A Novel Class of Defensive Compounds in Harvestmen: Hydroxy-γ-Lactones from the Phalangiid Egaenus convexus

Article abstract of DOI:10.1021/acs.jnatprod.0c00277

When threatened, the harvestman Egaenus convexus (Opiliones: Phalangiidae) ejects a secretion against offenders. The secretion originates from large prosomal scent glands and is mainly composed of two isomers of 4-hydroxy-5-octyl-4,5-dihydro-3H-furan-2-one (1), a β-hydroxy-γ-lactone. The compounds were characterized by GC-MS of their microreaction derivatives, HRMS, and NMR. After the synthesis of all four possible stereoisomers of 1, followed by their separation by chiral-phase GC, the absolute configurations of the lactones in the Egaenus secretion was found to be (4S,5R)-1 (90%) and (4S,5S)-1 (10%). Hydroxy-γ-lactones represent a new class of exocrine defense compounds in harvestmen.

Full text of DOI:10.1021/acs.jnatprod.0c00277

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A Novel Class of Defensive Compounds in Harvestmen: Hydroxy-γ-
Lactones from the Phalangiid Egaenus convexus
,
∇
∇
Gu
Stefan Do
̈
nther Raspotnig,* Felix Anderl, Olaf Kunert, Miriam Schaider, Adrian Bru
̈
ckner, Mario Schubert,
̈
tterl, Roman Fuchs, and Hans-Jorg Leis
̈
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sı Supporting Information
ABSTRACT: When threatened, the harvestman Egaenus convexus
Opiliones: Phalangiidae) ejects a secretion against offenders. The
(
secretion originates from large prosomal scent glands and is mainly
composed of two isomers of 4-hydroxy-5-octyl-4,5-dihydro-3H-
furan-2-one (1), a β-hydroxy-γ-lactone. The compounds were
characterized by GC-MS of their microreaction derivatives,
HRMS, and NMR. After the synthesis of all four possible
stereoisomers of 1, followed by their separation by chiral-phase
GC, the absolute configurations of the lactones in the Egaenus
secretion was found to be (4S,5R)-1 (90%) and (4S,5S)-1 (10%).
Hydroxy-γ-lactones represent a new class of exocrine defense
compounds in harvestmen.
hemical defense in harvestmen (arachnid order Opi-
liones) is associated with large prosomal scent glands
We focus here on the secretions of a first representative of
the phalangiid subfamily Opilioninae, Egaenus convexus, a
massively built species that is widespread in Central and
Southeastern Europe. When threatened, individuals of
E. convexus eject a “jet” from scent glands toward offenders
1
C
from which these arachnids discharge repellent secretions
2
against predators. Exudates from these glands have been
3
studied for more than 50 years, representing a rich source of
rare, unusual, and new natural products. Current knowledge
indicates that scent gland exudates comprise compounds such
as naphthoquinones, chloro-naphthoquinones, and aliphatic
methyl ketones in the harvestman suborder Cyphophthalmi,
nitrogen-containing substances, terpenes, aliphatic ketones,
and phenolics in the suborder Insidiatores, alkylated phenolics
and benzoquinones in the suborder Grassatores, and secretions
(
Figure 1).
Individual secretions directly dabbed from gland openings of
adults of both sexes constantly showed two major peaks (A, B)
by GC-MS. The peaks exhibited nearly identical EI-mass
spectra, indicating isomeric compounds, with an earlier-eluting
major isomer or mixture of isomers (peak A: RI = 1878 on a
ZB-5MS column) and a later-eluting minor isomer or mixture
of isomers (peak B: RI = 1901). Peaks A and B were present in
a ratio of about 9:1 (Figure 2).
2
,4−6
of rather miscellaneous chemistry in the Palpatores.
The
latter group, the Palpatores, is subdivided into suborders
7
Eupnoi and Dyspnoi and includes more than 2100 species of
A molecular ion for these compounds was very weak in EI-
commonly occurring and conspicuous harvestmen. Despite
this species richness, the chemistry of eupnoan and dyspnoan
secretions has remained largely enigmatic.
MS but confirmed to be at m/z 214 by PCI-MS (positive ion
+
chemical ionization; using methane as reagent gas: MH at m/
+
+
z 215; M + C H at m/z 243; M + C H at m/z 255).
2
5
3
5
In Dyspnoi, the secretions of only four species have hitherto
+
HRESI-MS revealed an exact monoisotopic mass for [M + H]
been analyzed, showing naphthoquinones along with methyl-
5
,8
at m/z 215.1642, corresponding to a molecular formula of
and ethyl-ketones. For Eupnoi, representatives of Scleroso-
matidae appear to rely on acyclic compounds such as ethyl
C H O and thus two sites of unsaturation/rings, respec-
1
2
22
3
9
tively.
ketones (and derivatives), whereas initial investigations of the
other large eupnoan family, the Phalangiidae (with only two
species of the subfamily Phalangiinae studied), indicate
naphthoquinones (Phalangium, Rilaena), 1,4-benzoquinone,
Received: May 20, 2020
6
,10
and medium chain fatty acids (Rilaena).
However,
dyspnoan and eupnoan secretions appear to be key elements
in a comprehensive, phylogenetically founded picture of
4
harvestmen scent gland chemistry.
