Asymmetric synthesis and structure-activity studies of the fungal metabolites colletorin A, colletochlorin A and their halogenates analogues

Article abstract of DOI:10.1016/j.tet.2018.05.077

The first asymmetric total synthesis of both enantiomers of the natural products colletorin A and colletochlorin A is presented. The proposed methodology is based on the coupling reaction between highly substituted aromatic Gilman cuprates and optically active allyl bromides, in turn obtained by Sharpless asymmetric dihydroxylation. The latter ensured a high degree of regio- and stereocontrol in the enantioselective step of the synthesis. The same synthetic strategy has been also applied for the preparation of differently halogenated synthetic analogues of colletochlorin A in high enantiomeric purity. The enantioselective synthesis of colletorin A and colletochlorin A allows to reliably assign their absolute configuration. Preliminary assessment of their herbicidal and insecticidal properties evidence the possibility to modulate the bioactivity of these compounds, highlighting its dependence on both the absolute stereochemistry and the halogen nature.

Full text of DOI:10.1016/j.tet.2018.05.077

Accepted Manuscript
Asymmetric synthesis and structure-activity studies of the fungal metabolites
colletorin A, colletochlorin A and their halogenates analogues
Giulia Marsico, Barbara A. Pignataro, Marco Masi, Antonio Evidente, Francesca
Casella, Maria Chiara Zonno, Jun-Hyung Tak, Jeffrey R. Bloomquist, Stefano
Superchi, Patrizia Scafato
PII:
S0040-4020(18)30636-7
10.1016/j.tet.2018.05.077
TET 29584
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 12 April 2018
Revised Date: 22 May 2018
Accepted Date: 26 May 2018
Please cite this article as: Marsico G, Pignataro BA, Masi M, Evidente A, Casella F, Zonno MC, Tak J-
H, Bloomquist JR, Superchi S, Scafato P, Asymmetric synthesis and structure-activity studies of the
fungal metabolites colletorin A, colletochlorin A and their halogenates analogues, Tetrahedron (2018),
doi: 10.1016/j.tet.2018.05.077.
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Graphical abstract
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Asymmetric Synthesis and Structure-Activity
Studies of the Fungal Metabolites Colletorin A,
Colletochlorin A and their Halogenates Analogues
Giulia Marsicoa, Barbara A. Pignataroa, Marco Masib, Antonio Evidenteb, Francesca Casellac, Maria Chiara
Zonnoc, Jun-Hyung Takd, Jeffrey R. Bloomquistd, Stefano Superchia and Patrizia Scafatoa*
aDepartment of Science, University of Basilicata, Viale Ateneo Lucano, 10, 85100 Potenza, Italy.
bDepartment of Chemical Sciences, University of Naples Federico II, Via Cintia 4, 80126 Napoli, Italy
cInstitute of Sciences of Food Production, CNR, Via Amendola 122/O, 70125 Bari, Italy
dDepartment of Entomology and Nematology, Emerging Pathogens Institute, University of Florida, Gainesville,
FL 33610, USA

ACCEPTED MANUSCRIPT
Asymmetric Synthesis and Structure-Activity Studies of the Fungal
Metabolites Colletorin A, Colletochlorin A and their Halogenates
Analogues
Giulia Marsicoa, Barbara A. Pignataroa, Marco Masib, Antonio Evidenteb, Francesca Casellac, Maria
Chiara Zonnoc, Jun-Hyung Takd, Jeffrey R. Bloomquistd, Stefano Superchia and Patrizia Scafatoa*
aDepartment of Science, University of Basilicata, Viale Ateneo Lucano, 10, 85100 Potenza, Italy.
bDepartment of Chemical Sciences, University of Naples Federico II, Complesso Universitario
Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy
cInstitute of Sciences of Food Production, CNR, Via Amendola 122/O, 70125 Bari, Italy
dDepartment of Entomology and Nematology, Emerging Pathogens Institute, University of
Florida, Gainesville, FL 33610, USA
*e-mail: patrizia.scafato@unibas.it
Abstract
The first asymmetric total synthesis of both enantiomers of the natural products colletorin A and
colletochlorin A is presented. The proposed methodology is based on the coupling reaction between
highly substituted aromatic Gilman cuprates and optically active allyl bromides, in turn obtained by
Sharpless asymmetric dihydroxylation. The latter ensured a high degree of regio- and stereocontrol
in the enantioselective step of the synthesis. The same synthetic strategy has been also applied for
the preparation of differently halogenated synthetic analogues of colletochlorin A in high
enantiomeric purity. The enantioselective synthesis of colletorin A and colletochlorin A allows to
reliably assign their absolute configuration. Preliminary assessment of their herbicidal and
insecticidal properties evidence the possibility to modulate the bioactivity of these compounds,
highlighting its dependence on both the absolute stereochemistry and the halogen nature.
Keywords: colletochlorin A, colletorin A, bioactive fungal metabolites, asymmetric
dihydroxylation, coupling reactions, asymmetric synthesis.
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Introduction
Fungi, being able to produce a large number of bioactive secondary metabolites, represent an
excellent source of pharmaceuticals, antifungal, and herbicidal compounds.1 Colletorins and
colletochlorins are two groups of interesting bioactive metabolites isolated from Colletotrichum
nicotianae, a fungus causing anthracnose disease of tobacco plants.2 These compounds share as
common structural feature a multi-substituted polyphenolic ring joined to a prenyl or diprenyl side
chain. Some of them have this terpene moiety partly oxidized at the terminal double bond,
displaying one or two hydroxylated stereogenic centers. This is the case of colletorin A (1a) and
colletochlorin A (1b) (Figure 1), which possess a chiral diol moiety.3 Phytotoxin 1b has been also
recently isolated from Colletotrichum higginsianum4 and Colletotrichum gloeosporioides.5 The
structurally-related colletochlorin B (2b) (Figure 1) displays promising pharmaceutical activity
being able to readily induce the differentiation of human promyelocytic cells (HL-60),6 associated
with the onset of diseases such as acute promyelocytic leukemia and neutrophilic leukopenia.
Compound 2b also shows inhibitory activity toward the enzymes acetylcholinesterase (AChE) and
β-glucuronidase, as well as some toxicity toward human lung fibroblasts.7 Moreover, a recent study
has also revealed the phytotoxic activity of 1b against Ambrosia artemisiifolia,5 allowing to
envisage its possible use as natural herbicidal for biocontrol of this weed responsible for serious
allergies in humans. In this latter study, the (R) absolute configuration at the hydroxylated
stereocenter of naturally occurring (+)-1b has been also determined by application of the modified
Mosher’s method,5 while the absolute configuration of 1a is still unknown. However, due to the
scarce availability of these compounds from natural sources (indeed fungi can produce these
metabolites in small amounts), only a few studies have been undertaken to investigate their
biological properties. These reasons prompted us to attempt the development of a new
stereoselective synthesis of both enantiomers of 1a and 1b, as well as their brominated and
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fluorinated analogues 1c and 1d, with the aim to carry out a structure activity relationship study to
define the role of both absolute stereochemistry and halogen nature on the biological properties of
this class of phytotoxins.8 Indeed, the role of the absolute configuration on the bioactive properties,
well known for the majority of chiral compounds,9 has been clearly recognized also in fungal
metabolites,10 and the influence of the presence and nature of halogen atoms on the same properties
is well established too.8,11
Few approaches for the synthetic preparation of 1a and 1b are reported in the literature, none
providing them in an optically active form. To the best of our knowledge, a single synthesis of
racemic 1b is described,12 which employs, as a key step, a lithium-mediated C-alkylation of 1,5-
dimethoxy-3-methyl-1,4-cyclohexadiene with the dihydroxylated side chain, in turn obtained from
geranyl acetate. However, this strategy provided the desired compound in a very low yield and was
not applicable neither to the synthesis of 1a nor to the preparation of the halogenated analogues 1c
and 1d.
Fig. 1. Structure of colletorin A and B (1a and 2a), colletochlorin A and B (1b and 2b), and their
synthetic analogues 1c and 1d.
