3.1 mL/mmol of 5 or 6, 1.2
equiv. of TMEDA, reflux, argon
b
Conversion evaluated as ratio between the product area and the s.m. area
2
.2.4. Negishi Cross-Coupling reaction on supported catalysts
c
Product purified by flash chromatography
At this stage of the study, for the identification of olive fruit fly
pheromones, it was not necessary a multigram scale up of the
synthesis, which will be however implemented, for the
development of semio-chemical traps for Bactrocera oleae.
As highlighted in the introduction, despite the several
advantages of supported-catalysts, their use for the synthesis of
biologically interesting molecules is still limited, and there are
currently no examples of Negishi supported catalysis between an
alkyl electron donor and an alkenyl electron acceptor. Therefore,
we tested three different commercially available supported
catalysts, mainly PdCl (PPh ) /PS, an analogue of PdCl (PPh )
3 2
2
.3 Transesterification of methyl alkenyl esters
To complete the list of compounds that we hypothesized, from
2
3 2
2
in which Pd (II) is complexed through phosphines in a cross-
linked system, FibreCat 1007, in which the phosphonic groups
the fragmentation analysis of female rectal gland extracts, we
synthesized the missing three ethyl alkenyl esters, with the same
method described in part 2.1, starting from commercially
available methyl (Z)-octadec-11-enoate (12) and (Z)-hexadec-9-
enoic acid (13), as well as from the previously prepared methyl
TM
coordinating Pd(II) are attached to the end tails of polymeric
TM
fibers, and Pd En Cat 40, where Pd(II) is locked inside a
polyurea matrix. Optimal conditions were investigated again on
the synthesis of 9, with the results reported in Table 5.
Stereoselectivity was still retained with a Z/E ratio of 99:1, with
all the three catalysts, even though, generally, longer reaction
times were required to reach completion. However, with
(Z)-hexadec-7-enoate (10). The corresponding ethyl alkenyl
esters, ethyl (Z)-octadec-11-enoate (14), ethyl (Z)-hexadec-7-
enoate (15) and ethyl (Z)-hexadec-9-enoate (16), were obtained
with high yield and purity as shown in Table 7.
TM
PdCl (PPh ) /PS and FibreCat 1007, we were able to decrease
2
3 2
the catalyst loading down to 0.1 mol%, with good yields, in 14
hours reaction time, which is fairly acceptable for supported
catalysts, owing to the numerous advantages of heterogeneous
Table 7. Synthesis of ethyl alkenyl esters
41
O
O
I
2
4 mol%
Reflux
catalysis already described in part 1.2.
R'
+
EtOH
O
R
O
R
n
n
1
1
1
2: n = 9, R' = Me, R = CH
0: n = 5, R' = Me, R = CH (CH ) -
3
(CH
2
)
5
-
3 2 3
14: n = 9, R = CH (CH ) -
3
2 7
15: n = 5, R = CH (CH ) -
3
2 7
3: n = 7, R' = H, R = CH (CH ) -
16: n = 7, R = CH (CH ) -
3 2 5
3
2
5

5
Time
GC
Conv.
b
1
day
4
days
9
days
13
days
17
days
21
ACCEPTED MANUSCRIPT
Starting
Product
a
Yield
Comp.
(h)
days
1
1
2
0
Ethyl (Z)-octadec-11-enoate (14)
Ethyl (Z)-hexadec-7-enoate (15)
8
97%
91%
0.1
± 0.02)
1.2
(± 0.03)
10.4
(± 0.4)
30.1
(± 2.3)
33.6
(± 2.7)
4.6
(± 0.08)
0.2
(± 0.02)
4.5
(± 0.4)
3.2
(± 0.09)
4.2
(± 0.07)
4.0
(± 0.1)
7.8
(± 0.3)
5.7
(± 0.7)
29.6
(± 1.2)
86.1
(± 3.6)
83.3
(± 1.7)
5.0
(± 0.5)
1.0
(± 0.05)
16.6
(± 0.8)
12.3
(± 1.1)
5.8
(± 0.4)
16.3
(± 1.0)
12.9
(± 1.9)
7.2
(± 0.5)
40.8
(± 1.6)
111
(± 3)
121
(± 3)
4.1
(± 0.2)
1.0
(± 0.1)
24.1
(± 2.4)
16.7
(± 0.7)
4.9
(± 0.3)
12.8
(± 0.6)
19.0
(± 0.8)
11.8
(± 0.6)
79.6
(± 1.4)
186
(± 3)
176
(± 4)
8.4
(± 0.4)
2.4
(± 0.1)
9.3
(± 0.5)
32.7
(± 2.4)
31
(± 1.8)
43.4
(± 1.6)
41.7
(± 2.2)
1
2
3
4
0
(
8
4
93%
86%
1
.4
(± 0.02)
.1
± 0.1)
.8
± 0.3)
.4
0
0
0
0
0
0
0
0
0
0
1
3
Ethyl (Z)-hexadec-9-enoate (16)
100%
87%
4
a
Conversion evaluated as ratio between the product area and the s.m. area
Product purified by flash chromatography
(
(
b
2
0
1
1
1
1
0
1
2
4
2
.4. Identification and quantification of the unknown compounds
in female B. oleae gland extracts.