©
XXXX American Chemical Society
https://dx.doi.org/10.1021/acs.jnatprod.0c00277
A
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Figure 1. (A) A female individual with the position of the ozopores (gland openings) indicated (arrows). (B) Ozopores (right ozopore is shown:
arrow) are located near-dorsal to the coxa of leg I, are oval-shaped with a dimension of about 340 μm × 150 μm, and are surrounded by a cuticular
rim (C, D). The flat central bottom is of smooth cuticle, whitish, thin, and membrane-like in appearance. This bottom is movable and trapdoor-like,
and secretion can be released through a slit (D). Details of the right ozopore: (B) light microscopic photograph; note the whitish and membranous
structure of the bottom of the pore; (C, D) scanning electron micrographs; note that the pore is slightly opened (arrow in (D)).
Figure 2. Total ion chromatogram of scent gland secretion of an individual of Egaenus convexus, showing main peaks A and B, and their EI-mass
spectra (a, b). The secretion was dabbed from ozopores in the moment of extrusion (compare Figure S23).
B
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Silylation of the extract with MSTFA (N-methyl-N-
trimethylsilyl)trifluoracetamide) led to the TMS (trimethyl-
silyl)-derivatives 2 of the original compounds (Scheme 1). EI-
addition of two TMS moieties to two hydroxy groups. This is
consistent with a methyl-ester bis-TMS-ether structure or a
carboxyl group plus two hydroxy groups after saponification.
(
Reduction of the original compounds using LiAlH led to
4
+
Scheme 1. Procedure of the Identification of 4-Hydroxy-5-
octyl-4,5-dihydro-3H-furan-2-one (1) by GC-MS of
Derivatives Obtained by Microderivatization
compound 7 with M at m/z 218, indicating the addition of
four hydrogens, as confirmed by subsequent MSTFA
a
+
derivatization (8: M at m/z 434; m/z 218 plus 3 × 72). By
using LiAlD , two hydrogens and two deuterium atoms were
4
added, resulting in a TMS product 8b of molecular weight at
M = 436 g/mol (Scheme 1). Thus, the parent structure was
indicated to carry two ketone/aldehyde functions with no ring
or was a lactone.
EI-MS of the original compound 1 as well as the
fragmentation of the TMS-ether 2 were consistent with the
1
1
structure of isomeric β-hydroxy-γ-alkyl-lactones. In detail, a
β-hydroxy-γ-octyl lactone was supported by (i) characteristic
fragment ions from the rearrangement of the lactone leading to
m/z 143/142 (= furan oxygen plus an octyl group:
+
+
Me(CH ) CHO and Me(CH ) CH O , respectively); (ii)
2
7
2 7
2
ions at m/z 124/125 (elimination of H O from the latter),
2
together with (iii) m/z 44 (= OH-bearing moiety of the
+
lactone ring: C H O ). In the TMS derivative 2, the
2
4
corresponding fragment ions were recorded at m/z 116 (44
72: C H OSi) and m/z 215 (143 + 72: Me(CH ) CHO-
+
5
12
2 7
+
3
SiMe ). The latter ion at m/z 215 arises by rearrangement of
3
-O-TMS-alkano-4-lactones, supporting the octyl group and
the OH group in the γ- and β-positions on the lactone ring,
respectively. With this information given, the mass spectra of
the remaining microderivatives 3−8 were interpreted (Scheme
1
), finding full accordance with a 4-hydroxy-5-octyl-4,5-
dihydro-3H-furan-2-one structure: saponification of 1 led to
the opening of the lactone ring and to the generation of a 3,4-
dihydroxydodecanoic acid (4), which could be converted into
trimethylsilyl 3,4-bis((trimethylsilyl)oxy)dodecanoate (5) by
methylation and silylation. On the other hand, the reduction of
1
led to lactone-ring opening and to the generation of 1,3,4-
dodecanetriol (7), which could be converted into 1,3,4-
tris((trimethylsilyl)oxy)dodecane (8) by silylation. Thus,
peaks A and B were proposed to be isomers or mixtures of
isomers of 4-hydroxy-5-octyl-4,5-dihydro-3H-furan-2-one (1).
Because the quantity of 1 in extracts of single individuals was
too low to perform NMR analyses, a pooled extract containing
the secretions of 400 individuals of both sexes was prepared.
The major isomer of 1 from the pooled extract (corresponding
to peak A in the chromatograms) was purified by column
chromatography, followed by preparative gas chromatography.
a
(
a) Silylation (MSTFA, MTBSTFA); (b) saponification (NaOCH );
3
(
c) reduction (LiAlH ); (d) methylation (diazomethane). For details,
4
see the Supporting Information.
mass spectra showed the addition of a single TMS moiety to
the original molecules (plus 72 mass units), with diagnostic
ions at m/z 271 (M-15) and a very weak molecular ion at m/z
2
86 (intensity < 1%). The TMS products were subsequently
analyzed by PCI-MS, confirming the molecular ion at m/z 286
+
+
5
1
(
MH at m/z 287, M + C H at m/z 315). Accordingly, the
One dimensional H NMR of 1 (Figure 3; Table 1) fully
2
derivatization with MTBSTFA (N-methyl-N-(tert-
butyldimethylsilyl)trifluotoacetamide) indicated the addition+
of one butyldimethylsilyl group, leading to adduct 3, with M
at m/z 328 (plus 114 mass units), as evidenced by PCI-MS
supported the proposed hydroxy-lactone structure, revealing
the presence of a linear alkyl (octyl) residue, two protons
located on carbon atoms carrying oxygen substituents, and two
diastereotopic protons present next to a carbonyl group.