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We herein describe the first asymmetric synthesis of both enantiomers of 1a and 1b as well as that
of the brominated and fluorinated unnatural analogues 1c and 1d. A preliminary investigation of the
phytotoxic and insecticidal properties of the prepared compounds is also described.
Results and Discussion
The synthetic approach described herein is based on the disconnection of the structures of 1a-1d
into an aromatic synthon and an optically active side chain (Scheme 1), having both all the
functional groups protected (Scheme 1) and where the aromatic formyl group is replaced by a less
reactive carbomethoxy precursor. A similar approach has been reported for the preparation of
unsaturated analogues colletorin B (2a) and colletochlorin B (2b) (Figure 1), by alkylation of an
aromatic mixed cuprate precursor with geranyl bromide.13 In our case, we envisaged the possibility
to obtain the chiral side chain in optically active form by a regio- and stereoselective Sharpless
asymmetric dihydroxylation of geranyl acetate.14 To avoid oxidizing conditions to the polyphenolic
aromatic precursor, the diol moiety was introduced on the side chain before the cross-coupling
reaction between the two moieties.
Scheme 1. Retrosynthetic approach
In Scheme 2 the synthetic steps for the preparation of the aromatic precursors are summarized.
Cyclohexenone 3, prepared by Robinson annulation,15 was brominated and aromatized with either 2
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or 3 equivalents of bromine to achieve benzoates 4a and 4c, respectively. Then, the unsubstituted
position of 4a was smoothly chlorinated by reaction with a sulfuryl chloride solution. Pure 4b was
obtained in quantitative yields after solvent removal under controlled pressure and temperature to
prevent product sublimation. Conversely, the introduction of fluorine on the aromatic ring was
rather difficult. Various unsuccessful attempts in different experimental conditions were made, by
employing reagents already known for electrophilic fluorination of phenols, such as N-
fluoropyridinium
triflate16
and
N-chloromethyl-N’-fluorotriethylendiammonium-bis
(tetrafluoborate) (Selectfluor®).17 No conversion was obtained with the pyridinium salt even at high
temperature and long reaction time. Finally, a moderate 50% conversion of 4a in 4d was obtained
by reaction with Selectfluor® in methanol at 50°C. Higher temperatures and different solvents led to
lower conversions and/or to the formation of unwanted quinones-type by-products. Through this
synthetic sequence we then obtained all the aromatic precursors 4a-d with all substituents in the
correct position.
Scheme 2. Preparation of functionalized aromatic moieties 4a-d.
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Scheme 3. Protection of the phenolic moieties.
Finally, to compare the effect of protecting groups on the following synthetic steps, the phenolic
groups of compounds 4a-d were protected as either SEM- or MEM-ethers 5 and 6, respectively
(Scheme 3). The SEM-derivatives 5a, 5b, and 5d were obtained in quantitative yield, while the
protection with the cheaper MEM-chloride provided products 6a and 6c in a lower yield (80%).
Both enantiomers of the side chain were prepared in high enantiomeric purity by Sharpless
asymmetric dihydroxylation starting from geranyl acetate (Scheme 4). This procedure allows the
formation of both enantiomers of the required diol 7 in high yield, high stereoselectivity (94-98%
ee, see below), and full regioselectivity.14 In this reaction, the temperature control is extremely
important because at T < 0°C a very low conversion occurs, whereas at T > 4°C oxidation of both
double bonds is observed. The absolute configuration of the obtained diols was preliminarily
assigned14a on the basis of the predictions provided by the empirical Sharpless rule.18 Accordingly,
the oxidation reaction was expected to provide diol (R)-7 when carried out with the ADmixβ
reagent and diol (S)-7 when ADmixα is employed. Diols (R)- and (S)-7 were then transformed into
ketals (R)-8 and (S)-8, respectively, and the ester groups were hydrolyzed to give alcohols (R)- and
(S)-9. Notably, all steps of the synthetic sequence, starting from geranyl acetate, provided very high
yields (≥90%).
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Scheme 4. Asymmetric synthesis of the side chain
Although the empirical Sharpless rule18 is commonly employed to predict the absolute
configuration of the diols obtained by alkene dihydroxylation with osmium tetraoxide as oxidant
and either dihydroquinine or dihydroquinidine derivatives as chiral ligands, some cases have been
described in which such a rule fails to predict the correct assignment.19 Therefore, for a more sound
configurational assignment, we turned out to the use of Electronic Circular Dichroism (ECD)
spectroscopy. In particular, we employed flexible biphenyl chiroptical probes for this purpose,
following an approach described by our group which proved to be very straightforward and reliable
for the absolute configuration assignment to chiral diols,20 acids,21 and amines.22 Following this
approach, to assign the absolute configuration to a chiral diol it must be transformed in the
corresponding biphenyl dioxolane and the ECD spectrum of the latter analyzed. A positive Cotton
effect at 250 nm (A band) in the ECD spectrum reveals a (R) absolute configuration of the diol
while a negative band is allied to an (S) configuration. Therefore, (−)-7, obtained from ADmixα,
was transformed to the corresponding biphenyl dioxolane20 13 (Scheme 5) and ECD spectrum of
the latter was recorded. The appearance of a clear negative Cotton effect at 250 nm in the ECD
spectrum (Figure 2) allows us to assign (S) absolute configuration to the diol (−)-7, then reliably
and independently confirming the tentative assignment based on the empirical Sharpless rule. This
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also allowed to establish the absolute configuration of compounds 8-10 derived from 7. Therefore,
the (R)/(-), (S)/(+) relationship was established for the ketals 8 and 9, while the instability of
bromide 10 did not allow measurement of its optical rotation. Moreover, this result allows to correct
the optical rotation sign of (R)-9 erroneously reported in the literature.23
Scheme 5. Synthesis of the biphenyl dioxolane derivative 13
10
8
20
∆ε
ε.10−3
15
6
4
2
0
10
5
-2
-4
-6
-8
-10
0
200
220
240
260
λ (nm)
280
300
320
Fig. 2. ECD and UV spectra of biphenyldioxolane 13 in THF.
To determine the enantiomeric excess of alcohol 9 by chiral HPLC analysis, both enantiomers were
transformed into the corresponding benzoates and separated on Chiralcel OD chiral stationary
phase, providing 98% ee for (R)-9 and 94% ee for the (S)-9 enantiomer. Alcohols (R)-9 and (S)-9
were then transformed, by treatment with 0.5 equivalent of PBr3 at −7°C in anhydrous diethyl ether,
into the corresponding bromides (R)-10 and (S)-10, respectively. After work-up, pure bromides (R)-
1
and (S)-10 were isolated in 90% yield, as confirmed by their HNMR spectra. Bromides 10 were
8

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used within 3-4 days of their preparation because they turned out to be very light sensitive slowly
transforming, even if stored in the dark at low temperature and under inert atmosphere, into a
diastereoisomeric mixture of tetrahydrofuran 11 (Scheme 4).14a The same behavior was shown by
the triflates of alcohols (R)- and (S)-9. In fact, by treating the alcohols 9 with triflic anhydride at
−78°C, the corresponding triflate was obtained in only 43% yield and a significant amount of
tetrahydrofuran 11 was isolated.
