Additional vinyl ketones and their pyranyl ketones in gonyleptid harvestmen (Arachnida: Opiliones) suggest these metabolites are widespread in this family

Article abstract of DOI:10.1021/np4001569

Four species of gonyleptid harvestmen, Acanthogonyleptes pulcher, Gonyleptes saprophilus (Gonyleptinae), Sodreana barbiellini, and Sodreana leprevosti (Sodreaninae), were examined by GC-MS and ¹H NMR. All of these species release vinyl ketones, and three of them produce the corresponding pyranyl ketones, which are presumed hetero-Diels-Alder (HDA) dimers. The vinyl ketones 5-methyl-1-hexen-3-one, rac-4-methyl-1-hexen-3-one, and (S)-4-methyl-1-hexen-3-one were synthesized. Natural 4-methyl-1-hexen-3-one is present as a single stereoisomer and has the R-configuration. Vinyl ketone dimers (HDA dimers) were also observed in the scent gland exudate and characterized by HRMS, ¹³C NMR, and ¹H NMR chemical shifts of the pyranyl moiety.

Full text of DOI:10.1021/np4001569

Article
pubs.acs.org/jnp
Additional Vinyl Ketones and Their Pyranyl Ketones in Gonyleptid
Harvestmen (Arachnida: Opiliones) Suggest These Metabolites Are
Widespread in This Family
Felipe C. Wouters,† Daniele F. O. Rocha,† Caroline C. S. Goncalves,† Glauco Machado,‡
̧
and Anita J. Marsaioli*,†
†Chemistry Institute, State University of Campinas, Campinas, SP, Brazil
‡Departamento de Ecologia, Instituto de Biocien
̂
cias, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
̃
̃
S
* Supporting Information
ABSTRACT: Four species of gonyleptid harvestmen, Acan-
thogonyleptes pulcher, Gonyleptes saprophilus (Gonyleptinae),
Sodreana barbiellini, and Sodreana leprevosti (Sodreaninae),
1
were examined by GC-MS and H NMR. All of these species
release vinyl ketones, and three of them produce the
corresponding pyranyl ketones, which are presumed hetero-
Diels−Alder (HDA) dimers. The vinyl ketones 5-methyl-1-
hexen-3-one, rac-4-methyl-1-hexen-3-one, and (S)-4-methyl-1-
hexen-3-one were synthesized. Natural 4-methyl-1-hexen-3-
one is present as a single stereoisomer and has the R-configuration. Vinyl ketone dimers (HDA dimers) were also observed in the
1
scent gland exudate and characterized by HRMS, 13C NMR, and H NMR chemical shifts of the pyranyl moiety.
and seven different HDA dimers in the scent gland exudates of
three of the four harvestmen (Table 1).
piliones, also known as harvestmen, are arachnids with
Olateral exocrine glands located on the cephalothorax that
release volatile compounds1 that are known to shield
harvestmen against natural predators, such as ants, frogs,
lizards, and spiders.2,3 In the suborder Laniatores, the
composition of scent gland exudates can contain benzoqui-
nones, alkyl phenols, and/or vinyl ketones.4−6 We have recently
reported the dimer of the α,β-unsaturated ketone 1-hepten-3-
one, 1-(6-butyl-3,4-dihydro-2H-pyran-2-yl)pentanone (1),
which was detected in the defensive secretions of two
harvestman species (Iporangaia pustulosa and Neosadocus
maximus) and is proposed to be a hetero-Diels−Alder
(HDA) dimer.4 Such dimerization generally requires specific
conditions (i.e., high pressure and temperature) not present in
harvestman scent glands, thus suggesting an enzymatically
controlled HDA reaction in harvestmen.4 Dimeric natural
products containing pyranyl rings that are present in plant
species, such as Arnica sachalinensis (Asteraceae)7 and Isodon
rubescens var. rubescens,8 are also proposed to be HDA dimers.