A COSY spectrum indicated that these protons where
located on adjacent carbon atoms (Figure S2). This is realized
within a 5-membered lactone. Moreover, the observed
chemical shifts and coupling constants were remarkably similar
to the reported values for 4-hydroxy-5-methylbutyrolactone
+
+
+
(
MH at m/z 329, along with C H and C H5 adducts at m/
2 5 3
z 357 and 369, respectively). Upon saponification (NaOMe),
an addition of 18 mass units to the original compounds was
+
observed (4: M at m/z 232). Subsequent derivatization with
+
MSTFA led to compound 5 with M at m/z 448 (as confirmed
1
2,13
by PCIMS), now showing the addition of three TMS moieties
and 4-hydroxy-5-octylbutyrolactone in the literature.
(
232 plus 3 × 72 = 448). These data are consistent with three
Additionally, a comparison to these γ-butyrolactones of defined
configuration tentatively suggested that the major isomer of 1
was a 4,5-anti-diastereomer, as indicated by coupling constants
of the isolated H-3a and H-3b resonances (Figure 4).
Because 4-hydroxy-5-octyl-4,5-dihydro-3H-furan-2-one (1)
possesses two stereogenic centers, four stereoisomers are
hydroxy groups or two hydroxy groups and one carboxyl group
after saponification. Treatment with diazomethane following
saponification and subsequent derivatization with MSTFA
produced compound 6 with M at m/z 390, suggesting (i) the
addition of a methyl group (to a carboxyl group) plus (ii) the
+
C
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1
Figure 3. Comparison of the characteristic regions of the H NMR spectrum of the authentic sample with the same regions of the synthetic
compounds. The resonances of the minor component in the authentic sample are marked with stars. The spectra were recorded in CDCl at 600
3
MHz for the authentic sample and at 700 MHz for the synthetic compounds (imp. = impurities).
Table 1. NMR Chemical Shift Values of the Authentic Sample and Corresponding Synthetic Compounds (4S,5R)-1/(4R,5S)-1
a
and (4S,5S)-1/(4R,5R)-1 with the Same Relative Configuration
(
4S,5R)-1/(4R,5S)-1
δH (J in Hz)
(4S,5S)-1/(4R,5R)-1
δH (J in Hz)
atom
authentic sample major component δH (J in Hz)
δC, type
δC, type
2
3
174.8, C
37.7, CH2
176.6, C
39.6, CH2
2.85 dd, (18.0, 6.7)
2.84, dd (17.9, 6.7)
2.52, dd (17.9, 3.8)
4.28, ddd (6.5, 4.0, 3.0)
4.34, ddd (8.1, 5.4. 3.1)
1.64, m
2.80, dd (17.8, 5.6)
2.54, d (17.8)
4.47, m
4.37, ddd (8.2, 5.7, 3.7)
1.84, m
2
.52 dd (18.0, 3.8)
4
5
4.29, ddd (6.5, 4.0, 3.0)
4.32, ddd (8.1, 5.4., 3.1)
1.61−1.64, m
71.8, CH
87.7, CH
33.2, CH2
69.0, CH
85.2, CH
28.3, CH2
1′
1
.61, m
1.49, m
.41, m
1.72, m
1.50, m
1.38, m
2
′
1.29−1.44
25.2, CH2
29.2, CH2
29.3*, CH2
29.4*, CH2
31.8, CH2
22.7, CH2
14.1, CH3
25.6, CH2
1
3′
4′
5′
6′
7′
8′
1.29−1.44
1.29−1.44
1.29−1.44
1.29−1.44
1.29−1.44
0.88, t (7.5)
1.28, m
1.28, m
1.29, m
1.26, m
1.29, m
0.88, t (7.2)
29.2, CH2
29.5, CH2
29.5, CH2
31.9, CH2
22.7, CH2
14.1, CH3
1.29, m
1.29, m
1.29, m
1.27, m
1.27, m
0.88, t (7.0)
a
Resonances marked with an “∗” could be either C-4′ or C-5′. Data were recorded in CDCl ; TMS was used as the internal standard. J values are in
3
Hz.
possible, (4R,5S)-1, (4S,5R)-1, (4R,5R)-1, and (4S,5S)-1
determine which of the isomers actually were present in the
Egaenus extract and to elucidate their absolute configuration,
all four stereoisomers of 1 were synthesized in enantiopure
(Chart 1). The original extract showed two peaks (A, B) on
an apolar nonchiral-phase column, indicating the presence of at
least two (but potentially all four) stereoisomers of 1. To
D
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Figure 4. Observed chemical shifts, multiplicities, and coupling constants of the major component 1 of the authentic sample (red) in comparison
12,13
with previously reported values of synthesized γ-butyrolactones with a defined configuration (blue).
Chart 1. Stereogenic Centers and Possible Stereoisomers of
-Hydroxy-5-octyl-4,5-dihydro-3H-furan-2-one (1)
Both enantiomers of the syn diastereomer of 1 [(4R,5R)-1
and (4S,5S)-1] were prepared in two steps from n-decanal
4
13
according to an already described procedure. Knoevenagl-
type condensation of n-decanal and monoethyl malonate
yielded skipped ester 10. The Sharpless asymmetric dihydrox-
ylation of this intermediate delivered both enantiomers of 1 in
good yield and excellent optical purity. As the analogous route
was not applicable to the corresponding anti-isomers [(4S,5R)-
1
and (4R,5S)-1], these were achieved in a different manner.