(
± 0.04)
0.05
± 0.01)
0.6
(
Extracts from virgin female rectal glands after 1, 4, 9, 13, 17
and 21 days after the fly emergence, obtained as described in the
experimental part 4.8., were analyzed by GC and GC/EI-MS,
using for each age three different samples. To safely assign the
right retention times and fragmentation patterns, we used two GC
columns with different polar stationary phase. The exact
superimposition of chromatograms and fragmentations allowed
us to unambiguously confirm the presence in the rectal glands of
(± 0.03)
.1
± 0.06)
.7
± 0.2)
.0
2
(
2
15
(
4
1
1
6
7
(
± 0.1)
0.7
(± 0.03)
1
, 2, 3, 4, 9, 10, 11, 14, 15, 16 and the commercially available
methyl (Z)-octadec-9-enoate (17). None of these compounds
were previously identified in female extracts, apart a generic cis-
42
As previously observed for the other esters previously found in
hexa-decenoate. The eleven compounds were quantified in
nanograms per gland (ng/fly), for each age investigated, through
absolute GC calibration curves, as shown in Table 8. Means and
standard deviations (SD) were calculated using absolute
calibration curves, by injecting pure standards (each injection
was replicated three times). Differences in the age-related
production of chemicals were analyzed using the generalized
15
13,14
females, and muscalure for males,
the amount of the
identified esters increases with the age, until a maximum of
secretion after 21 days from fly emergence. The linear trend
estimation of the new identified sex-specific components is
clearer in Figure 1. After 21 days, the production range of the
esters varied from a minimum of 2.4 ng/fly for 11 to a maximum
of 186 ng/fly for 3. These values are low compared to the
production of olean for female flies, that in the same condition
43
linear model described by Benelli et al. with two factors, i.e.,
the fly’s age (j=1-6) and identified chemical (j=1-10). Averages
were separated by the Tukey’s HSD test (P<0.05).
15
fluctuate between 3000 and 4000 ng/fly, but it’s proportionate
1
3,14
to muscalure production in males (50 ng/fly after 21 days).
Noteworthy, the amounts of compounds 3 and 4 look to
th
exponentially increase after the 4 day. Considering that sexual
maturation arises after 8 days for female flies, these compounds
could play an important role in sexual communication between
the insects. Anyway, this hypothesis will be evaluated only in
future, through behavioral and electrophysiological experiments.
Table 8. Quantification in ng/fly of extract components at
different ages

6
Tetrahedron
ACCEPTED MANUSCRIPT
Figure 1. Productions of identified components as a function of age after fly emergenc
In a typical experiment, in a 2-neck round-bottom flask,
equipped with a reflux condenser, one equivalent of the
corresponding acid or ester was dissolved in the opportune
alcohol (0.60 mL/mmol), and 2 or 4% of Iodine were added. The
solution was let stirring at reflux and monitored by GC until
complete conversion. The crude solution was concentrated at
reduced pressure and treated with a saturated solution of
Na S O . The aqueous phase was then extracted three times with
3
. Conclusion
This work represents a significant step forward into the guided
control of olive fruit fly, through the identification of 11 new sex-
specific components in female rectal glands. To effectively
develop, from these data, a semiochemical-based olive-fly fight,
2
2
3
diethyl ether and the reunited organic fractions were washed with
deionized water. The solution was dried over sodium sulfate,
filtered and concentrated under reduced pressure. The obtained
crude was then purified by flash chromatography.
further analyses are needed, especially to confirm the
reproducibility in different years and in different periods of the
year. Moreover, for the synthesis of alkenyl esters, a supported-
catalyst Negishi coupling, between an alkyl electron donor and
an alkenyl electron acceptor, has been developed, which is a
novelty in the field of supported cross-coupling.
4
.2.1 Isobuyl decanoate (1):
Following general procedure (A), to
a
mixture of
commercially available ethyl decanoate (1.93 mL, 8.29 mmol)
and 5 mL of isobuyl alcohol, 4 mol% of molecular iodine I (91.4
2
mg, 0.436 mmol) were added. The resulting solution was then
stirred at reflux for 8 hours. The excess of alcohol was removed
under reduced pressure and the residue was dissolved in diethyl
ether. The solution was washed with a solution of sodium
thiosulfate and distilled water, dried over anhydrous sodium
sulfate and concentrated in vacuo to yield the crude product,
which was purified by flash chromatography on silica gel
4
. Experimental section
4
.1 General Information
All reactions where performed under argon atmosphere,
handling solvents and reagents using hypodermic syringes
conveniently stored in a desiccator. Dichloromethane was dried
over calcium hydride and distilled while tetrahydrofuran was
dried over potassium and distilled. When specified, solvents
(Hex/Et O 90:10). The product was obtained as a colourless oil
2
with 88% yield (purity 99% by GC). Spectroscopic data in
44 1
agreement with literature. H NMR (400 MHz, CDCl ) δ 3.86
3
where purged through argon bubbling. Catalysts PdCl (dppf),
(d, J = 6 Hz, 2H), 2.31 (t, J = 8 Hz, 2H), 1.93 (m, 1H), 1.63 (m,
2H), 1.21-1.36 (bs, 12H), 0.93 (d, J = 8 Hz, 6H), 0.88 (t, J = 6
Hz, 3H); C NMR (100 MHz, CDCl3) δ 174.1, 70.5, 34.5, 32.0,
2
TM
PdCl (PPh ) , PdCl (PPh ) /PS (Pd: 1.00 mmol/g), FibreCat
2
3 2
2
3 2
TM
13
1
007 (Pd: 0.72 mmol/g), Pd En Cat 40 (Pd: 0.42 mmol/g)
where purchased by Sigma-Aldrich. Zinc powder had a particle-
size distribution < 10 µm (Sigma-Aldrich, code 209988, lot.