Here we describe the chemical characterization of the
exudates of four harvestman species belonging to the family
Gonyleptidae. The chemical composition of the exudate was
recently used as characteristic to construct the phylogeny of this
large family.6 The examined species produce a variety of vinyl
ketones. The vinyl ketones 5-methyl-1-hexen-3-one (2) and 4-
methyl-1-hexen-3-one (3) were synthesized and fully charac-
terized by GC-MS and NMR spectroscopy. We also report
spectroscopic evidence of the presence of four minor vinyl
ketones (1-hexen-3-one (4), 4-methylhexan-3-one (5), 4,5-
dimethylheptan-3-one (6), and 4-methyl-1-hexen-3-one (7))
RESULTS AND DISCUSSION
■
MS was the major tool to discover 5-methyl-1-hexen-3-one (2)
in Sodreana barbiellini (Mello-Leitao) and Sodreana leprevosti
̃
(B. Soares & H. Soares) and 4-methyl-1-hexen-3-one (3) in
Gonyleptes saprophilus (Mello-Leitao) and Acanthogonyleptes
̃
pulcher (Mello-Leitao) defensive secretions (Table 1, Figure 1).
̃
The MS spectrum of vinyl ketone 2 showed a molecular ion at
m/z 112 and fragments at m/z 55 and 70 arising from a ketone
α-cleavage and a McLafferty rearrangement, respectively.
Likewise, vinyl ketone 3 possessed a molecular ion at m/z
112 but was distinguished by a fragment at m/z 84 rationalized
as a McLafferty rearrangement. NMR characterization of 3 was
not available in the literature; however, the G. saprophilus
exudate was clean enough to make a full NMR spectroscopic
1
analysis (Table 2). The H NMR spectrum showed signals at
5.77, 6.27, and 6.44 ppm assigned to hydrogens on C-1 and C-2
with characteristic couplings of a terminal double bond (Table
2). The two diastereotopic hydrogens at C-5 appear as two
ensembles of seven peaks interpreted as partially overlapped
double doublets of quartets. The relative and absolute
configuration of this ketone was assessed by first establishing
a protocol to discriminate the enantiomers of a racemic
synthetic sample. The same synthetic pathway was established
for 2 and 3. The 3-methylbutanoic acid and 2-methylbutanoic
Received: February 22, 2013
Published: August 26, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
1559
dx.doi.org/10.1021/np4001569 | J. Nat. Prod. 2013, 76, 1559−1564

Journal of Natural Products
Article
Table 1. Compounds Detected in Defensive Secretion of Gonyleptid Harvestmen
a
b
Retention index obtained from linear regression from the other compounds. HREIMS obtained by CG-TOF-MS.
acid acyl chlorides were transformed into N-methoxy-N-3-
dimethylbutanamide derivatives, which produced 2 and 3,
respectively, by reaction with vinyl magnesium chloride. The
same synthetic pathway was applied to obtain (S)-3 using (S)-
2-methylbutanoic acid as starting material. Thus (S)-3 and
(rac)-3 were analyzed by GC-FID using a chiral phase column,
and their co-injections revealed the retention times of (S)-3 and
(R)-3, 11.6 and 12.0 min, respectively. Co-injection of G.
1560
dx.doi.org/10.1021/np4001569 | J. Nat. Prod. 2013, 76, 1559−1564

Journal of Natural Products
Article
Figure 1. GC-MS analyses of gonyleptid harvestmen exudate containing vinyl ketones and their hetero-Diels−Alder dimers. Peaks labeled with *
refer to solvent impurities. A. pulcher and G. saprophitus exudates were analyzed under two different conditions, but vinyl ketone 3 was confirmed by
co-injection with a standard compound and MS.
saprophilus and A. pulcher exudates with the racemic standard
showed that (R)-3 is the enantiomer present in >99%
enantiomeric excess (Figure 2). The absolute S-configuration
of analogous 4-methyl-3-ketones from arthropods was
previously determined for the (S)-4-methylheptan-3-one from
the ant Atta texana9 and (S)-4-methyl-1-hepten-3-one from the
walking stick Agathemera elegans.10
HDA dimers 8−14 detected in G. saprophilus, S. leprevosti,
and S. barbiellini (Figure 1, Table 1) were characterized by
HREIMS and MS spectra according to the molecular ions and
the fragmentation patterns of α-carbonyl cleavage followed by
1561
dx.doi.org/10.1021/np4001569 | J. Nat. Prod. 2013, 76, 1559−1564

Journal of Natural Products
Article
The presence of possible HDA dimers in several harvestman
exudates raises the issue of a hetero-Diels−Alderase. The only
confirmed Diels−Alderase has been isolated from the
bacterium Saccharopolyspora spinosa,12 and there is no report
of an isolated and characterized hetero-Diels−Alderase.