Commercial enantiopure benzyl glycidyl ether was treated with
n-heptylmagnesium bromide in the presence of catalytic
amounts of copper salts to yield benzyl ether 11. TBS
protection of the resulting hydroxy group led to compound 12,
and the subsequent hydrogenolysis of the primary benzyl ether
delivered monoprotected diol 13. TEMPO-catalyzed oxidation
of the said alcohol provided aldehyde 14, which was subjected
directly to an Evans aldol reaction with the boron enolate
derived from 16. The ensuing product 15 was isolated as a
single diastereomer in fair yield. Finally, the fluoride-mediated
form (Scheme 2; Supporting Information) and analyzed by
NMR as well as by chiral-phase gas chromatography.
a
Scheme 2. Synthesis of (4S,5S)-1 and (4S,5R)-1
a
Conditions: (a) monoethyl malonate, Et N, 42%; (b) AD-mix α, tBuOH/H O, 70%, (c) n-C H MgBr, CuCN (5 mol %), THF, 75%; (d)
3
2
7
15
TBSCl, imidazole, CH Cl , 89%; (e) H , Pd/C, EtOAc, quant.; (f) TEMPO (20 mol %), PhI(OAc) ; (g) 4-(S)-3-acetyl-4-benzyl-oxazolidin-2-one,
2
2
2
2
Bu BOTf, Et N, CH Cl , 50% over 2 steps; (h) Bu NF, THF, 71%. TBS: tert-butyldimethylsilyl; TEMPO: tetramethylpiperidineoxy radical.
2
3
2
2
4
E
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Figure 5. Identification of peaks A and B of the Egaenus extract as (4S,5R)-1 and (4S,5S)-1. Chromatography was performed on a chiral ß-
cyclodextrin phase. (A) Chromatographic comparison of synthetic (4R,5S)- and (4S,5R)-isomers of 4-hydroxy-5-octyl-4,5-dihydro-3H-furan-2-one
(1) to Egaenus compounds A and B. (4R,5R)- and (4S,5S)-isomers of 1 remained inseparable under these conditions. (B) Comparison of MSTFA-
derivatized (4R,5R)- and (4S,5S)-isomers of 1 to derivatized Egaenus extract.
removal of the TBS group resulted in spontaneous
lactonization with the expulsion of the auxiliary, thus
completing the synthesis of (4S,5R)-1. The opposite antipode
In contrast, the lactone motif itself is frequently present in
16,18,19
exocrine exudates of arthropods as well as vertebrates.
γ-
Lactones, for instance, are known from butterflies and beetles
16
(
4R,5S)-1 was prepared analogously in comparable yields and
where they may serve as sex pheromones. Similar to large-
20,21
optical purities. Therefore, (S)-glycidyl benzyl ether and (R)-3-
acetyl-4-benzyl-2-oxazolidinone were used instead of their
respective optical antipodes. Moreover, in the first step of the
ringed macrolides,
γ- and δ-lactones have also been
22
described as antimicrobial agents2, and a few additionally
3−25
possess a role in predator defense.
Regarding harvestmen, scent gland exudates have generally
sequence, Cu(OAc) was substituted for CuCN as the catalyst
2
1
,2
been considered defensive, even though additional functions
for the epoxide opening reaction.
26
may have evolved in particular taxa. In some harvestmen
species, chemical defense is indeed obvious: secretions are
readily expelled as sprays or jets, reaching an offender at a
Synthetic (4R,5S)-1 and (4S,5R)-1 appeared to be well
separable on a chiral ß-cyclodextrin phase, whereas (4R,5R)-1
and (4S,5S)-1 could only be separated as their TMS derivatives
2
distance of several centimeters, deterring invertebrates as well
(Figure 5). Consequently, an Egaenus extract (underivatized
27
as small vertebrates. Concurringly, specimens of E. convexus
forcefully eject secretion upon mechanical disturbance. This
mode of secretion transfer is known from other harvestmen
and MSTFA-derivatized) was chromatographed on the chiral
phase, clearly showing the presence of only two naturally
occurring stereoisomers of 1. A chromatographic comparison
of all four synthetic stereoisomers to the Egaenus compounds
proved the identities of peaks A and B as (4S,5R)-1 and
2
species and is called “jetting”, addressing the discharge of a
fine, directed splash against offenders. So far, jetting has been
mainly described for certain Laniatores whereas “spraying”,
defined as a fine, vaporized spray, is known from a group of
(
4S,5S)-1, respectively (Figure 5).
Lactones are quite frequent in nature and are well-known as
2
sclerosomatid Eupnoi. On the basis of our observations, the
scent/aroma compounds in fruits and milk and as flavoring
secretion of Egaenus also spreads over the body surface of
jetting individuals, hence impregnating the body surface.
Lactone amounts per individual were found to be highly
variable, obviously depending on the filling status of the scent
glands. In specimens extracted immediately after collection, we
found 12.5 ± 7.9 μg/per individual with no obvious differences
between the sexes. These amounts appear rather low, possibly
indicating that the lactones are dissolved in a carrier matrix of
currently unknown chemistry. In acyclic compound-producing
compounds in alcoholic beverages (i.e., whiskey and cognac
1
4,15
lactones
). However, among arthropods, the γ-lactones
found in the scent gland secretion of Egaenus are exceptional.