The coupling between 3-bromo-benzoates 5 and 6 and the optically active bromides 10 was first
attempted by Suzuki-Miyaura cross-coupling. Initially, various attempts were carried out to
transform 5 and 6 or even the unprotected analogues 4a-d into the boronic esters required for the
Suzuki-Miyaura coupling, but these reactions failed, probably as a consequence of the high steric
hindrance at the halogenated position on the aromatic moieties. Therefore, we turned to employ
mixed cuprate chemistry for the coupling. Accordingly, SEM-protected benzoates 5 were lithiated
at C-3 with n-butyllithium at −78°C in THF and treated at the same temperature with a freshly
prepared solution of (3-methyl-3-methoxy-1-butynyl)copper in THF and HMPA to provide the
mixed cuprate.24 Then, (R)- or (S)-bromide 10 in THF was added to this solution and the mixture
was slowly warmed at room temperature stirring for 18-20 h (Scheme 6). After quenching with a
pH 8 solution of ammonia/ammonium chloride the crude product was purified by column
chromatography affording benzoates 14 in 40-72% yield (Scheme 6). The same procedure provided
benzoate 15 starting from the MEM-protected bromoesters 6. As shown in Table 1, the reactions do
not appear to be influenced by the nature of the protecting group on the aromatic ring, since the
SEM-benzoate 5a and its MEM-analogue 6a gave the coupling products 14a and 15a, respectively,
in comparable yields (entry 1 vs 2). By working in a slight excess of bromides 10 the yield is
increased. In fact, in this case the coupling provided the 5-haloesters 15c and 14b in 70% yield,
starting from 6c and 5b, respectively (entries 3 and 4). No significant influence by the nature of the
halogen atom on the aromatic ring was found. A larger amount of electrophile did not improve the
yield but made the cross-coupling side reaction between the butynyl copper and bromide 10 more
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significant. Notably, in these reaction conditions the metalation of 3,5-dibromobenzoate 6c is
completely regioselective, since no trace of products resulting from either lithiation at C-5 position
of the substrate or from a double coupling reaction was detected. Conversely, a lower scale seems to
have negative effects on yield, because the same substrate 5b provided the product 14b in only 40%
yield when the reaction was carried out on 0.3 mmol solution (entry 5). From the coupling of 5-
chlorobenzoate 5b with both bromides (R)- and (S)-10, both enantiomers of the precursor 14b of
colletochlorin A (1b) were prepared, while the fluorinated analogue was also obtained in moderate
yield (entry 6) from 5-fluorobenzoate 5d.
Scheme 6. Cross-coupling reaction. See Table 1 for reaction yields.
Table 1. Coupling reactions: synthesis of benzoates 14-15
Entrya
Substrate
X
H
PG
10 (eq.)
Product
(S)-14a
(R)-15a
(R)-15c
(S)-14b
(R)-14b
(R)-14d
Yieldb (%)
1
2
SEM
MEM
MEM
SEM
SEM
SEM
(S)-10 (1.0)
(R)-10 (1.0)
(R)-10 (1.2)
(S)-10 (1.2)
(R)-10 (1.2)
(R)-10 (1.2)
42
40
70
72
40
46
5a
6a
6c
5b
5b
5d
H
3
Br
Cl
Cl
F
4
5c
6
a) All reactions were carried out using 0.5 mmol of substrate. b) Isolated yield. c) Performed with 0.3 mmol of 5b.
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The ester moiety of all compounds 14 and 15 was then transformed into the aldehyde in high
overall yields by means of a LiAlH4 reduction followed by oxidation of the resulting alcohol with
pyridinium chlorochromate (PCC) (Scheme 7).
Scheme 7.
The final step of the total synthesis involves the deprotection of the phenolic and diol moieties
(Scheme 8). The procedure reported in the literature to cleave SEM and MEM protecting groups by
P2I4 in dichloromethane which, thanks to the mild conditions employed, is claimed to be the best for
resorcinols, was instead unsuccessful in our case, providing no reaction at room temperature and a
mixture of unidentified products by treatment at higher temperature or for longer reaction time.
Instead, reaction of 16a with TBAF in THF/HMPA provided the removal of both SEM groups,
giving ketal 18a (Scheme 8). The latter was then hydrolized with H2SO4/H2O providing colletorin
A (S)-1a in 49% overall yield (Table 2, entry 1, method A). As regards to the deprotection of MEM
ethers we discarded the use of the most common reagents, namely Lewis acids such as zinc or
magnesium bromide, as it is known that acidic conditions induce ring closing and chroman or
chromene formation and are not applicable to highly functionalized phenol ethers like 17.13a,25
Therefore, we decided to test the same procedure used above to cleave the SEM groups, even if
only a single example reported its use to remove MEM.26 We however observed that TBAF was
usuitable to remove both MEM ethers, providing only the 4-monoprotected 2-
hydroxybenzaldehyde. After several attempts, it was found that treatment of the solution of MEM
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ethers 17a,c in THF with 6M HCl provides the simultaneous deprotection of both the phenol and
diol moieties, giving colletorin (R)-1a as well as the brominated analogue (R)-1c in moderate to
good yields (Table 2, entries 2-3, method B). For comparison, the two enantiomeric benzaldehydes
(S)-16b and (R)-16b were deprotected according to the method A and B, respectively, to provide
both enantiomers of colletochlorin A, (S)-1b and (R)-1b, (entries 4-5). Although the procedure with
HCl (method B) was simpler and shorter, while providing the simultaneous cleavage of both SEM
protecting group and ketal moiety, a better overall yield was achieved using the two-step sequence
based on the selective deprotection of phenolic and diol moieties (method A). This protocol was
also used as final step in the total synthesis of the fluorinated analogue (R)-1d (entry 6). The
enantioselective synthesis of 1a-d allows to establish to all these compounds the (R)/(+), (S)/(-)
relationship between the absolute configuration and the optical rotation sign, then providing a
reliable absolute configuration assignment to the naturally occurring colletorin A (1a) and
colletochlorin A (1b). In the latter case such assignment agrees with that carried out by application
of the Mosher’s method.5
Scheme 8. Final steps of total synthesis. See Table 2 for reaction yields.
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Table 2: Deprotection of phenol and diol moieties.
Entry
Substrate
(S)-16a
(R)-17a
(R)-17c
(S)-16b
(R)-16b
(R)-16d
X
H
PG
Method
Product
(S)-1a
(R)-1a
(R)-1c
(S)-1b
(R)-1b
(R)-1d
Total Yield (%)
1
2
3
4
5
6
SEM
MEM
MEM
SEM
SEM
SEM
49
30
55
46
37
30
A
B
B
A
B
A
H
Br
Cl
Cl
F
Finally, the synthesized compounds were subjected to bioactivity assays in order to investigate their
biological properties. In particular, we wanted to evaluate their phytotoxicity and insecticidal
activity. The phytotoxic activity was assayed on Ambrosia artemisifolia and Sonchus arvensis, two
weedy plants, on which the toxicity of the naturally occurring 1b was recently reported with
comparable results,4,5 by using a leaf puncture assay. Droplets of solutions (10 µl) containing the
metabolites (20 µg) were added to detached leaves previously wounded with a needle, kept in
moistened chambers, thus observing the eventual appearance of necrotic lesions. The effect on
chlorophyll content was tested on an aquatic model plant, Lemna minor, by adding the test solutions
to wells containing fronds of the plant, and then determining by spectrophotometer the eventual
reduction of the chlorophyll content in comparison with an untreated control. The results,
summarized in Table 3, indicate that both enantiomers of 1b exhibit the strongest phytotoxicity on
S. arvensis and, among thm, the natural enantiomer (R)-1b shows a slightly higher toxicity than its
enantiomer on A. artemisifolia plants. Conversely, the other tested compounds namely, both (R)-
and (S)-1a and the halogenated unnatural analogues (R)-1c and (R)-1d cause only weak effects.
Moreover on the L. minor assay, all the tested molecules cause a reduction in chlorophyll content,
even though to a different extent, with (S)-1a being the most active compound and the fluorinated
analogue (R)-1d the least. In conclusion, the presence of a chlorine atom seems to impart to these
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componds higher phytotoxicity against weeds, in respect to the other halogens. The same enhanced
phytotoxicity is observed with the naturally occurring (R) absolute configuration at the
hydroxylated stereocenter. Conversely, halogenated compounds displayed lower phytotoxicity
against Lemna minor. These results highlight the relevant effects that both the halogen nature and
the absolute configuration have on the bioactivity of these fungal metabolites.
Table 3. Phytotoxic activity assays
Compound
Sonchus
arvensisa
Ambrosia
artemisiifolia
Lemna
minorb
(R)-1a
(S)-1a
(R)-1b
(S)-1b
(R)-1c
(R)-1d
++
+
+
+
62.7
71.6
59.1
43.5
59.5
16.0
+++
+++
++
++++
+++
+
++
+
aNecrotic symptoms expressed as diameter of the necrosis in the leaf puncture
assay, by using a visual scale from “-“ = no symptoms, to “++++” = necrosis
b
around 1 cm diameter. Reduction of chlorophyll content expressed as percentage
in comparison with the untreated control.