SHBF7346V). Reactions were followed by GC and GC/EI-MS
on small reaction samples (30-100 µL), treated with the same
work-up described in general procedures. Purifications of the
reaction crude were performed by flash chromatography using
Silica Gel 60 (40-63 µm, Sigma Aldrich), or by fractional
distillation. TLC silica gel plates were purchased by Merck
29.5, 29.4, 27.9, 25.2, 22.8, 19.2, 14.2. IR (neat): 2958, 2924,
2857, 1740, 1466, 1393, 1376, 1348, 1245, 1164, 1110, 1060,
1004, 940, 912, 733 cm ; MS, m/z (%): 228(1), 173(67,
155(100), 143, (4), 129(20), 116(7), 97(5), 57(60), 41(30)
-
1
4
.2.2 Isobutyl dodecanoate (2):
Following general procedure (A), to
a
mixture of
commercially available ethyl laurate (1.10 mL, 4.14 mmol) and
(Merck 60 F254). Gas chromatography (GC) analyses were
2.5 mL of isobuyl alcohol, 4 mol% of molecular iodine I (45 mg,
2
performed on a Dani GC 1000 with PTV injector, FID detector
and two bonded FSOT columns (Alltech, 30 m×0.25 mm i.d.,
0
.18 mmol) were added. The resulting solution was then stirred at
reflux for 8 hours. The excess of alcohol was removed under
reduced pressure and the residue was dissolved in diethyl ether.
The solution was washed with a solution of sodium thiosulfate
and distilled water, dried over anhydrous sodium sulfate and
concentrated in vacuo to yield the crude product, which was
0
.25 ꢀm): AT-5 (column 1) and AT-35 (column 2). Gas
chromatography–mass spectrometry (GC/MS) analyses were
performed with an Agilent apparatus: mass selective detector
5
973 Network, 6890 N Network GC system and HP-5MS bonded
column (30 m×0.25 mm i.d., 0.25 ꢀm, column 3). NMR spectra
were recorded on Bruker 400 MHz, Bruker Avance II DRX 400
spectrometer and on Varian INOVA 600 MHz.
purified by flash chromatography on silica gel (Hex/Et O 90:10).
2
The product was obtained as a colourless oil with 88% yield
(purity 99% by GC). Spectroscopic data in agreement with
45 1
literature. H NMR (400 MHz, CDCl ) δ 3.86 (d, J = 8 Hz, 2H),
3
4
.2. General Procedure (A) for esterification and
2
1
.31 (t, J = 8 Hz, 2H), 1.93 (m, 1H), 1.63 (m, 2H), 1.37-1.21 (bs,
6H), 0.93 (d, J = 6 Hz, 6H), 0.88 (t, J = 6 Hz, 3H); C NMR
transesterification with molecular iodine
13
(
100 MHz, CDCl ) δ 174.0, 70.5, 34.5, 32.0, 29.7, 29.6, 29.5,
3

7
1
3
2
2
1
1
9.4, 29.3, 27.9, 25.2, 22.8, 19.2, 14.2. IR (n
A
ea
C
t):
C
2
E
95
P
2,
T
2
E
92
D
4, MA2H
N
),
U
1,
S
40
C
–1
R
,1
I
9 (m, 23H), 0,88 (t, J = 6,9 Hz, 3H); C NMR
P
T
852, 1737, 1466, 1418, 1379, 1281, 1256, 1234, 1172, 1110,
(100 MHz, CDCl ) δ 174.1, 130.0, 129.9, 60.3, 34.5, 31.9, 29.9,
29.6, 29.5, 29.4, 29.2, 29.1, 27.3, 25.1, 22.8, 14.4, 14.2. IR
(neat): 2924, 2852, 1737, 1460, 1373, 1343, 1303, 1250, 1175,
116, 1097, 1045, 722 cm ; MS, m/z (%): 310(8), 265(40),
22(27), 180(20), 123(22), 97(59), 88(63), 83(63), 69(79),
3
-
1
013, 909, 803, 730, 722 cm ; MS, m/z (%): 256(2), 201(84),
83(100), 171 (6), 157(13), 129(16), 116(9), 57(65), 41(30).
-
1
1
2
4
.3.3 Isobutyl tetradecanoate (3):
Following general procedure (A), to
commercially available ethyl myristate (2.47 mL, 8.28 mmol)
a
mixture of
55(100), 41(47). [Found: C, 77.40; H, 12.38. C20H38O2 requires
C, 77.36; H, 12.33%].
and 5.0 mL of isobuyl alcohol, 4 mol% of molecular iodine I (85
2
4
.3.6. Ethyl (Z)-hexadec-7-enoate (15):
mg, 0.33 mmol) were added. The resulting solution was then
stirred at reflux for 8 hours. The excess of alcohol was removed
under reduced pressure and the residue was dissolved in diethyl
ether. The solution was washed with a solution of sodium
thiosulfate and distilled water, dried over anhydrous sodium
sulfate and concentrated in vacuo to yield the crude product,
which was purified by flash chromatography on silica gel
Following general procedure (A), to a mixture of synthesised
methyl (Z)-hexadec-7-enoate (10) (266 mg, 0.991 mmol) and
0.60 mL of ethyl alcohol, 4 mol% of molecular iodine I (10 mg,
2
0.039 mmol) were added. The resulting solution was then stirred
at reflux for 8 hours. The excess of alcohol was removed under
reduced pressure and the residue was dissolved in diethyl ether.
The solution was washed with a solution of sodium thiosulfate
and distilled water, dried over anhydrous sodium sulfate and
concentrated in vacuo to yield the crude product, which was
(
Hex/Et O 90:10). The product was obtained as a colourless oil
2
with 85% yield (purity 98% by GC). Spectroscopic data in
44 1
agreement with literature. H NMR (400 MHz, CDCl ) δ 3.86
3
(d, J = 7 Hz, 2H), 2.31 (t, J = 8 Hz, 2H), 1.93 (m, 1H), 1.63 (m,
purified by flash chromatography on silica gel (Hex/Et O 95:5).