First, they represent a novel class of compounds for the
chemical inventory of scent gland secretions in harvestmen.
Regarding the chemistry of Eupnoi, the compounds are similar
neither to the acyclic compounds found in sclerosomatid
4
Eupnoi (“sclerosomatid compounds” [sensu ]) nor to the
6
,10
28
aromatic/quinonic compounds from Phalangiinae.
Second,
leiobunines, this matrix is aqueous.
the particular β-hydroxy-γ-lactones as seen in Egaenus have not
been found in any other arthropod yet. While lactones with
OH groups on the side chains appear to be present in at least
Considering secretion discharge following a disturbance, the
mode of secretion application (i.e., a directed jet against
offenders), and self-wetting of the body−surface, a defensive
and antimicrobial role of the Egaenus secretion appears to be
likely. These newly discovered lactones add an unexpected
component to the overall picture of harvestmen-secretion
chemistry. It will be a logical next step to investigate their
16
some insect species, only one example for an exocrine
lactone carrying a hydroxy group in the β-position (a 3-
hydroxy-γ-decalactone = 5-hexyl-4-hydroxy-dihydro-furan-2-
17
one from a tephritid fly) has recently been reported.
F
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evolutionary origin and taxonomic distribution across the
Opiliones.
4418, 4455, 4456, 4458, 4459, 5402−5411, 5414). A freshly emitted
secretion was collected by dabbing the secretion on filter paper pieces
(2 × 2 mm) directly from the gland openings (ozopores). “Loaded”
filter papers were extracted in hexane (100 μL) for 15 min and gave
extracts of pure secretion (Figure 2). Alternatively, individual whole-
body extracts were prepared (500 μL of hexane; 15 min). The latter
method was more feasible with respect to handling and resulted in
equal or higher quantities of secretion per extract but showed
additional, nonsecretion compounds in the extracts since some
cuticular hydrocarbons were coextracted (Figures S24 and S25).
Prepurification by Flash Chromatography. Initial fractiona-
tion was performed on silica gel (40−63 μm) using solvents of >99%
purity or p.a. grade. Hexane extracts, each containing the secretions of
40 individuals (in ∼10 mL), were concentrated in a stream of
nitrogen to a volume of ∼300 μL. Concentrated extracts were purified
on silica gel columns packed with 500 mg of unmodified SiOH
(Chromabond, 3 mL, Machery-Nagel) using solvents of >99% purity
or p.a. grade. Purification of these solutions by flash chromatography
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations of
compounds were measured using a Jasco P-2000 polarimeter at 25
C in chloroform. IR spectra were recorded on an Alpha-ATR FTIR
spectrometer (Bruker Biospin). NMR spectroscopy was performed on
either a Bruker Avance III HD 600 spectrometer or Bruker Avance III
HD 700, respectively. Frozen traps from pcGC (preparative capillary
gas chromatography, see below) containing the purified authentic
°
compound 1 were eluted with 500 μL of CDCl containing 0.03%
3
TMS for reference (99.8 atom %D, Armar, Germany). Spectra were
measured with either a cryoprobe at 298 K or with a quadruple
1
13
15
31
resonance probe (QXI H/ C/ N/ P) at 293 K. Chemical shift
assignment was achieved with COSY and TOCSY spectra (120 ms
mixing time). Raw data were processed in Topspin 3.2 (Bruker
Biospin), and 2D data was analyzed using Sparky (T. D. Goddard and
D. G. Kneller, SPARKY3, University of California, San Francisco).
COSY, HSQC, and HMBC spectra of the synthetic compounds were
(100% hexane → 100% CH
Cl ) provided the compounds of interest
2
2
in the CH Cl fractions. In detail, columns were washed with six
2
2
column equivalents (CE) of hexane before adding the extracts.
Subsequently, the columns were eluted with 10 CE of hexane to
remove the cuticular hydrocarbons. Finally, the purified polar fraction
recorded in 720 μL of CDCl at 25 °C with TMS as the internal
3
standard on a Bruker Avance III HD 700 spectrometer. Data were
processed with the MestReNova software package. Analytical GC-MS
was performed on a Trace GC-DSQ I system (electron impact
spectra; EI) and an ISQ Single Quadrupole mass spectrometer
was eluted with 10 CE CH Cl . Fractions of 10 columns
2 2
(corresponding to the secretion of 400 individuals) were combined
and carefully concentrated in a stream of nitrogen. The residue was
redissolved in hexane and subjected to preparative gas chromatog-
raphy.
(
positive ion chemical ionization (PCI), using methane as reagent
gas); both systems were from Thermo Fisher. Aliquots of extracts (1.5
μL) or accordingly diluted compounds were directly subject to GC-
MS. First, we used an apolar ZB-5 capillary column (30 m × 0.25 mm
0.25 μm; Phenomenex) and a temperature program (50 °C for 1
min with 10 to 300 °C for 5 min isotherm). EI and CI spectra were
Preparative Capillary Gas Chromatography (pcGC). The final
purification of the major compound of the prepurified extracts (“peak
A”) was accomplished by pcGC using a preparative fraction collector
(PFC). The GC-PFC system consisted of a gas chromatograph
equipped with a flame ionization detector (FID: Agilent 7890A), a
PFC device (Gerstel), and a ZB-5 fused silica capillary column (30 m
× 0.32 mm ID, 0.25 μm) from Phenomenex. Hydrogen was used as
carrier gas with a flow rate of 3 mL/min. The column was split at the
end by a μFlow splitter (Gerstel) into two columns leading to the FID
(2 m × 0.15 mm ID) and the PFC (1 m × 0.2 mm ID), respectively.
