Sigillin A, a unique polychlorinated arthropod deterrent from the snow flea ceratophysella sigillata

Article abstract of DOI:10.1002/anie.201501719

The snow flea Ceratophysella sigillata, a winter-active species of springtail, produces unique polychlorinated octahydroisocoumarins to repel predators. The structure of the major compound, sigillin A, was elucidated through isolation, spectroscopic analysis, and X-ray crystallography. Sigillin A showed high repellent activity in a bioassay with predatory ants. A promising approach for the total synthesis of members of this new class of natural compounds was also developed. Inedible: The snow flea Ceratophysella sigillata produces unique polychlorinated octahydroisocoumarins to repel predators. The structure of the major representative, sigillin A, was elucidated through isolation, spectroscopic analysis, and X-ray crystallography. A promising synthetic approach for the total synthesis of this class of compounds was developed, in which α-vinylation of a lactone and ring-closing metathesis are the key steps. (Image: F. Kohl)

Full text of DOI:10.1002/anie.201501719

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201501719
German Edition: DOI: 10.1002/ange.201501719
Chlorinated Repellents
Sigillin A, a unique polychlorinated arthropod deterrent from the snow
flea Ceratophysella sigillata**
Witali Schmidt, Thies M. Schulze, Gregor Brasse, Edyta Nagrodzka, Michael Maczka,
Jꢀrg Zettel, Peter G. Jones, Jçrg Grunenberg, Monika Hilker, Ute Trauer-Kizilelma, Ute Braun,
and Stefan Schulz*
[
4]
Abstract: The snow flea Ceratophysella sigillata, a winter-
active species of springtail, produces unique polychlorinated
octahydroisocoumarins to repel predators. The structure of the
major compound, sigillin A, was elucidated through isolation,
spectroscopic analysis, and X-ray crystallography. Sigillin A
showed high repellent activity in a bioassay with predatory
ants. A promising approach for the total synthesis of members
of this new class of natural compounds was also developed.
and 3-hydroxy-4,5-dimethoxy benzoic acids to ward off
attacks. Other known defense compounds from collembola
[5]
include 2-aminophenol from Neanura muscorum, and
unique pyrido[2,3-b]pyrazines contained in the pseudocelluar
fluid of Tetrodontophora bielanensis and also produced by
[6]
[
7]
Onychiurus scotarius and O. circulans.
To identify the repellent principle of C. sigillata, the
Collembola were collected during the major winter activity
period and extracted and analyzed, but none of the known
compounds were detected. The distinct odor that Collembola
release when handled could be identified by headspace
analysis to be (À)-geosmin, a known sesquiterpenoid released
T
he snow flea Ceratophysella sigillata (Hypogastruridae) is
a member of the taxon Collembola (springtails), which are
wingless, soil-inhabiting hexapods. The feeding-active indi-
viduals of C. sigillata can be found from December to March
in forests, especially north of the alps; they feed at temper-
atures down to À28C on microorganisms, with a strong
[8]
by many actinobacteria, cyanobacteria, and myxobacteria.
Nevertheless, geosmin proved to be ineffective in repelling
predators.
[
1]
preference for coccal algae. In February and March, the
springtails may form big colonies with millions of individuals
migrating like superorganisms on snow or litter.
Therefore, whole-body methyl acetate extracts were
analyzed by GC–MS. Besides common compounds such as
fatty acids, cholesterol, and cuticular hydrocarbons, a major
compound A with an unusual mass spectrum was detected
(Figure 1). GC–HRMS revealed its molecular formula to be
C H Cl O (m/z obs. 453.9315, calc. 453.9311), with five
double-bond equivalents. The isotope pattern of five Cl atoms
can be recognized in the otherwise mostly uncharacteristic
mass spectrum. The base peak at m/z 43 and the ion [MÀ60]
hinted at the presence of an acetoxy group. The rigorous
exclusion of any chlorine source during work-up excluded the
possibility that compound A was a work-up artefact.