2
2
H), 1.36-1.22 (bs, 20H), 0.94 (d, J = 7 Hz, 6H), 0.89 (t, J = 7
The product was obtained as a colourless oil with 86% yield
13
1
Hz, 3H) C NMR (100 MHz, CDCl ) δ 174.1, 70.5, 34.5, 32.1,
(purity 97% by GC). H NMR (600 MHz, CDCl ) δ 5,34 (dt, J =
3
3
2
9.81, 29.77, 29.60, 29.5, 29.4, 29.3, 27.9, 25.2, 22.8, 19.2, 14.2
6,5 Hz, 2H), 4,12 (q, J = 7,1 Hz, 2H), 2,28 (t, J = 7,6 Hz, 2H),
IR (neat): 2958, 2924, 2852, 1737, 1465, 1396, 1376, 1376,
2,04-1,94 (m, 4H), 1,66-1,58 (m, 2H), 1,39–1,20 (m, 19H), 0,87
-
1
13
1
2
5
348, 1242, 1169, 1113, 1012, 914, 732 cm ; MS, m/z (%):
84(4), 229(100), 211(98), 185(20),129(25), 116(12), 97(14),
7(91), 41(36).
(t, J = 7,0 Hz, 3H); C NMR (100 MHz, CDCl ) δ 174.0, 130.4,
3
129.6, 60.3, 34.5, 32.1, 29.9, 29.7, 29.5, 29.4, 28.9, 27.3, 27.2,
25.0, 22.8, 14.4, 14.3. IR (neat): 2997, 2924, 2857, 1737, 1460,
1
371, 1348, 1301, 1250, 1178, 1116, 1032, 937, 859, 783, 722
4
.3.4 Isobutyl hexadecanoate (4):
-1
cm ; MS, m/z (%): 282(18), 236(87), 194(63), 152(49), 101(75),
96(77), 88(84), 84(71), 69(66), 55(100), 41(63). [Found: C,
76.57; H, 12.15. C18H34O2 requires C, 76.54; H, 12.13%]
Following general procedure (A), to
a
mixture of
commercially available methyl palmitate (844 mg, 3.12 mmol)
and 1.88 mL of isobuyl alcohol, 4 mol% of molecular iodine I2
4
.3.7 Ethyl (Z)-hexadec-9-enoate (16):
(33 mg, 0.13 mmol) were added. The resulting solution was then
stirred at reflux for 8 hours. The excess of alcohol was removed
under reduced pressure and the residue was dissolved in diethyl
ether. The solution was washed with a solution of sodium
thiosulfate and distilled water, dried over anhydrous sodium
sulfate and concentrated in vacuo to yield the crude product,
which was purified by flash chromatography on silica gel
Following general procedure (A), to
commercially available (Z)-hexadec-9-enoic acid (13) (100 mg,
0.393 mmol) and 0.25 mL of ethyl alcohol, 4 mol% of molecular
a
mixture of
iodine I (4.0 mg, 0.016 mmol) was added. The resulting solution
2
was then stirred at reflux for 4 hours. The excess of alcohol was
removed under reduced pressure and the residue was dissolved in
diethyl ether. The solution was washed with a solution of sodium
thiosulfate and distilled water, dried over anhydrous sodium
sulfate and concentrated in vacuo to yield the crude product,
which was purified by flash chromatography on silica gel
(
Hex/Et O 90:10). The product was obtained as a colourless oil
2
with 87% yield (purity 99% by GC). Spectroscopic data in
45 1
agreement with literature. H NMR (400 MHz, CDCl ) δ 3.86
3
(d, J = 7 Hz, 2H), 2.31 (t, J = 7 Hz, 2H), 1.93 (m, 1H), 1.63 (m,
2
3
2
1
1
2
4
H), 1.34-1.22 (bs, 24H), 0.93 (d, J = 7Hz, 6H), 0.88 (t, J = 6 Hz,
(Hex/Et2O 95:5). The product was obtained as a colourless oil
13
1
H); C NMR (100 MHz, CDCl3) δ 174.1, 70.5, 34.5, 32.0,
9.81, 29.77, 29.71, 29.6, 29.5, 29.4, 29.3, 27.9, 25.2, 22.8, 19.2,
4.2. IR (neat): 2952, 2919, 2852, 1740, 1466, 1393, 1379, 1351,
with 87% yield (purity 99% by GC). H NMR (600 MHz, CDCl )
3
δ 5,38 (dt, J = 10,8 Hz, 2H), 4,12 (q, J = 7,1 Hz, 2H), 2,28 (t, J =
7,6 Hz, 2H), 2,04-1,94 (m, 4H), 1,66-1,58 (m, 2H), 1,39–1,20 (m,
-
1
13
242, 1172, 1113, 1013, 733, 722 cm ; MS, m/z (%): 312(4),
57(77), 239(68), 213(11), 129(18), 116(12), 97(13), 56(100),
1(32).
19H), 0,87 (t, J = 6,9 Hz, 3H); C NMR (100 MHz, CDCl ) δ
3
174.1, 130.1, 129.9, 60.3, 34.5, 32.0, 29.9, 29.8, 29.3, 29.24,
29.22, 29.1, 27.34, 27.28, 22.8, 14.4, 14.3. IR (neat): 2924, 2852,
1
9
737, 1463, 1371, 1348, 1301, 1245, 1178, 1115, 1094, 1035,
12, 853, 808, 727 cm ; MS, m/z (%): 282(14), 237(50), 219 (3),
4
.3.5 Ethyl (Z)-octadec-11-enoate (14):
-1
Following general procedure (A), to
commercially available methyl (Z)-octadec-11-enoate (12) (67.5
mg, 0.227 mmol) and 265 µL of ethyl alcohol, 4 mol% of
a
mixture of
194(43), 152(35), 101(61), 88(71), 69(81), 55(100), 41(58).