Nitrogen makeup gas with a flow rate of 25 mL/min was applied to
the splitter. The PFC was connected with the GC oven via a heated
transfer line, which was connected to seven transfer capillaries with an
eight port zero-dead volume valve via the deactivated column (for
further information about the setup, see refs 31 and 32). Four μL
sample aliquots were injected via a multimode inlet (MMI) (Agilent)
and heated to 320 °C. The temperature of the GC oven was raised
from 40 to 270 °C with a heating rate of 25 °C per minute. The
sampling time was 1 min, and the transfer line of the PFC was heated
to 270 °C. The volatile traps were self-made microliter glass tubes
filled with 50 mg of Carbotrap B (mesh 20−40, Supelco) and
deactivated glass wool. The traps were fixed in a handmade closed
cylindrical glass pipe with a screw coupling with a sealing ring
(SciLabware Ltd. Stone). The glass pipe with the trap used for
fraction collection was placed in a self-made cooling block and chilled
to −20 °C. After the preparative fractionated collection of the main
compound (from 9.5 to 9.9 min), the traps were frozen at −20 °C
until further processing.
×
2
9
taken at 70 eV; detailed MS conditions are described elsewhere.
Retention indices (RIs) were calculated according to Van den Dool
30
and Kratz. Compounds of interest (i.e., peaks A and B) eluted at tR
17.54−17.61 min and t = 17.80−17.85 min, respectively (RI
=
1
=
peak A
R
878; RI
= 1901). For the separation of stereoisomers and
peak B
enantiomers in particular, we used a chiral CycloSil-B capillary
column (30m × 0.25 mm × 0.25 μm), coated with 30% heptakis(2,3-
di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin (DIME-β-
CD) in DB-101 (Agilent J&W) and two different temperature
programs: (1) For separation of the R,S- and S,R-isomers, the oven
was programmed from 160 °C (1 min) with 1 °C/min to 200 °C and
with 10 °C/min to 230 °C (5 min isotherm). (4S,5R)-1 and (4R,5S)-
1
eluted at 38.97 and 39.27 min, respectively (Figure 5a). Both the
R,R- and S,S-isomers eluted in one peak at 41.26 min. (2) For the
separation of the R,R- and S,S-isomers, TMS derivatives were
prepared (see below) and analyzed using the following temperature
program: 100 °C (1 min) followed by an increase of 1 °C/min to 190
°
C and then with 15 °C/min to 230 °C (5 min isotherm). (4S,5S)-1-
TMS and (4R,5R)-1-TMS eluted at tR = 80.83 and 81.06 min,
respectively (Figure 5b). HRMS spectra were recorded on a Q-
exactive high-resolution Orbitrap MS with a heated electrospray
source coupled to an Accela 1250 HPLC-pump (Thermo Fisher).
Analytical thin layer chromatography (TLC) was carried out on
precoated 0.25 mm silica gel 60 (F254) plates from Macherey-Nagel.
The visualization of substances was performed under UV light (254
nm) and/or by staining with either aqueous potassium permanga-
nate/potassium carbonate solution (KMnO4 stain) or 5% (w/v)
phosphomolybdic acid solution in ethanol (PMA stain).
Compounds in Extracts. (4S,5R)-4-Hydroxy-5-octyl-4,5-dihy-
1
dro-3H-furan-2-one (4S,5R-1). H NMR, Table 1. EIMS (70 eV): m/
z 196 [M − 18] (1), 143 (27), 142 (39), 125 (11), 124 (30), 115 (6),
111 (5), 102 (7), 101 (10), 98 (10), 97 (15), 96 (11), 95 (11), 89
(7), 88 (6), 84 (10), 83 (56), 82 (23), 81 (11), 71 (13), 69 (100), 68
(12), 67 (10), 57 (41), 55 (47), 44 (31), 43 (37), 41 (24). PCIMS
m/z 255 [M + C H ] (17), 243 [M + C H ] (13), 215 [MH] (100),
All solvents and reagents were obtained from ABCR, Carl Roth,
and Sigma-Aldrich and were used as received unless stated otherwise.
Preparation of Extracts. 400 Adult individuals of both sexes of
Egaenus convexus were collected by hand from May to July 2013 at the
3
5
2
5
197 (20), 195 (10), 179 (36), 161 (16), 155 (68), 137 (23).
+
“
Rosenhain”, Graz, Austria (N47.084838; E15.449741). An additional
HRESIMS m/z 215.1642 [M + H] (calcd for C H O , 215.1647);
1
2
23
3
5
5 individuals (from the same location) were from collections in July
019 and July 2020, respectively. All specimens were deposited in the
gas chromatographic retention index (ZB-5) 1878.
2
(4S,5S)-4-Hydroxy-5-octyl-4,5-dihydro-3H-furan-2-one (4S,5S-1).