[
2,3]
Although
C. sigillata can escape arthropod predators by using its furca
to propel itself away from them in a manner typical for
collembola, they also show a second line of defense that relies
on chemical substances. Several arthropod predators are
repelled, including spiders (Neriene peltata), pseudoscorpions
1
4
15
5
6
(
Neobisium muscorum), centipedes (Lithobius forficatus),
predatory insects (Stenus sp., Microvelia reticulata), and mites
Pergamasus barbarus), although tolerance has also been
(
[2]
observed. We became interested in deciphering the chem-
ical nature of this avoidance behavior. Previous work has
shown that other Collembola successfully use chemical
defense to repel predators. C. denticulata produces syringic
By using preparative HPLC, one mg of compound A was
isolated from about 10 g springtails with 78% purity. The gas-
phase infrared spectrum showed significant absorptions at
À1
À1
3
554 cm and at 3465 cm , which indicate the presence of
À1
À1
two hydroxy groups. Absorptions at 1764 cm and 1220 cm
[
*] Dr. W. Schmidt, Dr. T. M. Schulze, Dr. G. Brasse, Dr. E. Nagrodzka,
M. Maczka, Prof. Dr. J. Grunenberg, Prof. Dr. S. Schulz
Institut fꢀr Organische Chemie, TU Braunschweig
Hagenring 30, 38106 Braunschweig (Germany)
E-mail: stefan.schulz@tu-bs.de
hinted at an ester or lactone group. Several microscale
derivatization reactions were performed to obtain more
information on the functional groups present in A. No
reaction was observed with diazomethane, thus excluding the
presence of an acid functionality. Reaction with N-methyl-N-
Homepage: http://www.oc.tu-bs.de/schulz/index.html
Prof. Dr. P. G. Jones
Institut fꢀr Analytische und Anorganische Chemie
TU Braunschweig, Hagenring 30, 38106 Braunschweig (Germany)
(
trimethylsilyl)trifluoroacetamide (MSTFA) led to a monosi-
lylated compound. IR spectroscopy of this product showed
that only one of the two hydroxy groups was silylated, thus
indicating a hindered OH group. Saponification of the natural
sample with sodium methoxide followed by silylation with
MSTFA led to two major compounds with molecular masses
of 660 and 588. These values can only be explained by the
opening of a lactone ring to a methyl ester, cleavage of an
acetate group, and finally silylation of two or all three OH
groups now present. It followed that compound A should be
a lactone with three additional OH groups, of which one is
Prof. Dr. J. Zettel
Speichergasse 8, 3150 Schwarzenburg (Switzerland)
Prof. Dr. M. Hilker, U. Trauer-Kizilelma, U. Braun
Institut fꢀr Biologie, Freie Universitꢁt Berlin
Haderslebener Strasse 9, 12163 Berlin (Germany)
[**] We thank the Deutsche Forschungsgemeinschaft for financial
support by a grant, DFG Schu984/8-1.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201501719.
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Communications
Scheme 1. Possible structures of natural compound A that are consis-
tent with the obtained NMR and MS data. The correct structure (1) is
highlighted.
DFT (density functional theory) calculations of the gas
phase IR spectra of compounds 1–4 were performed using the
B3LYP hybrid functional in combination with a standard 6-
Figure 1. a) EI mass spectrum of unknown compound A, identified to
be sigillin A (1). b) Total ion chromatogram of a methyl acetate extract
of Ceratophysella sigillata. Fatty acids (FA), cholesterol (CH), hydro-
carbons (HC), and minor sigillin derivatives (B–I) of major compound
A (1, sigillin A), are present in the extract. The structures of sigillins
B–I (B–I; compounds 5–12) are shown in Scheme 2.
3
1(d) basis set (Figure S16 in the Supporting Information).
The sum of the difference of the nine important lines of the
calculated versus natural spectrum was markedly lower for
1
compared to all of the others, thus pointing to structure 1 as
the one that most likely represents the natural product.
At this stage, a new work-up procedure, including
extraction of live colonies and immediate work-up, was
developed (see the Supporting Information), which resulted
in an increase of the yield of A and related metabolites to
about 20 mg per 10 g fresh weight. Evidently, the concen-
tration of A in C. sigillata is very high (0.2% or higher of fresh
weight). From this material, a single crystal suitable for X-ray
analysis was obtained. The result confirmed the proposed
structure (Figure 2) and established the absolute configura-
acetylated; five chlorine atoms; and two additional double-
bond equivalents. No similar compound is known from the
literature.
1
3
1
The C NMR and H NMR spectra revealed a total of 14
carbon atoms and 15 hydrogen atoms in agreement with the
molecular formula (see Table S1 in the Supporting Informa-
1
3
tion). The C NMR resonances were assigned to two
2
carbonyl groups (d 170.9, 168.4 ppm), two sp quaternary
3
carbons (d 130.5, 121.0 ppm), three sp quaternary carbons (d
3
3
9
9.3, 73.4, 50.0 ppm), one sp methine (d 82.7 ppm), two sp
3
oxomethine (d 74.5, 73.0 ppm), two sp methylene (d 31.9,
7.0 ppm), and two sp methyl (d 20.3, 14.9 ppm) carbon
3
2
atoms. The remaining two double bond equivalents showed
the presence of an additional ring and one double bond in A.
Signals for two protons (d 5.14, 3.4 ppm) could be eliminated
1
from the H NMR spectrum by adding [D ]MeOH and were
4
thus assigned to two hydroxy groups.
Figure 2. Structure of sigillin A as determined by X-ray crystallography.
H atoms are omitted for clarity in the structure on the right.