[Found: C, 76.40; H, 12.8. C18H34O2 requires C, 76.54; H,
12.13%].
molecular iodine I (2.4 mg, 0.095 mmol) were added. The
2
4
.3. General procedure (B) for the synthesis of (Z)-1-bromo-1-
resulting solution was then stirred at reflux for 8 hours. The
excess of alcohol was removed under reduced pressure and the
residue was dissolved in diethyl ether. The solution was washed
with a solution of sodium thiosulfate and distilled water, dried
over anhydrous sodium sulfate and concentrated in vacuo to yield
the crude product, which was purified by flash chromatography
alkenes
In a typical experiment, in a 3-neck round-bottom flask,
equipped with a dropping funnel and a reflux condenser, one
equivalent of the corresponding alkyne and one equivalent of
catecholborane were mixed, under argon. The mixture was stirred
at 70°C for 3 hours, and afterwards cooled down at room
temperature and diluted with dichloromethane (0.25mL/mmol).
The solution was then cooled down at -10°C. A solution
containing 2 equiv. of bromine in dichloromethane (5 M) was
on silica gel (Hex/Et O 95:5). The product was obtained as a
2
1
colourless oil with 91% yield (purity 99% by GC). H NMR (600
MHz, CDCl ) δ 5,34 (tm, J = 5,5 Hz, 2H), 4,11 (q, J = 7,1 Hz,
3
2
H), 2,27 (t, J = 7,6 Hz, 2H), 2,08 – 1,93 (m, 4H), 1,65-1,57 (m,

8
Tetrahedron
as MA
added dropwise, over a period of one hour.
A
Th
C
e
C
so
E
lu
P
tio
T
n
E
w
D
w
N
as
U
ag
S
ain
C
w
R
a
I
rmed up to reflux and the reaction was followed by
P
T
then let stirring for an additional hour, at -10°C. The mixture was
warmed up at 0°C, and 68 mL of NaOH 2N aqueous solution
were added dropwise, over a period of 15 minutes. The reaction
was let stirring for an additional hour at 0 °C. The obtained
GC, until complete conversion. Once the reaction went to
completion, the flask was let cooling down at room temperature
and zinc was let precipitating over 6 hours. The titration of
organozinc compound was carried out by iodometry, using a
literature method.
solution was extracted
3
times (3
x 100 mL) with
dichloromethane and the collected organic phases washed with
brine until neutral pH. The solution was dried over sodium
sulfate, filtered and concentrated under reduced pressure. The
crude was purified by flash chromatography or distillation.
4
.5. General procedure (D) for Negishi coupling between
organozinc halides and alkenyl halides
In a 2-neck round bottom flask, equipped with a reflux
condenser and under inert atmosphere, was added the catalyst, in
the form of PdCl (PPh ) or one of its supported version,
4
.3.1. (Z)-1-bromohex-1-ene (5):
2
3 2
TM
TM
Following general procedure (B), 1.13 mL of 1-hexyne (9.84
PdCl (PPh ) /PS, FibreCat 1007 and Pd En Cat 40 , in the
2 3 2
mmol), and 1.05 mL of catecholborane (9.84 mmol) were stirred,
under argon, at 70 °C. Once cooled down, the mixture was
diluted with 2.5 mL of dichloromethane and added dropwise with
specific amounts indicated in Tables 4-7, for each product
synthesized. Taking into account, the volume of solvent already
used to prepare the organozinc halide solution, following
procedure (C), dry THF was added calculating a final dilution of
about 3 mL/mmole of alkenyl halide. Afterwards, 1.2 equivalents
of TMEDA, 1 equivalent of alkenyl halide and 1.2 equivalents of
organozinc halide, were added to the solution, that was then
warmed up to reflux until completion. Reaction was monitored
4
.0 mL of a dichloromethane solution containing bromine (5.0
M). The reaction was quenched as described in the general
procedure (B). The crude was purified by fractional distillation
(226 mbar), obtaining the product as a colourless oil with a yield
of 51% (purity 88% by GC, Z/E, 99:1). Once identified the
product by NMR, the intermediate was used directly for the
by GC. At last, 10 mL of saturated solution of NH Cl were
4
following4 steps. Spectroscopic data in agreement with
added, to quench the reaction. The aqueous phase was extracted 3
times with diethyl ether, the collected organic fractions were
dried over sodium sulphate, filtered and concentrated at reduced
pressure. The crude was purified by flash chromatography.
6 1
literature. H NMR (400 MHz, CDCl ) δ 6.13 (d, J = 4 Hz), 6.10
3
(
m, 1H), 2.21 (m, 2H), 1.33-1.44 (m, 4H), 0.93 (t, J = 4 Hz, 3H).