EIMS (70 eV): m/z 196 [M − 18] (3), 143 (23), 142 (41), 136 (11),
131 (7), 125 (11), 124 (28), 115 (11), 112 (11), 111 (13), 110 (12),
102 (15), 101 (8), 98 (15), 97 (18), 96 (13), 95 (15), 89 (11), 84
collection of the Institute of Biology, Division of Zoology, University
of Graz, Austria (voucher numbers RG 4240−4261, 4266−4275,
4277, 4288, 4325, 4349, 4356, 4365−4383, 4387, 4408−4411, 4416−
G
https://dx.doi.org/10.1021/acs.jnatprod.0c00277
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
(
4
12), 83 (74), 82 (35), 71 (23), 70 (28), 69 (100), 57 (53), 55 (68),
4 (39), 43 (37), 41 (26). HRESIMS m/z 215.1642 [M + H] (calcd
μL) was added to an aliquot of extract containing 1 (50 μL in hexane)
+
at ambient temperature. After 30 min, H O (2 mL) was added
2
for C H O , 215.1647); gas chromatographic retention index (ZB-
carefully. After the exothermic reaction has subsided, the resulting
12
23
3
5
) 1901.
Microderivatization. Aliquots of extracts were subjected to
slurry was extracted with Et O (3 × 1 mL). An aliquot was checked
2
for 1,3,4-dodecanetriol (7). The combined organic layers were
concentrated in a stream of nitrogen before adding MSTFA reagent
(60 μL). The resulting mixture was incubated for 40 min at 60 °C,
resulting in 8. An aliquot of 8 was used directly for GC-MS analysis:
silylation, saponification, reduction, and methylation, respectively. For
silylation, we used (i) MSTFA (N-methyl-N-(trimethylsilyl)-trifluor-
acetamide in pyridine 2:1 containing 1% trimethylchlorosilane) and
(ii) MTBSTFA (N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide
EIMS (70 eV) m/z 419 ([M − CH ] (<1), 344 (1), 329 (2), 219
3
containing 1% tert-butyldimethylchlorosilane), respectively. Saponifi-
cation was performed using NaOCH3 in MeOH; reduction was
performed with lithium aluminum hydride and methylation, with
diazomethane.
(23), 215 (39), 147 (28), 115 (18), 103 (80), 75 (32), 73 (100).
PCIMS m/z 435 [MH] (16), 433 (13), 420 (40), 419 (97), 391 (6),
346 (23), 345 (76), 343 (51), 329 (91), 321 (16), 289 (7), 256 (25),
255 (199), 219 (29), 215 (38), 165 (6).
Microderivatization of 4-Hydroxy-5-octyl-4,5-dihydro-3H-
furan-2-one (1). Preparation of 5-Octyl-4-(trimethylsilyloxy)-
dihydro-3H-furan-2-one (2). MSTFA (50 μL) was added to an
aliquot of the scent gland extract in hexane (50 μL). The reaction
mixture was incubated at 55 °C. After 30 min, an aliquot of the
mixture (1.5 μL) was directly used for GC-MS analysis. EIMS (70
eV) m/z 286 (<1), 271 (6), 229 (78), 227 (7), 215 (100), 143 (9),
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00277.
■
*
1
Synthesis of reference materials; NMR spectra ( H,
1
29 (11), 117 (42), 116 (95), 101 (75), 75 (25), 73 (45), 69 (5), 59
1
COSY, and TOCSY spectra for authentic material, H
(12). 55 (12), 43 (18), 41 (17). PCIMS m/z 327 [M + C H ] (6),
13
3
5
and C spectra for (4R,5S)-1, (4R,5R)-1, 11, 12, 13, 15;
15 [M + C H ] (14), 287 [MH] (100), 269 (10), 229 (20), 215
2 5
COSY, HSQC, and HMBC spectra for (4R,5S)-1); EI-
mass spectra of microderivatives (compounds 2, 3, 5, 6,
8a, 8b); comparison of sampling techniques (direct
sampling of secretion vs whole body extraction: total ion
Preparation of 4-(tert-Butyldimethylsilyloxy)-5-octyldihydro-3H-
furan-2-one (3). MTBSTFA (50 μL) was added to an aliquot of the
secretion extract in hexane (50 μL). The reaction mixture was
incubated at 55 °C. After 30 min, an aliquot of the mixture (1.5 μL)
was directly used for GC-MS analysis: EIMS (70 eV) m/z 271 [M −
C H ] (5), 230 (15), 229 (78), 192 (2), 143 (5), 129 (15), 117 (14),
1
chromatograms and H spectrum of whole-body extract)
(PDF)
4
9
1
11 (8), 101 (20), 97 (13), 84 (8), 81 (12), 75 (100), 73 (14), 55
AUTHOR INFORMATION
(
(
(
89), 41 (11). PCIMS m/z 369 [M + C H ] (13), 357 [M + C H ]
3
5
2
5
■
28), 329 [MH] (100), 327 (13), 311 (8), 271 (15), 229 (43), 225
7), 203 (8), 197 (20), 179 (23), 161 (3), 179 (23), 161 (3), 137 (7).