However, the presence of this enzyme in harvestmen and/or
their symbiont microorganisms13 seems to be responsible for
the production of pyranyl ketones from the vinyl ketones, as
this reaction typically requires forcing conditions to occur
chemically. Additionally, the occurrence of the dimers depends
on the presence of the vinyl ketones, while the inverse is not
necessarily true, as observed for A. pulcher (Figure 1). The
racemic nature of 1 however is not readily explained by an
enzymatic reaction, and we have no other evidence than the
fact that dimer formation is not spontaneous.4
Table 2. NMR Spectroscopic Data (CDCl3) for Natural 4-
Methyl-1-hexen-3-one (3) from Gonyleptes saprophilus.
position
1
δC, type
δH (J in Hz)
128.1, CH2
6.27, dd (17.5, 1.4)
5.77, dd (10.5, 1.4)
6.44, dd (17.5, 10.5)
2
3
4
5
135.5, CH
204.4
45.2, CH
26.2, CH2
2.42, qdd (7.5, 7.1, 7.1)
a
1.72, ddq, (14.0, 7.1, 6.9)
a
1.42, ddq, (14.0, 7.1, 6.9)
6
7
11.8, CH3
16.1, CH3
0.89, t (7.5)
1.10, d (6.9)
a
These signals are partially superimposed double doublets of quartets
that look like septets.
The chemical profiles of A. pulcher, G. saprophilus, S.
barbiellini, and S. leprevosti defensive secretions are consistent
with those of other species of the suborder Laniatores,6
showing vinyl ketones as major components (Table 1). Vinyl
ketones were detected in representatives of the family
Gonyleptidae, mainly in the subfamilies Gonyleptinae,
Hernandariinae, Sodreaninae, Progonyleptoidellinae, and Cae-
lopyginae.4,5
This work confirms the structure and absolute configuration
of (R)-3 in G. saprophilus and reports the presence of pyranyl
ketones in three additional species of harvestmen (S. barbiellini,
G. saprophilus, and S. leprovosti). Such data suggest that
representatives of the family Gonyleptidae possess hetero-
Diels−Alderases responsible for the biosynthesis of these
secondary metabolites. Further molecular biology studies are
necessary to prove the existence of this novel enzyme in
harvestmen. The chemical profile of the defensive secretions
and the relationships between the components give information
about the chemical defense evolution and possible biosynthetic
routes of harvestman secondary metabolites.
Figure 2. GC-FID analysis of 4-methyl-1-hexen-3-one (3). (A)
racemic, (B) S-enantiomer, (C) S- and rac-, (D) natural sample, (E)
natural and racemic sample.
EXPERIMENTAL SECTION
General Experimental Procedures. NMR spectra were acquired
■
with either an 11 T Varian Inova instrument, operating at 499.88 MHz
CO loss (Figure 3). Their structures were proposed
considering the presence of vinyl ketones in the same exudate
and all alternatives of the HDA reactions between diene and
dienophile. Compounds 9 and 11 present in G. saprophilus
were suggested as heterodimers of 3 and 4 and were
distinguished by their base peak at m/z 139 and 125,
respectively, and the presence of the monomeric vinyl ketones.
The structures of the heterodimers 10 and 12 found in S.
barbiellini and S. leprevosti were suggested taking into
consideration the presence of the vinyl ketones 2 and 4 and
their base peak fragments at m/z 139 and 125, respectively. The
presence of the pyranyl moiety in the homodimer 14 from the
S. barbiellini exudate was confirmed by comparing the NMR
1
for H NMR and 125.71 MHz for 13C NMR, or a 5.87 T Bruker
1
Avance DPX, at 250.13 MHz for H NMR and 62.89 MHz for 13C
NMR. CDCl3 was used as the solvent, and tetramethylsilane (TMS) as
the internal reference (0.0 ppm). GC-MS analyses were performed
using an Agilent 6890-5973 system using a DB-5 fused silica capillary
column (30 m × 0.25 mm × 0.25 μm). EIMS spectra were recorded at
70 eV at a scanning speed of 3.54 scans s−1 from m/z 40 to 400. The
oven temperature ranged from 50 to 200 °C at 10 °C min−1 and then
to 290 °C at 16 °C min−1. Natural samples (1 μL) were injected in the
splitless mode and synthetic in split (1:10) mode. The injector
temperature was 250 °C and the detector was 280 °C with helium as
the carrier gas. The retention index (RI)14 was determined using
splitless mode and two different temperature programs: (A) G.