MS, m/z (%): 164(43), 162(42), 121(18), 119(17), 83(50),
7(15), 56(100), 41(85). bp = 76-77 °C/226 mbar (lit. 96 °C/226
mbar)
6
4
.5.1 Methyl (Z)-dodec-7-enoate (9):
Following general procedure (D), to a flask containing 1
4
.3.2. (Z)-1-bromodec-1-ene (6):
mol% (or other amounts reported in Table 4) of PdCl (PPh )
2
3 2
Following general procedure (B), 1.80 mL of 1-decyne (10.0
(10.5 mg), (or the supported catalysts reported in Table 6), (Z)-1-
mmol), and 1.07 mL of catecholborane (10 mmol) were stirred,
under argon, at 70 °C. Once cooled down, the mixture was
diluted with 2.5 mL of dichloromethane and added dropwise with
bromohex-1-ene (5) (1.50 mmol, 244 mg), 1mL of THF and 270
µL of TMEDA (1.80 mmol) were added. The synthesized
organozinc
intermediate,
5-methoxycarbonyl-1-pentylzinc
4
mL of a dichloromethane solution containing bromine (5.0 M).
The reaction was quenched as described in the general procedure
B). The crude was purified by flash chromatography on silica
bromide (7), was added as a THF solution (3.2 mL of a 0.56M
solution, 1.8 mmol) previously titrated following general
procedure (C). The reaction was warmed up at reflux for 3 hours
and quenched as reported in general procedure (D). The product
(
gel, using hexane as a solvent, obtaining the product as a
colourless oil with a yield of 85% (purity 98% by GC, Z/E, 99:1).
Once identified the product by NMR, the intermediate was used
directly for the following steps. Spectroscopic data in agreement
was isolated by flash chromatography on silica gel (Hex/Et O,
2
95:5), as a colourless oil with the yields reported in Table 4 and
48 1
6. Spectroscopic data in agreement with literature. H NMR
(600 MHz, CDCl3) δ 5,33 (dt, J = 10 Hz, 2H), 3.66 (s, 3H), 2.30
(t, J = 7 Hz, 2H), 1.65-1.61 (m, 4H), 1.36-1.30 (bs, 8H), 0.89 (t, J
47 1
with literature. H NMR (400 MHz, CDCl ) δ 6.14 (d, J = 4 Hz,
3
1
0
1
4
H), 6.09 (m, 1H), 2.20 (m, 2H), 1.42 (m, 2H), 1.30 (m, 10H),
.89 (t, J = 4 Hz, 3H). MS, m/z (%): 220(9), 218(10), 150(11),
48(11), 121(16), 119(16), 97(48), 83(100), 69(51), 57(56),
1(51).
13
= 7 Hz, 3H); C NMR(100 MHz, CDCl ) δ 174.3, 130.2, 129.5,
3
51.5, 34.1, 31.9, 29.4, 28.8, 27.0, 26.9, 24.9, 22.4, 14.0; IR
(neat): 3003, 2930, 2852, 1740, 1460, 1432, 1376, 1359, 1315,
-
1
1
258, 1200, 1172, 1116, 1077, 1013, 912, 733 cm ; MS, m/z
4
.4. General procedure (C) for the preparation of organozinc
(%): 212(7), 180(30), 138(45), 123(24), 110(24), 96(57), 74(82),
halides through oxidative addition to alkyl bromides
5
5(100), 41(55)
In a 2-neck round-bottom flask equipped with a reflux
condenser, under inert atmosphere, one equiv. of LiCl was added.
The system was warmed up to 150-170°C, at reduced pressure
4
.5.2. Methyl (Z)-hexadec-7-enoate (10):
Following general procedure (D), to a flask containing 0.05
(0.1 mbar), for 20 minutes. Two equiv. of zinc powder were
mol% of PdCl (PPh ) (1.6 mg), (or the supported version of the
2 3 2
added, and the system was again warmed up to 150-170°C, at
reduced pressure (0.1 mbar), for 20 minutes. The flask was let
cooling down to room temperature and treated with 3 cycles of
vacuum-argon. Afterwards dry THF (1 mL/mmol of
corresponding alkyl bromide) and 10 mol% of 1,2-
dibromoethane were added. The mixture was let stirring at 65°C
for 15 minutes and cooled down at room temperature. Depending
on substrate, 1 or 5 mol% of trimethylsilyl chloride were added,
and the solution was warmed up to 65°C for 15 minutes and then
cooled down to room temperature. The suitable alkyl bromide (1
equiv.) was then added dropwise and the solution stirred at reflux
for 3 hours. The solution was cooled down to room temperature
and 10 mol% of 1,2-dibromoethane were added. The solution
catalyst, as reported in Table 7), (Z)-1-bromodec-1-ene (6) (4.50
mmol, 986 mg), 4.3 mL of THF and 810 µL of TMEDA (5.40
mmol) were added. The synthesized organozinc intermediate, 5-
methoxycarbonyl-1-pentylzinc bromide (7), was added as a THF
solution (9.5 mL of a 0.57 M solution, 5.4 mmol) previously
titrated following general procedure (C). The reaction was
warmed up at reflux for 3 hours and quenched as reported in
general procedure (D). The product was isolated by flash
chromatography on silica gel (Hex/Et O, 95:5), as a colourless oil
2
1
with the yields reported in Table 5 or 7. H NMR (600 MHz,
CDCl ) δ 5,33 (dt, J = 10,8 Hz, 2H), 3,66 (s, 3H), 2,30 (t, J = 7,6
3
Hz, 2H), 2,00-1,95 (m, 4H), 1,63-1,58 (m, 2H), 1,39-1,20 (m,
13
16H), 0,87 (t, J = 7,0 Hz, 3H); C NMR (100 MHz, CDCl ) δ
3

9
1
2
1
74.4, 130.3, 129.5, 51.6, 34.1, 32.0, 29.8, 29.6
7.3, 27.1, 25.0, 22.8, 14.3 IR (neat): 2924, 2852, 1743, 1460,
435, 1362, 1317, 1253, 1200, 1172, 1119, 1079, 1010, 727 cm ;
A
, 2
C
9.
