Frogolide – An Unprecedented Sesquiterpene Macrolactone from Scent Glands of African Frogs

Article abstract of DOI:10.1002/ejoc.201800199

Some amphibians use chemical signals in addition to optical and acoustical signals to transmit information. Males of mantellid frogs from Madagascar and hyperoliid frogs from Africa emit complex, species- and sex-specific bouquets of volatiles from their femoral or gular glands. We report here on the identification, synthesis, and determination of the absolute configuration of a macrocyclic lactone occurring in several species of both families, (S)-3,7,11-dodec-6,10-dien-12-olide (S-14, frogolide). Macrolides are a preferred compound class of frog volatiles. Nevertheless, frogolide is the first macrocyclic lactone obviously derived from the terpene pathway, in contrast to known frog macrolides that are usually formed via the fatty acid biosynthetic pathway.

Full text of DOI:10.1002/ejoc.201800199

DOI: 10.1002/ejoc.201800199
Full Paper
Volatile Macrolactones
Frogolide – An Unprecedented Sesquiterpene Macrolactone
from Scent Glands of African Frogs
Markus Menke,[a] Kristina Melnik,[a] Pardha S. Peram,[a] Iris Starnberger,[b] Walter Hödl,[b]
Miguel Vences,[c] and Stefan Schulz*[a]
Abstract: Some amphibians use chemical signals in addition of both families, (S)-3,7,11-dodec-6,10-dien-12-olide (S-14, frog-
to optical and acoustical signals to transmit information. Males olide). Macrolides are a preferred compound class of frog vola-
of mantellid frogs from Madagascar and hyperoliid frogs from tiles. Nevertheless, frogolide is the first macrocyclic lactone ob-
Africa emit complex, species- and sex-specific bouquets of vola- viously derived from the terpene pathway, in contrast to known
tiles from their femoral or gular glands. We report here on the frog macrolides that are usually formed via the fatty acid bio-
identification, synthesis, and determination of the absolute con- synthetic pathway.
figuration of a macrocyclic lactone occurring in several species
Introduction
Anuran amphibians (frogs and toads) communicate via acous-
tic, visual, and tactile signals. In addition to these traits, some
representatives of anuran families such as the Hyperoliidae and
Mantellidae use the chemical communication channel to trans-
mit information via volatile pheromones (Figure 1). The first
volatile pheromone compounds identified from frogs were
(R)-8-methylnonan-2-ol (3), and the macrolide phoracantholide
J (4). Both compounds induce directional movement in both
females and males of Mantidactylus multiplicatus, although the
exact biological function of the pheromone is still open to dis-
Figure 1. Compounds produced in scent glands of anuran amphibians. For
compounds 3 and 4, a species- and sex-specific, biological function has been
cussion.[1] M. multiplicatus belongs to the family Mantellidae,
endemic to Madagascar. Males of many mantellids possess
femoral glands on the ventral sides of their shanks that dissemi-
nate volatile compounds. The volatiles are mostly alcohols and
macrocyclic lactones, e.g. phoracantholide J (4),[1,2] gephyro-
mantolide A (1),[1] mantidactolides (5, 6),[3] or other macrolides
with even larger rings (2).[4,5] Further studies have provided
evidence that macrolactones can actually be perceived by the
olfactory system of mantellids[6] and that these frogs contain
species-specific mixtures of volatiles despite occasional large
variations within species.[2,6]
experimentally tested.
that is innervated during the mating season. On these sacs a
colorful gular gland is visible (Figure 2A) that releases a com-
plex mixture of volatile compounds during calling. An initial
screening study on several species revealed that these glands
contain some volatiles similar to those of the Mantellidae, but
additionally often sesquiterpenes, many of which not readily
identifiable using mass spectroscopic databases.[7]
Currently we work on the identification of these sesqui-
terpenes in the Hyperoliidae that occur in complex species-
specific blends. Due to the small amounts and the complex
composition of the gland contents, the isolation and structural
identification of unknown constituents is challenging. There-
fore, microderivatization and mass spectral information are
used to develop structural proposals. Finally, structure verifica-
tion has to be performed by synthesis.[8]
Another family obviously using volatile signals are the Afri-
can reed frogs of the family Hyperoliidae. They emit acoustic
and visual signals to attract females by inflation of a vocal sac
[a] Institute of Organic Chemistry, Technische Universität Braunschweig,
Hagenring 30, 38106 Braunschweig, Germany
E-mail: stefan.schulz@tu-braunschweig.de
http.//www.oc.tu-bs.de/schulz/index_en.html
[b] Department for Integrative Zoology,
Here we report on the identification of a novel unique ses-
quiterpene lactone, frogolide, by the approach described. This
compound was of particular importance, because it occurs in
several frog species of both mantellids and hyperoliids.
Frogolide is again a macrolactone, but originates from the
Althanstraße 14, 1090 Vienna, Austria
[c] Institute of Zoology, Technische Universität Braunschweig,
Mendelsohnstraße 4, 38106 Braunschweig, Germany
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejoc.201800199.
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Figure 4. Mass spectra of (A) natural compound A, and (B) hydrogenated