Insecticidal activity of both enantiomers of 1b and the brominated analogue (R)-1c was examined
via topical application and injection on Aedes aegypti, the yellow fever mosquito, which is a vector
for transmitting several tropical fevers.27 The results (Table 4) show that all three compounds
exhibit negligible insecticidal activity against the adult mosquitos in both test methods. Except for
(R)-1b at 2 µg/mosquito dose (3.3 ± 3.3% mortality), none of the compounds shows any mortality
via topical application. Moreover, (R)-1b shows greater mortality in the injection assay (13 ± 3.3%),
but this value is not statistically significant compared to controls. A pre-treatment with piperonyl
butoxide (PBO) slightly increases toxicity of all three tested compounds, but again the effect is not
statistically significant in a t-test compared to compound injected alone. Thus, mixed-function
oxidases do not seem to be a major route of metabolism in the tested compounds in A. aegypti, and
the result suggests that the observed low toxicity is due to limited intrinsic insecticidal potency.
Table 4. Insecticidal activity assays Aeades aegypti
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Topical application
Injection
knock-down
(% ± SE)
6.7 ± 3.3
dose
dose
(µg/insect)
0.1
Compd
knock-down
(% ± SE)a
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
mortality
(% ± SE)a
3.3 ± 3.3
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
mortality
(% ± SE)
13.3 ± 3.3
33.3 ± 8.8
6.7 ± 3.3
(µg/insect)
(R)-1b
(S)-1b
(R)-1c
control
2
1
2
1
2
1
0.1 + PBOb
0.1
20.0 ± 5.8
10.0 ± 5.8
16.7 ± 8.8
6.7 ± 3.3
0.1 + PBO
0.1
30.0 ± 10.0
10.0 ± 5.8
23.3 ± 3.3
10.0 ± 5.8
10.0 ± 5.8
0.1 + PBO
without PBO
with PBO
13.3 ± 3.3
10.0 ± 5.8
6.7 ± 3.3
aknock-down effect was recorded at 1h post-treatment, and 24h for mortality.
b For assay with a mono-oxygenase synergist, mosquitoes were pre-treated with piperonyl butoxide (PBO, at 0.5
µg/insect) via a topical application, and left for 4 hr.
Conclusions
Herein we have shown an efficient method for the highly enantioselective synthesis of colletorin A
(1a) and colletochlorin A (1b) and their brominated (1c) and fluorinated (1d) synthetic analogues.
This protocol allows to carry out the most critical steps of the whole synthetic pathway, namely the
coupling and deprotection reactions, in medium to good yields. Indeed it was possible to obtain the
precursors of such compounds in 40-72% yields by coupling reactions between highly substituted
aromatic mixed cuprates and optically active allylic bromides. Moreover, two different procedures
for the deprotection of both phenolic and ketal moieties have been tested, depending on the nature
of the phenolic protecting group. Finally, a high regio- and stereocontrol is also guaranteed by the
Sharpless asymmetric dihydroxylation of geranyl acetate, which constitutes the enantioselective
step of the total synthesis. We believe that this strategy could be successfully applied also to other
highly functionalized phenolic compounds bearing terpenoid side chains. The enantioselective
synthesis of 1a-d allows to reliably assign the absolute configuration to the natural compounds 1a
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and 1b. The preliminary tests of biological activity as herbicides and insecticides carried out
highlight the potential bioactivity of these compounds and allow sorting out the dependence on the
absolute stereochemistry and on the halogen nature on the compound bioactivity.
Experimental Section
General Information
1H (500 or 400 MHz) and 13C (125 or 100 MHz) NMR spectra were recorded in CDCl3 on a Varian
INOVA 500 or a Varian INOVA 400 spectrometer, using tetramethylsilane (TMS) as an internal
standard. 19F (376 MHz) NMR spectra were recorded in CDCl3 on a Varian INOVA 400
spectrometer, using CFCl3 as an internal standard. GC–MS spectra were recorded on a Hewlett
Packard 6890 gas chromatograph, equipped with a mass spectrometric detector HP-5973 type and a
capillary column HP-5MS 30 m x 0.25 mm. Optical rotations were measured on a JASCO DIP-370
polarimeter, with α values expressed in deg·cm3·g-1·dm-1 and concentration c in g·(100 mL)-1.
HPLC analyses were performed on a JASCO PU-1580 intelligent HPLC pump equipped with a
Varian 2550 UV detector and a CHIRALCEL OD column. Analytical thin layer chromatography
(TLC) was performed using silica gel 60 Macherey-Nagel sheets and visualized by ultraviolet
radiation and/or spraying the plates with a potassium permanganate solution; column
chromatography separations were carried out using silica gel 60 (70–230 mesh). Et2O and THF
were freshly distilled before their use on sodium benzophenone ketyl under nitrogen atmosphere.
N,N-diisopropylethylamine (DIPEA), trimethylamine, and CH2Cl2 were dried by distillation over
calcium hydride and stored under a nitrogen atmosphere. Commercially available n-butyllithium
(Aldrich) was a 1.6 M solution in hexane. The other analytical grade solvents and commercially
available reagents were used without further purification. 3-Hydroxy-4-carbomethoxy-5-
methylcyclohexenone 3 was prepared starting from methyl crotonate and methyl acetoacetate as
described.15 The ketal 12 was prepared as described20 and (3-methyl-3-methoxy-1-butynyl)copper
16

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was obtained starting from the commercially available (Aldrich) cuprous acetylide (2-methylbut-3-
yn-2-ol) as already described elsewere.24
Synthesis of the aromatic precursors (4a-d)
Methyl 3-bromo-2,4-dihydroxy-6-methylbenzoate (4a)
To a solution of cyclohexenone 3 (1.0 g, 5.4 mmol) in acetic acid (10 mL) 2 equivalents of bromine
(0.56 mL, 10.8 mmol) were added. The mixture was first stirred at room temperature for 30 min and
then heated at 100 °C for further 30 min. After cooling to rt, the mixture was poured into water/ice
and extracted with ethyl acetate. The organic layer was washed with a saturated sodium
metabisulphite solution and brine. The crude product was purified by column chromatography on
silica gel (hexane:diethyl ether 7:3) (772 mg, 55%).1H-NMR (500 MHz, CDCl3) δ (ppm): 2.49 (s,
3H); 3.96 (s, 3H); 6.00 (bs 1H); 6.47 (s, 1H); 12.56 (s, 1H). 13C-NMR (125 MHz, CDCl3) δ (ppm):
24.2; 52.3; 96.6; 106.2; 110.7; 142.6; 156.8; 161.1; 172.0. MS (EI): m/z 262 (M+, 30); 260 (M+,
30); 230 (98); 228 (100); 149 (20).
Methyl 3,5-dibromo-2,4-dihydroxy-6-methylbenzoate (4c)
To a solution of cyclohexenone 3 (1.0 g, 5.4 mmol) in acetic acid (10 mL) 3 equivalents of bromine
(0.84 mL, 16.2 mmol) were added. The mixture was first stirred at room temperature for 30 min and
then heated at 100°C for further 30 min. After cooling to rt, the mixture was poured into water/ice
and extracted with ethyl acetate. The organic layer was washed with a saturated sodium
metabisulphite solution and brine. The crude product was purified by crystallization from hexane
(826 mg, 45%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 2.66 (s, 3H); 3.98 (s, 3H); 6.48 (s 1H); 12.19
(s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 23.3; 52.8; 96.3; 105.5; 107.7; 140.5; 153.9; 159.4;
171.2. MS (EI): m/z 342 (M+, 12); 340 (M+, 24); 338 (M+, 12); 310 (50); 308 (100); 306 (50).
17

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Methyl 3-bromo-5-chloro-2,4-dihydroxy-6-methylbenzoate (4b)
To a solution of the monobromide 4a (0.524 g, 2.0 mmol) in diethyl ether (4 mL) a solution of
sulfuryl chloride (1 M in dichloromethane, 4 mL) was added and the mixture was stirred at room
temperature for 3 h. By solvent removal under reduced pressure (300-400 mbar, 40°C) the pure
product was obtained (560.5 mg, 95%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 2.61 (s, 3H); 3.98 (s,
13
3H); 6.46 (bs 1H); 12.23 (s, 1H). C-NMR (100 MHz, CDCl3) δ (ppm): 19.8; 52.7; 96.6; 107.3;
113.8; 138.7; 153.3; 159.0; 171.3. MS (EI): m/z 298 (M+, 6); 296 (M+, 24); 294 (M+, 18); 266 (26);
364 (100); 262 (78).