C
5,
E
29
P
.4
T
, 2
E
8
D
.9, MA
A
N
ck
U
no
S
wl
C
ed
R
ge
I
m
P
e
T
nts
-
1
This research was partially funded by the University of Pisa
on the project PRA 2016-50. A.C. is grateful to Prof. Gloria
Uccello Barretta and Dr. Federica Balzano for 600 MHz NMR
spectra. G.A. is grateful to GABA Therwil GmbH - A Colgate-
Palmolive Company for instruments donation.
MS, m/z (%): 268(8), 236(41), 194(33), 110(35), 96(68), 74(85),
9(64), 55(100), 41(69). [Found: C, 76.11; H, 12.06. C17H32O2
requires C, 76.07; H, 12.02%].
6
4
.5.3. Methyl (Z)-hexadec-11-enoate (11):
References
Following general procedure (D), to a flask containing 0.05
1
2
3
4
5
.
.
.
.
.
Benelli G, Daane KM, Canale A, Niu CY, Messing
RH, Vargas RI. J Pest Sci (2004). 2014; 87: 385–405.
mol% of PdCl (PPh ) (1.6 mg), (or the supported version of the
2
3 2
catalyst, as reported in Table 7), (Z)-1-bromohex-1-ene (5) (4.50
mmol, 733 mg), 4.4 mL of THF and 810 µL of TMEDA (5.40
mmol) were added. The synthesized organozinc intermediate,
Rice RE, Phillips P a, Stewart-leslie J, Sibbett GS.
Calif Agric. 2003; 57: 122–127.
(
10-methoxy-10-oxodecyl) zinc(II) bromide (8), was added as a
THF solution (8.6 mL of a 0.63 M solution, 5.4 mmol)
previously titrated following general procedure (C). The reaction
was warmed up at reflux for 3 hours and quenched as reported in
general procedure (D). The product was isolated by flash
Daane KM, Johnson MW. Annu Rev Entomol. 2010;
5
5: 151–169.
Yokoyama VY, Rend N PA, Sivinski J. Environ
Entomol. 2008; 37: 764–773.
chromatography on silica gel (Hex/Et O, 95:5), as a colourless oil
2
1
with the yields reported in Table 5 and 7. H NMR (600 MHz,
CDCl ) δ 5,34 (t, J = 5,5 Hz, 2H), 3,66 (s, 3H), 2,29 (t, J = 7,4
3
Bertolini E, Kistenpfennig C, Menegazzi P, Keller A,
Koukidou M, Helfrich-Förster C. Sci Rep. 2018; 8:
Hz, 2H), 2,05-1,95 (m, 4H), 1,65-1,57 (m, 2H), 1,36-1,21 (m,
13
1
1
2
1
6H), 0,94-0,84 (m, 3H); C NMR (100 MHz, CDCl ) δ 174.5,
3
8
16.
30.0, 51.6, 34.2, 32.1, 29.9, 29.6, 29.4, 29.3, 27.3, 27.0, 25.0,
2.5, 14.1. IR (neat): 2924, 2852, 1743, 1460, 1435, 1376, 1359,
-
1
6.
Varikou K, Garantonakis N, Birouraki A. Crop Prot.
015; 68: 60–66.
256, 1197, 1169, 1116, 1099, 1018, 915, 876, 803, 730 cm ;
2
MS, m/z (%): 268(9), 235(40), 194(28), 152(24), 96(45), 74(55),
9(59), 55(100), 41(49). [Found: C, 76.10; H, 12.07. C17H32O2
requires C, 76.07; H, 12.02%].
6
7
8
.
.
Yokoyama VY. J Insect Sci. 2014; 14: 1–5.
Varikou K, Garantonakis N, Birouraki A. Bull
Insectology. 2014; 67: 109–114.
4
.7. General procedure (F) for female olive fruit fly breeding and
preparation of extraction samples
Insects used in this study were obtained from pupae collected in a
Tuscan olive-mill during October 2016. The flies were reared
following the method described by Carpita et al. (13).
9.
Margaritopoulus. Pest Manag Sci. 2008; 1106: 1100–
1106.
1
0.
Kavallieratos NG, Athanassiou CG, Balotis GN, Tatsi
GT, Mazomenos BE. J Econ Entomol. 2005; 98:
4
.8. General procedure (G) for identification and quantification
of the sex-specific components in female olive fruit fly rectal
glands
1
499–505.
1
1
1
1
1.
2.
3.
4.
Baker R, Herbert R, Howse PE, Jones OT, Francke
W, Reith W. J Chem Soc Chem Commun. 1980: 52.
The solutions extracted from virgin female rectal glands, at 1,
, 9, 13, 17, and 21 days after the fly emergence (December
015) were analysed by GC and GC/EI-MS. For each age, 3
samples (each one from 10 virgin females) were analysed in
triplicates. Both GC and GC/EI-MS were performed with
splitless injection technique, injecting 3 µl of hexane solution of
the samples, using helium as carrier (1mL/min). The
programmed temperature for GC and GC/EI-MS was set as
following: 1 min. at 40°C, temp. ramp at 10°C/min until 250°C, 5
min. at 250°C, temp. ramp at 20 °C until 280°C, 30 min. at
4
2
Haniotakis GE, Kozyrakis E, Bonatsos C. J Appl
Entomol. 1986; 101: 343–352.
Carpita A, Canale A, Raffaelli A, Saba A, Benelli G,
Raspi A. Naturwissenschaften. 2012; 99: 77–81.
Canale A, Germinara SG, Carpita A, Benelli G,
Bonsignori G, Stefanini C, Raspi A, Rotundo G.
Chemoecology. 2013; 23: 155–164.