1
1
Two-dimensional NMR experiments ( H, H-COSY,
1
1
1
13
1
13
H, H-COSY-long range, H, C-HMBC and H, C-HSQC)
were performed to determine the connectivity. A CCl3-
CH(O)-CH -CH(C)(O)-CH -C= spin system was deduced
tion to be 3R,5R,6R,9S,10R. The large trichloromethyl group
prefers a quasi-equatorial position, thereby fixing the con-
formation of the lactone ring into a twisted boat conformation
typical for d-lactones, while the cyclohexane ring remains in
the common chair conformation. We propose the name
sigillin A for the natural compound A.
Careful analysis of the natural extract revealed the
presence of eight additional sigillin derivatives, named
sigillins B–I (Figure 1 and Scheme 2). The structures were
tentatively deduced from their mass spectra (see the Support-
ing Information). The level of chlorination, easily detectable
2
2
but it remained unclear whether the adjacent, obviously
chlorine-substituted double bond was endo- or exocyclic. No
1
3
[9]
reliable C NMR data are available for similar structural
elements because they have rarely been synthesized or
isolated. Furthermore, the trichloromethyl group and the
two hydroxyl groups were located on the same side, as
indicated by NOE NMR experiments, while the orientation of
the acetyloxy group could not be clarified. In conclusion, the
four structures 1–4 (relative configurations, Scheme 1)
seemed to be in accord with the spectroscopic data.
2
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The ecological effects of sigillin A were tested in a bioas-
say with the ant Myrmica rubra. This ant species is a generalist
predatory model insect that has been used in many bioassays
for testing the feeding deterrent activity of natural products
[13,14]
identified in hexapods.
Collembola are potential prey of
[
15]
M. rubra, which forages both on soil and in vegetation for
food. An aqueous suspension of crushed larvae of Drosophila
melanogaster containing 0.2% sigillin A was prepared to
simulate the natural concentration of 1 in the Collembola.
This preparation and a control sample consisting of crushed
D. melanogaster alone were offered simultaneously in an
arena containing 15 ants. The feeding behavior at test and
control samples was recorded for a period of 10 min. First
encounters of workers with the test and control samples
occurred randomly. When an ant encountered a test sample, it
first took a taste and started briefly to feed upon it. Hence, the
ant behavior did not indicate that test or control samples had
any repellent activity via the gas phase.
Scheme 2. The sigillins A–I (1, 5–12). The percentages shown describe
the peak area relative to that of 1.
However, soon after tasting the test samples, the ants
moved away from them. Significantly fewer ants fed upon the
test samples than upon the controls over the entire bioassay
period (Figure 3). Ant individuals started to feed at different
times during the assay. The bioassay showed that sigillin A
acts as strong feeding deterrent against a generalist predatory
insect. Therefore, the predator reaction described by Zettel
by the distinctive isotopic pattern of chlorine, differs between
the sigillins, with values of between four and six detected.
Deacetylated compounds accompany the acetates, usually in
lower amounts. Despite their relatively high numbers of
alcohol groups, the sigillins can be investigated by GC. The
presence of additional more polar derivatives was ruled out
using HPLC–UV/MS in ESI negative mode (see the Support-
ing Information). No additional derivatives were found, while
most of the sigillins were detected. Derivatives containing
a dichloromethylidene group proved to be UV active, while
the others were not, thus further supporting the structural
assignments. Samples from different years and regions always
showed the presence of sigillins, although the content varied.
The biosynthetic origin of the sigillin family is unknown.
The compounds are probably polyketides because they show
the appropriate arrangement of methyl and hydroxy groups
[
2]
et al. is most likely caused by sigillin.
(
Figure S17). The octahydrobenzopyranone ring system could
be formed from two malonate and three methylmalonate
units through ring closure. The C6 acetyloxy group requires
an additional oxidation. The chlorine atoms are probably
introduced by radical halogenation.
A sequence via
a dichloromethyl group that is transformed through halogen-
ation into the trichloromethyl group, followed by elimination
to the dichloromethenyl group seems to take place; all these
structural elements are found in the sigillins. There are known
examples of radical formation of a trichloromethyl group.
Unactivated methyl groups have been shown to be radically
chlorinated by non-heme iron halogenases in a reaction
Figure 3. Bioassay with the ant Myrmica rubra feeding on crushed
Drosophila melanogaster larvae without (black bar) or with (white bar)
0.2% sigillin A (1). The numbers of ants feeding on the two samples
were recorded every minute for ten minutes. Twelve arenas with 15
ants each were used. N: number of feeding ants; ** Pꢀ0.01
(Wilcoxon signed-rank test for paired differences).
[
10]
requiring a-ketoglutarate and oxygen. These enzymes have
[
11]
been investigated in detail in the biosynthesis of barbamide,
armentomycin, and 2-amino-4,4-dichlorobutanoic acid.