Methyl 3-bromo-2,4-dihydroxy-5-fluoro-6-methylbenzoate (4d)
To a solution of the monobromide 4a (0.930 g, 3.58 mmol) in methanol (25 mL) Selectfluor® (1.65
g, 1.3 eq.) was added and the mixture was heated to 50°C for 24 h. The reaction was monitored by
GC-MS analysis and quenched, when no further conversion occurred (about 50%), by cooling at
room temperature and removing the solvent (300 mbar, 40°C). Then the residue was treated with
water and extracted with diethyl ether. The pure product was obtained by column chromatography
on silica gel (dichloromethane) in 45% yield (448 mg). 1H-NMR (500 MHz, CDCl3) δ (ppm): 2.68
(s, 3H); 3.98 (s, 3H); 6.48 (s 1H); 12.19 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 13.6 (d, J =
7.5 Hz); 52.5; 96.8 (d, J = 2.2 Hz); 104.9; 126.7 (d, J = 14.4 Hz); 143.2 (d, J = 231.4 Hz); 147.0 (d,
J = 21.7 Hz); 157.0 (d, J = 2.3 Hz); 171.6 (d, J = 3.0 Hz). 19F (376 MHz, CDCl3) δ (ppm): -145.4.
MS (EI): m/z 280 (M+, 26); 278 (M+, 27); 248 (98); 246 (100).
General procedure for the protection of phenolic groups
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2,4-dihydroxybenzoates 4a-d (1.0 mmol) were dissolved in 4 mL of dry dichloromethane and
DIPEA (5.0 mmol, 1.04 mL) was added, followed by the slow addition of the SEM-Cl or MEM-Cl
(4.0 mmol). The mixture was stirred at room temperature for 2-4 h and then poured into water/ice
and extracted with diethyl ether. The crude products were purified by column chromatography on
silica gel (hexane:diethyl ether 7:3 for SEM-ethers and 1:1 for MEM-protected compounds).
Methyl 2,4-bis[2-(trimethylsilyl)ethoxymethoxy]-3-bromo-6-methylbenzoate (5a)
Following the general procedure the product was isolated in quantitative yield (521 mg) as a
colorless oil. 1H-NMR (500 MHz, CDCl3) δ (ppm): 0.01 (s, 9H); 0.04 (s, 9H); 0.96 (t, J = 8.5 Hz,
2H); 1.01 (t, J = 8.5 Hz, 2H); 2.29 (s, 3H); 3.79 (t, J = 8.5 Hz, 2H); 3.85 (t, J = 8.5 Hz, 2H); 3.90
(s, 3H); 5.15 (s, 2H); 5.30 (s, 2H); 6.83 (s 1H).
Methyl 2,4-bis[2-(trimethylsilyl)ethoxymethoxy]-3-bromo-5-chloro-6-methylbenzoate (5b)
Following the general procedure the product was isolated in quantitative yield (556 mg) as a
colorless oil. 1H-NMR (500 MHz, CDCl3) δ (ppm): 0.04 (s, 18H); 1.01 (m, 4H); 2.30 (s, 3H); 3.83
(t, J = 8.5, Hz, 2H); 3.92 (s, 3H); 4.00 (t, J = 8.5 Hz, 2H); 5.14 (s, 2H); 5.21 (s, 2H).
Methyl 2,4-bis[2-(trimethylsilyl)ethoxymethoxy]-3-bromo-5-fluoro-6-methylbenzoate (5d)
Following the general procedure the product was isolated in 95% yield (512 mg) as a colorless oil.
1H-NMR (500 MHz, CDCl3) δ (ppm): 0.04 (s, 18H); 1.19 (m, 4H); 2.34 (s, 3H); 3.85 (t, J = 8.5
Hz, 2H); 3.92 (s, 3H); 4.02 (t, J = 8.5 Hz, 2H); 5.15 (s, 2H); 5.21 (s, 2H).
Methyl 2,4-bis[(2-methoxyethoxy)methoxy]-3-bromo-6-methylbenzoate (6a)
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Following the general procedure the product was isolated in 82% yield (357 mg) as colorless oil.
1H-NMR (500 MHz, CDCl3) δ (ppm): 2.34 (s, 3H); 3.37 (s, 3H); 3.40 (s, 3H); 3.72 (m, 4H); 3.79
(m, 2H); 3.85 (m, 2H); 3.89 (s, 3H); 5.24 (s, 2H); 5.33 (s, 2H); 6.93 (s, 1H).
Methyl 2,4-bis[(2-methoxyethoxy)methoxy]-3,5-dibromo-6-methylbenzoate (6c)
Following the general procedure the product was isolated in 80% yield (413 mg) as colorless oil.
1H-NMR (400 MHz, CDCl3) δ (ppm): 2.32 (s, 3H); 3.38 (s, 3H); 3.39 (s, 3H); 3.58 (m, 4H); 3.70
(m, 2H); 3.91 (s, 3H); 4.09 (m, 2H); 5.18 (s, 2H); 5.24 (s, 2H).
Asymmetric synthesis of the side chain
(R,E)-6,7-Dihydroxy-3,7-dimethyloct-2-en-1-yl acetate ((R)-7)
AD-mix-β (14 g, 10 mmol) and methanesulfonamide (951 mg, 10 mmol) were added to a 1:1
mixture of t-BuOH:H2O (100 mL) and vigorously stirred for 15 min at room temperature. After
cooling to 0 °C, geranyl acetate (2.13 mL, 10 mmol) was added, the mixture was stirred at that
temperature for 24 h, then quenched with solid sodium metabisulfite (13.5 g). The mixture was
extracted several times with dichloromethane and the combined organic layers were washed with a
10% aq. NaOH solution and brine. The crude product was purified by column chromatography on
silica gel (hexane:ethyl acetate 7:3) to afford the diol (R)-7 in 96% yield (2.20 g) as a colorless oil.
25
23
[α]D = +26.1 (c = 0.7, CHCl3), lit.28 [α]D = +26.8 (c = 1.0, CHCl3). Following the same
25
procedure with AD-mix-α, the enantiomeric diol (S)-7 was isolated in 90% yield (2.07 g). [α]D
=
−23.9 (c = 0.9, CHCl3), lit.29 [α]D20 = −25.1 (c = 0.7, EtOH). 1H-NMR (500 MHz, CDCl3) δ (ppm):
1.17 (s, 3H); 1.22 (s, 3H); 1.45 (m, 1H); 1.62 (m, 1H); 1.73 (s, 3H); 1.91 (br s, 2H); 2.06 (s, 3H);
2.12 (m, 1H); 2.32 (m, 1H); 3.45 (dd, J = 10.5, 2.0 Hz, 1H) 4.59 (d, J = 7.0 Hz, 2H); 5.40 (br t, J =
7.0 Hz, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 16.4; 21.01; 23.2; 26.5; 29.4; 35.6; 61.3; 73.0;
20

ACCEPTED MANUSCRIPT
78.0; 118.7; 142.0; 171.1. MS (EI): m/z 170 (M+ - AcOH, 1); 152 (3); 111 (18); 94 (32); 81 (27); 68
(72); 59 (100); 43 (73).