2
80°C. For the comparison of retention times a slower program
was used: 1 min. at 40°C, temp. ramp at 10°C/min until 200°C,
temp. ramp at 6°C until 280°C, 30 min. at 280°C. Compounds
were identified correlating mass spectra and retention times, over
two different columns, with commercially available standards or
with synthesized molecules. The quantification of the identified
compounds was done through GC, using absolute calibration
curves obtained from pure standard injections at five different
concentrations (3 times), in range between 0.5 and 17.3 mg/L.
1
5.
Canale A, Benelli G, Germinara GS, Fusini G,
Romano D, Rapalini F, Desneux N, Rotundo G, Raspi
A, Carpita A. Chemoecology. 2015; 25: 147–157.
1
1
1
6.
7.
8.
Ramalinga K, Vijayalakshmi P, Kaimal TNB.
Tetrahedron Lett. 2002; 43: 879–882.
Xu P, Hooper AM, Pickett JA, Leal WS. PLoS One.
2
012; 7: 2–11.
Cui J, Lethu S, Yasuda T, Matsuoka S, Matsumori N,
Sato F, Murata M. Bioorganic Med Chem Lett. 2015;
2
5: 203–206.

1
0
Tetrahedron
enD MA34
1
9. Darwish TA, Luks E, Moraes G, Yep
PJ, Jamesa M. J Label Compd Radiopharm. 2013; 56:
20–529.
A
ur
C
i N
C
R
E
, H
P
o
T
ld
E
NUS
.
F
C
en
R
g
I
C, Cunningham DW, Easter QT, Blum SA. J
P
T
Am Chem Soc. 2016; 138: 11156–11159.
5
3
3
3
3
3
5.
6.
7.
8.
9.
Tamaru Y, Ochiai H, Nakamura T, Yoshida Z. Angew
Chemie Int Ed English. 1987; 26: 1157–1158.
2
2
0.
1.
Abe M, Nakano M, Kosaka A, Miyoshi H.
Tetrahedron Lett. 2015; 56: 2258–2261.
Bhuniya R, Nanda S. Tetrahedron. 2013; 69: 1153–
1165.
Kinjo Y, Pei B, Bufali S, Raju R, Richardson SK,
Imamura M, Fujio M, Wu D, Khurana A, Kawahara
K, Wong CH, Howell AR, Seeberger PH, Kronenberg
M. Chem Biol. 2008; 15: 654–664.
Yamanaka H, Sato K, Sato H, Iida M, Oishi T, Chida
N. Tetrahedron. 2009; 65: 9188–9201.
Krasovskiy A, Lipshutz BH. Org Lett. 2011; 13:
2
2.
Richards ML, Scott PJH. In Green Techniques for
Organic Synthesis and Medicinal Chemistry; W.
Zhang, and B. W. Cue, Eds.; John Wiley & Sons, Ltd:
Chichester, UK, 2012; pp 185–215.
3
818–3821.
Brown HC, Subrahmanyam C, Hamaoka T,
Ravindran N, Bowman DH, Misumi S, Unni MK,
Somayaji V, Bhat NG. J Org Chem. 1989; 54: 6068–
2
2
3.
4.
Doucet H. European J Org Chem. 2008: 2013–2030.
6
075.
Sato M, Miyaura N, Suzuki A. Chem. Lett., 1989, 8,
4
0.
Krasovskiy A, Knochel P. Synthesis (Stuttg). 2006:
1
405–1408.
8
90–891.
2
2
2
2
2
3
5.
6.
7.
8.
9.
0.
Zou G, Reddy YK, Falck JR. Tetrahedron Lett. 2001;
4
4
1.
2.
Yin, Liebscher J. Chem Rev. 2007; 107: 133–173.
Gariboldi P, Verotta L, Fanelli R. Experientia. 1983;
4
2: 7213–7215.
Bellina F, Anselmi C, Rossi R. Tetrahedron Lett.
3
9: 502–505.
2
001; 42: 3851–3854.
4
4
4
3.
4.
5.
Benelli G, Donati E, Romano D, Stefanini C, Messing
RH, Canale A. Sci Nat. 2014; 102.
Fall Y, Doucet H, Santelli M. Appl Organomet Chem.
2
008; 22: 503–509.
Rocha SM, Coutinho P, Barros A, Delgadillo I,
Coimbra MA. J Chromatogr A. 2006; 1114: 188–197.
Negishi E iehi, Valente LF, Kobayashi M. J Am Chem
Soc. 1980; 102: 3298–3299.
Sobolevsky TG, Revelsky AI, Revelsky IA, Miller B,
Oriedo V. J Chromatogr B Anal Technol Biomed Life
Sci. 2004; 800: 101–107.
Klatt T, Markiewicz JT, Sämann C, Knochel P. J Org
Chem. 2014; 79: 4253–4269.
Knochel P, Yeh MCP, Berk SC, Talbert J. J Org
Chem. 1988; 53: 2390–2392.
4
4
4
6.
7.
8.
Rossi R, Carpita A, Quirici MG, Gaudenzi ML.
Tetrahedron. 1982; 38: 631–637.
3
3
1.
2.
Huo S. Org Lett. 2003; 5: 423–425.
Knapp FF, Srivastava PC. Synth Commun. 1983; 13:
1027–1032.
Krasovskiy A, Malakhov V, Gavryushin A, Knochel
P. Angew Chemie - Int Ed. 2006; 45: 6040–6044.
Tour JM, Pendalwar SL, Kafka CM, Cooper JP. J Org
Chem. 1992; 57: 4786–4787.
3
3.
Yeung CS, Dong VM. J Am Chem Soc. 2008; 130:
7
826–7827.