The unique structural features of sigillin A make it
[
12]
A
a challenging target for total synthesis. We were interested
in obtaining derivatives for testing in various biological assays
to evaluate the activity profile of these halogenated octahy-
droisocoumarines. As a start, the dideoxy derivative 20 of
sigillin A served as synthetic target because it contains the
isocoumarin backbone, as well as the two chlorinated func-
tional groups.
similar enzyme might mediate a radical chlorination in the
biosynthesis of the sigillins. Because no polyketide synthases
are known from insects or other arthropods, it seems likely
that sigillin is produced by symbiotic bacteria or taken up
from the algal food. The release of geosmin further supports
an insect/bacterial symbiosis. Nevertheless, the large amounts
of sigillins present in C. sigillata are striking and might
indicate biosynthesis by the animal itself.
The synthesis started from key lactone 13, which was
obtained from chloral by building on a strategy introduced by
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[
16]
Shimizu et al., (see the Supporting Information) but can
alternatively be obtained through a ring-closing metathesis
transformed into the dichloromethylidene unit found in
sigillin A. This transformation was achieved, but with limited
success. Although model compounds such as 2-hydroxy- or 2-
acetyloxycyclohexanone could be converted with the stan-
[17]
(
RCM) approach.
a copper-catalyzed conjugated 1,4-addition reaction with the
use of 3-butenylmagnesium bromide and a copper bromide
It served as Michael acceptor in
[
24]
dard method using CCl /PPh ,
this reaction completely
4
3
[
18]
dimethylsulfide complex to furnish lactone 14 (Scheme 3).
failed with 19. The transformation of 19 or analogues with
a protected hydroxy group by various other methods, e.g., the
[
25]
Takeda method of dichloromethenylation with a titanocene
[
26]
or a magnesium/titanium catalyst, also proved unsuccessful.
Finally, the synthesis of 20 was achieved in very low yield by
treating 19 with hydrazine and subsequent addition of
[
27]
CBrCl , ammonia, and catalytic amounts of CuCl. Never-
3
theless, the target compound didesoxysigillin A (20) was
isolated and a crystal structure was obtained (see the
Supporting Information). The relative stereochemistry is
identical to that of 1.
While marine organisms are well known producers of
a wide variety of halogenated compounds, fewer complex
halogenated secondary metabolites are found in terrestrial
[
28]
organisms. Most are produced by bacteria and fungi, only
a few are produced by more complex organisms such as plants
[
28]
or arthropods.
dichloromethylidene functional groups are rare structural
Trichloromethylcarbinol or its ester and
[29]
elements in natural products. They occur in Muironolide A,
a marine sponge metabolite, citreochlorol, which is produced
by Penicillium sp., and the fungal products pinicoloform
[30]
[31]
[
32]
and KS-504 compounds.
In conclusion, sigillin A and its congeners represent a new
class of natural products. They function as strong feeding
deterrents against ants and seem to play an important role in
the ecology of springtails. A synthesis of the parent chlori-
nated ring system was developed, which allows the synthesis
of structural analogues. Currently, the bacteria associated
with Collembola are under investigation to establish the
biosynthetic origin of the sigillins. Furthermore, biological
testing and chemical synthesis of sigillin analogues are under
way.
Scheme 3. Synthesis of didesoxysigillin 20.
In the next step, a vinyl group had to be attached next to the
carbonyl group. The use of acetaldehyde as a vinyl cation
synthon proved to be unsuccessful. While the addition is
uneventful, the following elimination reaction, even when
using a good leaving group such as mesylate, results in
concomitant loss of HCl and a low yield. This problem was
circumvented through the addition of phenylselenoacetalde-
hyde to form the alcohol 15 and subsequent elimination under
Keywords: insect repellents · isocoumarine · natural products ·
organohalogen compounds · a-vinylation
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Published online: && &&, &&&&
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Communications
Chlorinated Repellents
Inedible: The snow flea Ceratophysella
sigillata produces unique polychlorinated
octahydroisocoumarins to repel preda-
tors. The structure of the major repre-
sentative, sigillin A, was elucidated
through isolation, spectroscopic analysis,
and X-ray crystallography. A promising
synthetic approach for the total synthesis
of this class of compounds was devel-
oped, in which a-vinylation of a lactone
and ring-closing metathesis are they key
steps. (Photo F. Kohl)
W. Schmidt, T. M. Schulze, G. Brasse,
E. Nagrodzka, M. Maczka, J. Zettel,
P. G. Jones, J. Grunenberg, M. Hilker,
U. Trauer-Kizilelma, U. Braun,
S. Schulz*
&&&& — &&&&
Sigillin A, a unique polychlorinated
arthropod deterrent from the snow flea
Ceratophysella sigillata
6
www.angewandte.org
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Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!