(R,E)-3-Methyl-5-(2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)pent-2-en-1-yl acetate ((R)-8)
To a solution of the diol (R)-7 (2.06 g, 9 mmol) in methanol (5 mL) dimethoxypropane (15 mL) and
p-toluensulfonic acid monohydrate (10 mg) were sequentially added and the mixture was stirred at
room temperature for 3 h. The mixture was quenched with pyridine (0.051 mL) and concentrated
under reduced pressure. The residue was dissolved in ethyl acetate and filtered on a silica gel pad to
25
give the pure ketal in 93% yield (2.26 g). [α]D = −2.0 (c = 1, CHCl3). Following the same
25
1
procedure, the ketal (S)-8 was isolated in 90% yield (2.19 g). [α]D = +2.0 (c = 1, CHCl3). H-
NMR (500 MHz, CDCl3) δ (ppm): 1.10 (s, 3H); 1.24 (s, 3H); 1.33 (s, 3H); 1.42 (s, 3H); 1.50 (m,
1H); 1.65 (m, 1H); 1.73 (s, 3H); 2.06 (s, 3H); 2.10 (m, 1H); 2.29 (m, 1H); 3.65 (dd, J = 9.5, 3.0 Hz,
1H) 4.59 (d, J = 7.5 Hz, 2H); 5.39 (dt, J = 7.0, 1.5 Hz, 1H). 13C-NMR (125 MHz, CDCl3) δ (ppm):
16.4; 20.9; 22.7; 25.8; 26.7; 27.1; 28.3; 36.4; 61.1; 79.9; 82.5; 106.4; 118.6; 141.3; 170.8. MS (EI):
m/z 255 (M+ - 15, 52); 153 (54); 135 (60); 85 (50); 81 (65); 71 (84); 43 (100).
(R,E)-3-Methyl-5-(2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)pent-2-en-1-ol ((R)-9)
To a solution of the ketal (R)-8 (2.16 g, 8 mmol) in methanol (27 mL) potassium carbonate (510
mg) was added and the mixture was stirred at room temperature. After 24 h the mixture was
quenched with 2 mL of a saturated aq. solution of NH4Cl and methanol was removed under reduced
pressure. The residue was dissolved in ethyl acetate and the organic phase was washed with brine.
The pure alcohol (R)-9 was isolated by evaporation of the solvent in 92% yield (1.68 g) as a
25
colorless oil. [α]D = −2.8 (c = 1, CHCl3) . Following the same procedure, the alcohol (S)-9 was
obtained in 92% yield (1.68 g). [α]D25 = +3.1 (c = 1, CHCl3). Both alcohols 9 were stored at - 20 °C
1
under argon atmosphere. H-NMR (500 MHz, CDCl3) δ (ppm): 1.10 (s, 3H); 1.25 (s, 3H); 1.33 (s,
21

ACCEPTED MANUSCRIPT
3H); 1.42 (s, 3H); 1.52 (m, 1H); 1.64 (m, 1H); 1.70 (s, 3H); 2.04 (s, 1H); 2.07 (m, 1H); 2.27 (m,
1H); 3.65 (dd, J = 9.2, 3.2 Hz, 1H) 4.17 (d, J = 7.0 Hz, 2H); 5.46 (br t, J = 7.0 Hz, 1H). 13C-NMR
(100 MHz, CDCl3) δ (ppm): 16.3; 22.9; 26.0; 26.9; 27.5; 28.6; 36.6; 59.3; 80.1; 82.9; 106.6; 123.6;
139.1. MS (EI): m/z 213 (M+ - 15, 46); 142 (48); 109 (22); 81 (100); 71 (92); 59 (60); 43 (88).
In order to determine the enantiomeric excesses by chiral HPLC analysis, a small portion of both
alcohols 9 was transformed into the corresponding benzoate as follows: anhydrous triethylamine
(21 µL, 0.15 mmol) and benzoyl chloride (12 µL, 0.1 mmol) were sequentially added to a solution
of the alcohol (23 mg, 0.1 mmol) in anhydrous diethyl ether at 0°C. The mixture was then stirred at
room temperature for 16 h, poured into icy water and extracted with ethyl acetate to give the
benzoate in 80% yield (25.8 mg). 1H-NMR (500 MHz, CDCl3) δ (ppm): 1.12 (s, 3H); 1.25 (s, 3H);
1.33 (s, 3H); 1.43 (s, 3H); 1.55 (m, 1H); 1.68 (m, 1H); 1.81 (s, 3H); 2.14 (m, 1H); 2.33 (m, 1H);
3.68 (dd, J = 9.5, 3.5 Hz, 1H) 4.87 (d, J = 7.5 Hz, 2H); 5.54 (br t, J = 7.5 Hz, 1H); 7.44 (t, J = 8.0
Hz, 2H); 7.56 (t, J = 8.0 Hz, 1H); 8.06 (d, J = 8.0 Hz, 2H). The enantiomers were separated by
HPLC on Chiralcel-OD column, hexane:2-propanol 98:2, 254 nm: tR(S) = 9.6 min, tR(R) = 11.7
min. The alcohol (R)-9 was found to possess 98% ee, while its enantiomer (S)-9 gave 94% ee.
(R,E)-5-(5-bromo-3-methylpent-3-en-1-yl)-2,2,4,4-tetramethyl-1,3-dioxolane ((R)-10)
To a solution of the alcohol (R)-9 (230 mg, 1.0 mmol) in anhydrous diethyl ether PBr3 (48 µL, 0.51
mmol) was slowly added at −7°C. After 20 min the mixture was diluted with diethyl ether and
poured into a cooled 10% aq. NaHCO3 solution. The separated organic phase was washed with
NaHCO3 and brine and the solvent removed under reduced pressure to give the pure bromide (R)-10
in 90% yield (262 mg). Bromide (S)-10 was obtained by the same procedure, starting from alcohol
(S)-9, in the same 90% yield (262 mg). These compounds are stable for about one week only if
stored at −20 °C under argon atmosphere. 1H-NMR (500 MHz, CDCl3) δ (ppm): 1.10 (s, 3H); 1.25
22

ACCEPTED MANUSCRIPT
(s, 3H); 1.33 (s, 3H); 1.42 (s, 3H); 1.50 (m, 1H); 1.63 (m, 1H); 1.75 (s, 3H); 2.12 (m, 1H); 2.31 (m,
1H); 3.66 (dd, J = 9.5, 2.5 Hz, 1H) 4.03 (d, J = 8.3 Hz, 2H); 5.59 (br t, J = 8.3 Hz, 1H).
(E)-5-(4’,4’-dimethyl-5,7-dihydrospiro[dibenzo[a,c][7]annulene-6,2’-[1,3]dioxolan]-5’-yl)-3-
methylpent-2-en-1-yl acetate (13)
To a solution of the ketal 12 (91.6 mg, 0.36 mmol) in anhydrous CHCl3 (3 mL) the diol (S)-7 (83
mg, 0.36mmol) and traces of p-toluenesulfonic acid mono-hydrate were added in the presence of 4-
Å molecular sieves. After 8 h of stirring at room temperature, the reaction mixture was filtered and
the solvent removed under reduced pressure. The crude product was purified by column
chromatography on silica gel (diethyl ether:petroleum ether 1:3) giving the biphenyl dioxolane 13
25
1
as a yellow oil (43.8 mg, 29% yield). [α]D = −70.0 (c = 1.75, CHCl3); H-NMR (500 MHz,
CDCl3) δ (ppm): 1.22 (s, 3H); 1.28 (s, 3H); 1.59 (m, 1H); 1.69 (m, 1H); 1.75 (s, 3H); 2.06 (s, 3H);
2.18 (m, 1H); 2.32 (m, 1H); 2.65 (m, 1H); 2.72 (m, 2H); 2.86 (m, 1H); 3.75 (br s, 1H); 4.64 (br s,
2H); 5.46 (br s, 1H); 7.24 (t, J = 7 Hz, 1H); 7.30 (m, 3H); 7.36 (t, J = 7 Hz, 2H); 7.44 (d, J = 7.0 Hz,
13
2H); C-NMR (125 MHz, CDCl3) δ (ppm): 10.9; 20.0; 21.1; 26.8; 35.9; 36.6; 43.9; 50.4; 61.4;
80.3; 82.5; 116.2; 119.2; 126.9; 127.3; 128.1; 128.5; 136.1; 140.2; 145.7; 171.1.