saprophilus, S. barbellini, and S. leprevosti had the highly volatile 1-
hexen-3-one (4), and the oven temperature ranged from 40 to 290 °C
at a rate of 4 °C min−1 and 7.07 psi; (B) for the other analyzed species,
the temperature ranged from 50 to 290 °C, at a rate of 4 °C min−1 and
7.62 psi. Separately, an alkane standard solution, C8−C20 (Fluka), was
injected in the same program (for 1-hexen-3-one (4) addition of
heptane was needed). Enantioselective analyses were conducted with a
GC-FID Agilent 6890 using stationary phase Chirasil-dex (25 m ×
0.25 mm × 0.25 μm), H2 at 0.5 mL min−1, injector and detector at 220
and 250 °C, respectively. A volume of 1 μL of the samples was injected
in the splitless mode. The temperature ranged from 40 to 100 °C at a
rate of 3 °C min−1, from 100 to 180 °C at a rate of 30 °C min−1, and
held at 180 °C for 20 min. HREIMS was performed on a Waters GCT
Premier at 20 scans s−1, resolution 7000 fwhm, sub-5 ppm RMS with
1
data (13C NMR and H NMR) of the exudate with those of 1
(Table 3). Additionally, several signals of methyl groups at
23.5−22.9 ppm are consistent with a mixture of compounds
with 3-methylbutan-1-one and isobutyl side chains.
Hara et al.5 previously reported the same composition in the
S. leprevosti (=Zortalia inscripta) exudate, but dimer 14 was
described as unknown. The ketone 2 was detected in
Neosadocus maximus, while 3 was present in Gonyleptes
curvicornis and Parampheres sp. defensive secretions.5 The
tenebrionid Amargmus tristis also produces 2 as a component of
its defensive secretion together with other defensive com-
pounds.11
1562
dx.doi.org/10.1021/np4001569 | J. Nat. Prod. 2013, 76, 1559−1564

Journal of Natural Products
Article
Figure 3. MS spectrum and fragmentation of HDA dimers 1, 13, and 14.
internal lock mass correction and electron impact (EI) of 70 eV. The
GC Agilent 7683 operated with an oven temperature ranging from 50
to 250 °C at 10 °C min−1 and an HP5-MS column (30 m × 0.25 mm
× 0.25 μm). The injection volume was 1 μL in splitless mode. The
injector temperature was 270 °C and the detector was 250 °C, using
helium as the carrier gas.
́
Fazenda Capricornio (23°22′ S; 45°04′ W), municipality of Sao Paulo,
̃
in February 2011. Finally, S. leprevosti was collected in the Parque
Estadual Intervales (24°14′ S; 48°04′ W), municipality of Ribeirao
̃
Grande, state of Sao Paulo, in November−December 2009. Voucher
̃
specimens of all studied species are deposited in the Museu de
Zoologia (MZSP), Universidade de Sao Paulo, Brazil.
̃
Secretion Sampling. Individuals of each species were collected in
different places of the Atlantic Forest in southeastern Brazil. A. pulcher
Individuals of all studied species were taken to the laboratory and
kept alive in plastic vials containing a piece of wet cotton to maintain
moisture. The defensive secretions were collected by pressing the
gland openings with cotton wool previously cleaned with doubly
distilled EtOAc. Each individual produced 1 to 5 μL of defensive
secretion mixed with enteric fluid. The liquid absorbed in the cotton
wool was washed with CDCl3 (2 mL) for NMR analyses and eluted
was collected in the Estaca
̧
o Biolog
̃
́
ica do Alto da Serra (23°46′ S;
̃
46°20′ W), municipality of Sao Paulo, in January 2010. G. saprophilus
was collected in the vicinity of the Parque Nacional do Itatiaia (22°20′
S; 44°35′ W), along the border of the states of Minas Gerais and Rio
de Janeiro, in November 2010. S. barbiellini was collected in the
1563
dx.doi.org/10.1021/np4001569 | J. Nat. Prod. 2013, 76, 1559−1564

Journal of Natural Products
Article
1
Table 3. Characteristic H and 13C NMR Signals for HDA
ASSOCIATED CONTENT
* Supporting Information
Spectroscopic characterization of all natural and synthetic
compounds is available free of charge via the Internet at http://
pubs.acs.org.