Preparation of Trimethylsilyl 3,4-Bis((trimethylsilyl)oxy)-
Corresponding Author
Gunther Raspotnig − Institute of Biology, University of Graz,
010 Graz, Austria; Research Unit of Osteology and Analytical
̈
8
dodecanoate (5). 300 μL of a solution of NaOCH3 in MeOH
25%) was added to an aliquot of hexane extract (50 μL), and the
Mass Spectrometry, Medical University, University Children’s
Hospital, 8036 Graz, Austria; orcid.org/0000-0002-6947-
(
resulting mixture was incubated for 16 h at 75 °C. The reaction was
stopped by the careful addition of 500 μL of H O, followed by
9
406; Email: guenther.raspotnig@uni-graz.at
2
acidification to pH 2−3 with 1 N HCl and extraction with 1 mL of
EtOAc. An aliquot was analyzed by GC-MS for 3,4-dihydroxydode-
Authors
Felix Anderl − Institute of Biology, University of Graz, 8010
canoic acid (4). The addition of H O during the workup leads to
2
warming of the solution and complete saponification of the
intermediately formed methyl ester by emerging NaOH. The
remaining solution was dried under a stream of nitrogen for further
derivatization. For silylation, MSTFA reagent (60 μL; containing 1%
TMCS (trimethylchlorosilane); 2:1 in pyridine)) was added, and the
resulting mixture was incubated for 40 min at 60 °C, resulting in 5. An
aliquot of the reaction mixture 5 was used directly for GC-MS
Graz, Austria
Olaf Kunert − Institute of Pharmaceutical Sciences, University of
Graz, 8010 Graz, Austria
Miriam Schaider − Institute of Biology, University of Graz,
8
010 Graz, Austria
Adrian Bruckner − Division of Biology and Biological
̈
Engineering, California Institute of Technology, Pasadena,
California 91125, United States of America
analysis: EIMS (70 eV) m/z 433 [M − CH ] (1), 343 (6), 306 (50),
3
2
7
15 (54), 190 (11), 147 (34), 133 (11), 116 (7), 103 (17), 83 (14),
Mario Schubert − Department of Biosciences, University of
Salzburg, 5020 Salzburg, Austria; orcid.org/0000-0003-
5 (28), 73 (100), 69 (20), 44 (16). PCIMS m/z 477 [M + C H ]
2
5
(
(
4), 449 [MH] (21), 447 (17), 433 (100), 387 (5), 359 (72), 343
48), 335 (12), 306 (62), 269 (18), 215 (41), 147 (3), 73 (3).
Preparation of Methyl 3,4-Bis((trimethylsilyl)oxy)dodecanoate
0
278-4091
Stefan Do
Salzburg, 5020 Salzburg, Austria
̈
tterl − Department of Biosciences, University of
(6). Diazomethane was prepared from Diazald (N-methyl-N-nitroso-
33
p-toluenesulfonamide; Sigma), and a solution of diazomethane (in
Roman Fuchs − Department of Biosciences, University of
Salzburg, 5020 Salzburg, Austria
̈
Hans-Jorg Leis − Research Unit of Osteology and Analytical
Mass Spectrometry, Medical University, University Children’s
Et O/MeOH 9:1, saturated, 1 mL) was added to 4. After 20 min at
2
ambient temperature, the reaction mixture was concentrated to
dryness in a stream of nitrogen. MSTFA reagent (60 μL; containing
1
% TMCS, 2:1 in pyridine) was added, and the resulting mixture was
Hospital, 8036 Graz, Austria
incubated for 40 min at 60 °C, resulting in 6. An aliquot of the
reaction mixture 6 was used directly for GC-MS analysis: EIMS (70
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00277
eV) m/z 375 [M − CH ] (2), 359 [M − OCH ] (3), 343 (1), 285
3
3
(
(
[
(
11), 248 (64), 215 (51), 175 (5), 159 (8), 147 (22), 133 (16), 116
21), 103 (21), 89 (18), 83 (23), 75 (40), 73 (100). PCIMS m/z 419
M + C H ] (7), 391 [MH] (31), 375 (100), 359 (22), 343 (5), 329
12) 301 (58), 285 (38), 248 (15), 215 (18), 211 (15).
Preparation of 1,3,4-Tris((trimethylsilyl)oxy)dodecane (8). A
Author Contributions
G.R. and F.A. contributed equally to this work.
Notes
2
5
∇
solution of lithium aluminum hydride (10 mg/mL in Et O, 200
The authors declare no competing financial interest.
2
H
https://dx.doi.org/10.1021/acs.jnatprod.0c00277
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
(
26) Holmberg, R. G. In Proceedings of the 9th International
ACKNOWLEDGMENTS
■
Arachnological Congress; Eberhard, W. G., Lubin, Y. D., Robinson, B.
C., Eds.; Smithsonian Institution Press: Washington, DC, 1986; pp
This work was supported by the Austrian Science Fund
(
FWF), project number P29699-B25. We are particularly
grateful to P. Fottinger and A. Bauer for their kind assistance in
many laborious night excursions to collect specimens of
Egaenus convexus and to I. Schaffler for supporting the
131−133.
̈
(27) Machado, G.; Carrera, P. C.; Pomini, A. M.; Marsaioli, A. J. J. J.
Chem. Ecol. 2005, 31, 2519−2539.
(28) Meinwald, J.; Kluge, A. F.; Carrel, J. E.; Eisner, T. Proc. Natl.
̈
Acad. Sci. U. S. A. 1971, 68, 1467−1468.
preparative GC analyses. We thank G. Kunz for providing a
photograph of a living specimen of Egaenus convexus. We
further thank C. O. Kappe, D. Dallinger, and Y. Chen for their
help regarding the initial steps in the synthesis of the β-
hydroxy-γ-lactones.
(
(
29) Bodner, M.; Raspotnig, G. J. J. Chem. Ecol. 2012, 38, 547−556.
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https://dx.doi.org/10.1021/acs.jnatprod.0c00277
J. Nat. Prod. XXXX, XXX, XXX−XXX