General procedure for coupling reactions: synthesis of benzoates 14 and 15
A solution of 3-methoxy-3-methyl-1-butyne (57 mg, 0.57 mmol) in THF (0.5 mL) was treated with
n-BuLi (1.6 M in hexane, 356 µL, 0.57 mmol) at 0°C, stirred for 5 min and added to a suspension
of copper iodide (109 mg, 0.57 mmol) in THF (0.5 mL), precooled to 0°C, giving a red-orange
solution which was stirred at that temperature for 30 min. Then HMPA (175 µL) was added and the
resulting solution of the cuprous acetylide was transferred to a solution of the desired lithium-aryl
reagent previously prepared as follows: the benzoates 5 (or 6) (0.5 mmol) were dissolved in THF (3
mL), the solution was cooled at −78°C, treated with n-BuLi (0.57mmol) and, after 15 min, with the
23

ACCEPTED MANUSCRIPT
solution of cuprous acetylide. The resulting clear solution was stirred at −78°C for 30 min and the
optically active bromide 10 (0.57 mmol) solution in THF (0.7 mL) was added. The mixture was
slowly warmed to room temperature and stirred for 16-20 h. The mixture was poured in a pH 8
aqueous ammonia/ saturated ammonium chloride solution and extracted with diethyl ether. The
organic phase was filtered on Celite and the crude products were purified by column
chromatography on silica gel.
(S,E)-methyl 6-methyl-3-(3-methyl-5-(2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)pent-2-en-1-yl)-2,4 -
bis((2-(trimethylsilyl)ethoxy)methoxy)benzoate ((S)-14a)
The product was isolated by column chromatography on silica gel (hexane:diethyl ether 3:1) in 42%
yield (137 mg). 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.00 (s, 9H); 0.02 (s, 9H); 0.97 (m, 4H); 1.06
(s, 3H); 1.21(s, 3H); 1.25 (m, 1H); 1.27 (s, 3H); 1.39 (s, 3H); 1.58 (m, 1H); 1.77 (s, 3H); 1.98 (m,
1H); 2.18 (m, 1H); 2.28 (s, 3H); 3.37 (d, J = 6.8 Hz, 2H); 3.61 (dd, J = 9.4, 3.4 Hz, 1H); 3.75 (m,
13
4H); 3.88 (s, 3H); 4.99 (s, 2H); 5.20 (br t, J = 7.0 Hz, 1H); 5.22 (s, 2H); 6.75 (s, 1H). C-NMR
(100 MHz, CDCl3) δ (ppm): -1.4; 16.3; 18.0;18.1; 19.9; 22.9; 23.2; 26.0; 26.7; 27.7; 28.5; 36.7;
52.0; 66.3; 67.5; 80.0; 82.9; 92.6; 99.0; 106.4; 111.8; 122.0; 123.2; 134.3; 135.2; 153.6; 157.0;
168.7.
(R,E)-methyl 2,4-bis((2-methoxyethoxy)methoxy)-6-methyl-3-(3-methyl-5-(2,2,5,5tetramethyl- 1,3-
dioxolan-4-yl)pent-2-en-1-yl)benzoate ((R)-15a)
The product was purified by column chromatography on silica gel (hexane:ethyl acetate 1:1). In this
case a further chromatographic purification was necessary (silica gel, chloroform) affording the
1
pure product in 40% yield (114 mg). H-NMR (500 MHz, CDCl3) δ (ppm): 1.08 (s, 3H); 1.21(s,
3H); 1.27 (s, 3H); 1.30 (m, 1H); 1.40 (s, 3H); 1.44 (m, 1H); 1.58 (m, 1H); 1.77 (s, 3H); 2.00 (m,
1H); 2.18 (s, 3H); 3.32 (d, J = 7.0 Hz, 2H); 3.37 (s, 3H); 3.39 (s, 3H); 3.55 (m, 4H); 3.62 (dd, J =
24

ACCEPTED MANUSCRIPT
9.5, 3.0 Hz, 1H); 3.72 (m, 2H); 3.79 (m, 2H); 3.88 (s, 3H); 4.83 (s, 2H); 5.03 (br t, J = 6.8 Hz, 1H);
5.22 (s, 2H); 6.84 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 16.2; 16.5; 22.9; 24.9; 26.0; 26.7;
27.6; 28.5; 36.6; 52.1; 59.0; 67.4; 67.7; 71.4; 71.5; 71.7; 80.0; 82.8; 92.3; 93.5; 94.0; 100.1; 106.4;
122.7; 123.4; 134.2; 135.2; 152.7; 156.1; 169.1.
(R,E)-methyl
3-bromo-4,6-bis((2-methoxyethoxy)methoxy)-2-methyl-5-(3-methyl-5-(2,2,5,5-
tetramethyl-1,3-dioxolan-4-yl)pent-2-en-1-yl)benzoate ((R)-15c)
The product was isolated in 70% yield (226 mg) by column chromatography on silica gel
(hexane:diethyl ether 1:1). 1H-NMR (500 MHz, CDCl3) δ (ppm): 1.07 (s, 3H); 1.21(s, 3H); 1.27 (s,
3H); 1.39 (s, 3H); 1.43 (m, 1H); 1.57 (m, 1H); 1.74 (s, 3H); 1.99 (m, 1H); 2.18 (dt, J = 10.0, 4.5 Hz,
1H); 2.32 (s, 3H); 3.38 (s, 3H); 3.39 (s, 3H); 3.48 (d, J = 6.0 Hz, 2H); 3.58 (m, 4H); 3.60 (dd, J =
10, 3.2 Hz, 1H); 3.83 (m, 2H); 3.90 (s, 3H); 3.97 (m, 2H); 5.04 (s, 2H); 5.14 (s, 2H); 5.22 (br t, J =
13
6.4 Hz, 1H). C-NMR (125 MHz, CDCl3) δ (ppm): 16.4; 20.8; 22.9; 24.6; 26.0; 26.7; 27.6; 28.5;
36.5; 52.4; 59.1; 66.7; 69.3; 69.6; 71.6; 71.7; 80.0; 82.7; 95.6; 98.7; 99.7; 106.4; 122.7; 127.1;
129.2; 134.5; 135.1; 152.3; 154.6; 167.9.
(S,E)-methyl 3-chloro-2-methyl-5-(3-methyl-5-(2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)pent-2-en-1-
yl)-4,6-bis((2-(trimethylsilyl)ethoxy)methoxy)benzoate ((S)-14b)
The product was isolated in 72% yield (247 mg) by column chromatography on silica gel (hexane:
diethyl ether 3:1). By the same procedure, starting from 0.3 mmol of substrate, the enantiomer (R)-
14b was obtained in 40% yield (82 mg). 1H-NMR (500 MHz, CDCl3) δ (ppm): 0.02 (s, 9H); 0.03 (s,
9H); 0.98 (m, 4H); 1.06 (s, 3H); 1.21 (s, 3H); 1.27 (s, 3H); 1.39 (s, 3H); 1.43 (m, 1H); 1.58 (m, 1H);
1.77 (s, 3H); 2.00 (m, 1H); 2.19 (dt, J = 10.5, 4.5 Hz, 1H); 2.29 (s, 3H); 3.37 (d, J = 6.8 Hz, 2H);
3.61 (dd, J = 9.5, 3.5 Hz, 1H); 3.76 (m, 2H); 3.89 (m, 2H); 3.91 (s, 3H); 4.99 (s, 2H); 5.09 (s, 2H);
13
5.25 (br t, J = 6.0 Hz, 1H). C-NMR (100 MHz, CDCl3) δ (ppm): −1.4; 16.5; 17.6; 18.1; 22.9;
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ACCEPTED MANUSCRIPT
24.4; 26.0; 26.7; 27.7; 28.5; 36.6; 52.4; 67.6; 67.8; 80.0; 82.8; 97.8; 99.1; 106.4; 122.8; 124.9;
126.9; 129.2; 132.7; 135.0; 151.6; 153.6; 168.0.