■
Adduct 14 in Sodreana barbiellini Exudate (C6D6) and 1-(6-
Butyl-3,4-dihydro-2H-pyran-2-yl)pentan-1-one (1, CDCl3,
ref 5)
S
AUTHOR INFORMATION
Corresponding Author
*Tel: +55-19-35213067. Fax: +55-19-35213023. E-mail: anita@
iqm.unicamp.br.
■
Notes
The authors declare no competing financial interest.
a
Results from 1H and 13C (fully decoupled DEPT-135) and 2D
ACKNOWLEDGMENTS
■
HSQC (1H−13C, J) spectra of the concentrated Sodreana barbiellini
exudate.
1
We acknowledge D. S. Caetano and B. A. Buzatto for helping to
collect the individuals in the field, Prof. C. H. Collins
(Chemistry Institute/Unicamp) for revising the text, and
CNPq, Petrobras
́
, and Fundaca
̧
o de Amparo a
̀
Pesquisa do
̃
with EtOAc (2 mL) for GC-MS analyses. All solvents were of high
analytical grade and doubly distilled before use. The number of
individuals of each species was as follows: A. pulcher (n = 5), G.
saprophilus (n = 15), S. barbiellini (n = 5), S. leprevosti (n = 11).
Synthesis. N-Methoxy-N-3-dimethylbutanamide (15). 3-Methyl-
butanoic acid (9.06 mmol, 1 mL) was mixed with dimethylformamide
(35 μL) and cooled in an ice bath. Thionyl chloride (10.42 mmol, 0.76
mL) was added dropwise, and the system was allowed to reach room
temperature (rt) and mixed for 1 h. The excess of HCl was removed
by following nitrogen flux, and CHCl3 (75 mL) and N,O-dimethyl
hydroxylamine (9.97 mmol, 0.972 g) were added. The system was
cooled, and pyridine (22.65 mmol, 2.2 mL) was added. After 1 h at rt,
the reaction mixture was washed with H2O and dried with MgSO4, and
the pyridine was removed as an azeotropic mixture with heptane. The
product was purified by column chromatography with silica gel and a
gradient of hexane/EtOAc as mobile phase, giving 0.68 g of a colorless
oil, in 52% yield. 1H NMR (250.13 MHz, CDCl3) δ 3.68 (3H, s), 3.18
(3H, s), 2.30 (2H, m), 2.09−2.25 (1H, m), 0.97 (6H, d, 3J = 7.5 Hz);
13C NMR (62.89 MHz, CDCl3) δ 174.1 (C, CO), 61.2 (CH3,OCH3),
40.7 (CH2), 25.1 (CH), 22.7 (CH3); EI-MS m/z 145 [M+] (13),
85(77), 61(40), 58(11), 57(100), 43(12), 41 (35); GC retention time
(min) 6.57.
Estado de Sao Paulo (GM 02/00381-0; 08/06604-7) for
̃
financial support.
REFERENCES
■
(1) Machado, G.; Pinto-da-Rocha, R.; Giribet, G. What are
harvestmen. In Harvestmen: The Biology of Opiliones; Pinto-Da-
Rocha, R., Machado, G., Giribet, G., Eds.; Harvard University Press,
2007.
(2) Eisner, T.; Rossini, C.; Gonzalez, A.; Eisner, M. J. Exp. Biol. 2004,
207, 1313−1321.
(3) Machado, G.; Carrera, P. C.; Pomini, A. M.; Marsaioli, A. J. J.
Chem. Ecol. 2005, 31, 2519−2539.
(4) Rocha, D. F. O.; Hamilton, K.; Gonca̧ lves, C. C. S.; Machado, G.;
Marsaioli, A. J. J. Nat. Prod. 2011, 74, 658−663.