(R,E)-methyl 3-fluoro-2-methyl-5-(3-methyl-5-(2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)pent-2-en -1-
yl)-4,6-bis((2-(trimethylsilyl)ethoxy)methoxy)benzoate ((R)-14d)
The product was isolated in 46% yield (154 mg) by column chromatography on silica gel
(hexane:diethyl ether 3:1) followed by further column chromatography on silica gel
(dichloromethane). 1H-NMR (500 MHz, CDCl3) δ (ppm): 0.03 (s, 9H); 0.04 (s, 9H); 0.99 (m, 4H);
1.08 (s, 3H); 1.22 (s, 3H); 1.29 (s, 3H); 1.41 (s, 3H); 1.43 (m, 1H); 1.61 (m, 1H); 1.78 (s, 3H); 2.02
(m, 1H); 2.20 (s, 3H); 2.21 (m, 1H); 3.44 (d, J = 6.0 Hz, 2H); 3.63 (br d, J = 9.5 Hz, 1H); 3.78 (t, J
= 8.5 Hz, 2H); 3.85 (t, J = 9.0 Hz, 2H); 3.91 (s, 3H); 4.98 (s, 2H); 5.18 (s, 2H); 5.24 (m, 1H). 13C-
NMR (125 MHz, CDCl3) δ (ppm): −1.4; 18.0; 18.1; 22.9 (d, J = 12.5 Hz); 24.0; 26.0 (d, J = 11.5
Hz); 26.7 (d, J = 9.5 Hz); 27.7; 28.5 (d, J = 7.6 Hz); 30.9; 31.0; 36.6; 52.3 (d, J = 20.0 Hz); 67.5;
67.6; 80.0; 82.9 (d, J = 19.0 Hz); 97.3 (d, J = 7.6 Hz); 99.2; 106.4; 121.9 (d, J = 19.5 Hz); 122.8 (d,
J = 24.0 Hz); 125.0; 128.4; 134.9; 149.2 (d, J = 119.3 Hz); 151.6; 167.5; 206.9.
General procedure for the synthesis of aldehydes 16 and 17
To a suspension of LiAlH4 (18 mg, 0.47 mmol) in diethyl ether (4 mL) a solution of benzoates 14
(or 15, 0.2 mmol) in diethyl ether (5 mL) was added at 0°C and the mixture was stirred at room
temperature for 2 h. Then it was diluted with diethyl ether (15-20 mL), treated with an aqueous
saturated solution of sodium sulfate (167 µL), filtered and the solvent removed under reduced
pressure. The crude alcohol was dissolved in dichloromethane (7 mL), treated with PCC (2 eq.) and
stirred at room temperature for 1h. Then, 2 more equivalents of PCC were added and the mixture
was stirred for further 2-3 hours, when a complete conversion to aldehyde was detected by TLC
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analysis. The mixture was diluted with diethyl ether, filtered on celite and subsequently on a silica
gel pad, eluting with diethyl ether. By removing the solvent at reduced pressure all pure aldehydes
were recovered in about 90% overall yield.
(S,E)-(6-methyl-3-(3-methyl-5-(2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)pent-2-en-1-yl)-2,4-bis
((2-
(trimethylsilyl)ethoxy)methoxy)benzaldehyde (16a)
(111 mg, 89%). 1H-NMR (500 MHz, CDCl3) δ (ppm): −0.03 (s, 9H); 0.02 (s, 9H); 0.97 (m, 4H);
1.07 (s, 3H); 1.20(s, 3H); 1.24 (m, 1H); 1.27 (s, 3H); 1.39 (s, 3H); 1.59 (m, 1H); 1.79 (s, 3H); 2.01
(m, 1H); 2.21 (m, 1H); 2.59 (s, 3H); 3.39 (d, J = 6.5 Hz, 2H); 3.63 (dd, J = 9.5, 4.0 Hz, 1H); 3.76
(m, 2H); 3.84 (m, 2H); 5.06 (s, 2H); 5.22 (br t, J = 6.8 Hz, 1H); 5.30 (s, 2H); 6.78 (s, 1H); 10.36 (s,
1H).
(R,E)-(2,4-bis((2-methoxyethoxy)methoxy)-6-methyl-3-(3-methyl-5-(2,2,5,5-tetramethyl-1,3-
dioxolan-4-yl)pent-2-en-1-yl)benzaldehyde (17a)
(112.5 mg, 88%). 1H-NMR (500 MHz, CDCl3) δ (ppm): 1.07 (s, 3H); 1.20 (s, 3H); 1.25 (s, 3H);
1.39 (s, 3H); 1.45 (m,1H); 1.58 (m, 1H); 1.78 (s, 3H); 2.01 (m, 1H); 2.18 (dt, J = 10.0, 4.5 Hz, 1H);
2.53 (s, 3H); 3.36 (d, J = 7.0 Hz, 2H); 3.38 (s, 3H); 3.39 (s, 3H); 3.57 (m, 4H); 3.61 (dd, J = 9.0,
3.5 Hz, 1H); 3.79 (m, 2H); 3.84 (m, 2H); 4.83 (s, 2H); 5.02 (br t, J = 6.8 Hz, 1H); 5.33 (s, 2H); 6.85
(s, 1H); 10.54 (s, 1H).
(R,E)-(3-bromo-4,6-bis((2-methoxyethoxy)methoxy)-2-methyl-5-(3-methyl-5-(2,2,5,5-tetra
methyl-1,3-dioxolan-4-yl)pent-2-en-1-yl)benzaldehyde (17c)
(111 mg, 90%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 1.07 (s, 3H); 1.21 (s, 3H); 1.27 (s, 3H); 1.39
(s, 3H); 1.44 (m,1H); 1.59 (m, 1H); 1.77 (s, 3H); 2.01 (m, 1H); 2.18 (m, 1H); 2.67 (s, 3H); 3.37 (s,
3H); 3.39 (s, 3H); 3.48 (d, J = 6.0 Hz, 2H); 3.55 (m, 2H); 3.60 (m, 2H); 3.63 (dd, J = 10.0, 3.6 Hz,
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1H); 3.86 (m, 2H); 3.98 (m, 2H); 5.09 (s, 2H); 5.21 (s, 2H); 5.23 (br t, J = 6.5 Hz, 1H); 10.34 (s,
1H).
(S,E)-(3-chloro-2-methyl-5-(3-methyl-5-(2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)pent-2-en-1-yl)-4,6-
bis((2-(trimethylsilyl)ethoxy)methoxy)benzaldehyde (16b)
(121 mg, 92%). 1H-NMR (500 MHz, CDCl3) δ (ppm): 0.03 (s, 18H); 0.99 (m, 4H); 1.07 (s, 3H);
1.21(s, 3H); 1.27 (s, 3H); 1.40 (s, 3H); 1.44 (m, 1H); 1.59 (m, 1H); 1.79 (s, 3H); 2.02 (m, 1H); 2.21
(m, 1H); 2.63 (s, 3H); 3.47 (d, J = 6.5 Hz, 2H); 3.63 (dd, J = 9.2, 3.2 Hz, 1H); 3.81 (m, 2H); 3.91
(m, 2H); 5.02 (s, 2H); 5.18 (s, 2H); 5.26 (br t, J = 7.0 Hz, 1H); 10.36 (s, 1H).
(R,E)-(3-fluoro-2-methyl-5-(3-methyl-5-(2,2,5,5-tetramethyl-1,3-dioxolan-4-yl)pent-2-en-1-yl)-4,6-
bis((2-(trimethylsilyl)ethoxy)methoxy)benzaldehyde (16d)
(113 mg, 88%). 1H-NMR (500 MHz, CDCl3) δ (ppm): 0.07 (s, 18H); 0.97 (m, 4H); 1.07 (s, 3H);
1.20 (s, 3H); 1.26 (s, 3H); 1.39 (s, 3H); 1.43 (m, 1H); 1.60 (m, 1H); 1.79 (s, 3H); 2.02 (m, 1H); 2.20
(m, 1H); 2.47 (s, 3H); 3.43 (d, J = 5.5 Hz, 2H); 3.62 (dd, J = 9.5, 3.5 Hz, 1H); 3.81 (m, 4H); 5.01 (s,
2H); 5.23 (br t, J = 6.0 Hz, 1H); 5.26 (s, 2H); 10.34 (s, 1H).
Deprotection reactions: synthesis of colletorin A, colletochlorin A and analogues
Method A: step 1 (general procedure)
To a solution of SEM-protected 2,4-dihydroxybenzaldehydes 16 (0.16 mmol) in HMPA (1,6 mL) a
TBAF solution (1M in THF, 10 eq., 1.6 mL) was added at room temperature. Then the reaction
mixture was warmed up to 70°C and stirred for 4-5 h. After removal of THF, the residue was
poured in a mixture of ethyl acetate and brine. The solution was extracted with ethyl acetate and the
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