(5) Hara, M. R.; Cavalheiro, A. J.; Gnaspini, P.; Santos, D. Y. A. C.
Biochem. Syst. Ecol. 2005, 33, 1210−1225.
(6) Caetano, D. S.; Machado, G. Cladistics 2013, DOI: 10.1111/
cla.12009.
(7) Passreiter, C. M.; Willuhn, G.; Weber, H.; Schleifer, K. J.
Tetrahedron 1999, 55, 2997−3006.
(8) Han, Q.; Lu, Y.; Zhang, L.; Zheng, Q.; Sun, H. Tetrahedron Lett.
2004, 45, 2833−2837.
N-Methoxy-N,2-dimethylbutanamide (16). Following the proce-
dure described above and using 2-methylbutanoic acid (9.06 mmol, 1
mL), 1 g of the title compound was obtained as a colorless oil in 76%
(9) (a) Riley, R. G.; Silverstein, R. M.; Moser, J. C. Science 1974, 183,
760−762. (b) Riley, R. G.; Silverstein, R. M. Tetrahedron 1974, 30,
1171−1174.
(10) (a) Schmeda-Hirschmann, G. Z. Naturforsch. 2006, 61c, 592−
594. (b) Espinoza-Moraga, M.; Cornejo-Morales, R.; Santos, L. S.
Tetrahedron: Asymmetry 2009, 20, 1062−1064.
(11) Gnanasunderam, C.; Young, H.; Hutchins, R. F. N. Insect
Biochem. 1982, 2, 221−224.
(12) (a) Kim, H. J.; Ruszczycky, M. W.; Choi, S.-H.; Liu, Y.-N.; Liu.,
H.-W. Nature 2011, 473, 109−112. (b) Hess, B. A., Jr.; Smentek, L.
Org. Biomol. Chem. 2012, 10, 7503−7509.
(13) Crawford, J. M. Chem. Commun. 2011, 47, 7559−7566.
(14) Van Den Dool, H.; Kratz, P. D. J. Chromatogr. 1963, 11, 463−
471.
1
yield. H NMR (250.13 MHz, CDCl3) δ 3.69 (3H, s), 3.19 (3H, s),
2.75−2.84 (1H, m), 1.58−1.81 (1H, m), 1.39−1.47 (1H, m), 1.11
(3H, d, 3J = 6.9 Hz), 0.89 (3H, t, 3J = 7.4 Hz); 13C NMR (62.89 MHz,
CDCl3) δ 171.3 (C, CO), 61.6 (CH3, OCH3), 37.0 (CH), 32.5 (CH3,
NCH3), 27.0 (CH2), 17.3 (CH3, HCCH3), 12.2 (CH3, H2CCH3); EI-
MS m/z 145(9) [M+], 85(45), 61(16), 57(100), 41(22); GC retention
time (min) 6.37.
5-Methyl-1-hexen-3-one (2). A solution of vinyl bromide
(approximately 3 mL) in anhydrous tetrahydrofuran (THF, 10 mL)
was added to a suspension of Mg0 (39.5 mmol, 0.96 g) in THF (10
mL). The reaction was heated to reflux, and after total consumption of
the magnesium, the solution was transferred to a vessel containing 15
(2.07 mmol, 300 mg) and 3 mL of THF. The reaction was stirred for
16 h at rt and quenched with saturated NH4Cl. After extraction with
ethyl ether, the organic phase was dried with anhydrous MgSO4 and
the solvent removed by fractional distillation. The final product was a
solution of the title compound in THF. Ketone 2 was first described in
ref 15.
(15) Hammen, P. D.; Braisted, A. C.; Northrup, D. L. Synth.
Commun. 1991, 21, 2157−2163.
4-Methyl-1-hexen-3-one (3). The procedure was similar to that
described above, substituting 16 for 15. The pure S-enantiomer was
obtained from (S)-16 using silylated glassware with freshly distilled
TMS-Cl. The product was obtained as a THF solution. Retention
times (Chirasil-dex column): (S)-16, 11.55 min; (R)-16, 12.0 min.
The 1H NMR spectrum had THF signals, and 3 did not endure
distillation; therefore we have used the solution in GC-MS analyses.
1564
dx.doi.org/10.1021/np4001569 | J. Nat. Prod. 2013, 76, 1559−1564