natural compound A. The shift of the M+-ion by 4 amu indicates the presence
of two C–C double bonds in A.
double-bond equivalents. Hydrogenation of the natural sample
furnished the mass spectrum shown in Figure 4B, most likely
derived from A. A mass of 240 amu was obtained, hinting at
two C–C double bonds in the molecule. We recently published
a ruleset for obtaining structural information from the mass
spectra of macrolactones.[8] A consecutive loss of two H2O units
together with the M-60 ion indicate a macrolide. The hydrogen-
ation product showed small ions at m/z 60 and 87, typical for
3-methyl-branched macrocyclic lactones. These ions arise by
McLafferty-type rearrangement followed by a second McLaf-
ferty rearrangement or a ꢀ-cleavage, respectively.[8] The mass
spectrum of A showed a strong ion m/z 168, a loss of 68 amu
from M+. This fragment is typical for the loss of an isoprene
unit, suggesting the unknown compound to be of terpenoid
origin.[9] In addition, a double bond near the alcohol end of A
becomes likely.
With these data in hand, we proposed A to be a macrolide
derived from partly hydrogenated, terminally oxidized farnesoic
acid. Often the 2,3-double-bond is hydrogenated in volatile
farnesoic acid derived signaling compounds.[10] Therefore,
3,7,11-dodec-6,10-dien-12-olide (14) was chosen as target for
our synthesis as being the most likely structure of A. The syn-
thesis was performed according to Scheme 1.
Figure 2. Frog species with male scent glands. (A) Hyperolius viridiflavus dur-
ing calling. The yellow gular gland is clearly visible (arrow) on the vocal sac.
(B) Male of Gephyromantis leucomaculatus. The femoral glands on the hind-
legs are not visible.
terpene pathway in contrast to other macrolactones produced
by these frogs.
Results and Discussion
GC/MS analysis of an extract of the gular gland of Hyperolius
viridiflavus (Figure 2A) revealed the presence of an unknown
compound A with a gas chromatographic retention index
I 1710 that occurred in several other frog species like e.g.
Gephyromantis leucomaculatus (Figure 2B) as well. Figure 3
shows the total ion chromatogram of the gular gland extract of
H. viridiflavus. The mass spectrum of compound A is shown in
Figure 4A.
Enantio- and regioselective hydrogenation of stereochemical
pure (E,E)-farnesol (7) according to Pfaltz et al. using commer-
cially available [Ru{(R)-tol-binap}](OAc)2 under 35 bar H2-atmos-
phere gave alcohol S-8 in 96 % yield.[11] The ee was 90 %, calcu-
lated back from final product 14. Subsequent stepwise oxid-
ation using Parikh–Doering and Pinnick–Lindgren oxidation[12]
followed by methylation[13] led to methyl ester 10 in 73 % yield
over three steps. This stepwise procedure proved to be more
effective than direct oxidation of alcohol 8 to the correspond-
Figure 3. Total ion chromatogram of a gular gland extract of H. viridiflavus.
The title compound A is indicated. X indicates a contaminant. All other peaks
are oxidized sesquiterpenes of unknown structure.
HR-MS delivered a molecular mass of m/z 236.1753 (calcd. ing acid with 2-azaadamantane N-oxyl (AZADO) and
[13]
236.1776) consistent with the composition C15H24O2 with four PhI(OAc)2 that gave lower yields. Also oxidation of alcohol 8
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Scheme 2. Proposed fragmentation of frogolide (14) leading to the character-
istic ions m/z 168 (17) and m/z 109 (18) of the mass spectrum (Figure 4A).
phase as shown in Figure 5. Coinjection of the racemic mixture
with the (S)-enantiomer proved the later eluting peak to be the
(S)-enantiomer. Synthetic S-14 had an ee of 90 % while syn-
thetic R-14 showed an ee of 85 %. Natural 14 was a 98:2 S/R
mixture.
Compound A was not only found in extracts of H. viridiflavus,
but also in other species, both hyperoliids and mantellids
(Table 1). These include H. cinnamomeoventris and several
Gephyromantis, Guibemantis, Mantella, and Spinomantis species.
In several of them only minor amounts occur, but extracts of
H. viridiflavus, Spinomantis aglavei and Guibemantis liber were
analyzed by chiral GC as well [see Supporting Information].
While H. viridiflavus contained a 98.5:1.5 S/R mixture, G. liber
produced only S-14. S. aglavei also contained S-14, but its low
abundance prohibited accurate ee determination.
Scheme 1. Synthesis of frogolide (S-14). The synthesis was also performed
with [Ru{(S)-tol-binap}](OAc)2 as catalyst, giving access to R-14.
using IBX led to acid catalyzed cyclization forming undesired
byproducts and thus reducing the yield significantly.[14] The fol-
lowing Riley-Oxidation of methyl ester 10 gave two major prod-
ucts, alcohols 11 and 12 in a 2:1 ratio.[13] These alcohols were
separable by column chromatography. Saponification of iso-
lated 11 using LiOH furnished acid 13 that was directly lacton-
ized with bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl)
and triethylamine according to Corey et al.[15] Target compound
S-14 was obtained as clean product by simple filtration of the
catalyst and removal of the solvent.
Gratifyingly, comparison of mass spectral and gas chromato-
graphic data proved the identity of natural compound A and
14. The synthetic sequence was then performed again using
[Ru{(S)-tol-binap}](OAc)2 as hydrogenation catalyst to obtain
R-14 as well.
Because of its abundance across species, we propose the
name frogolide for this new natural compound. Surprisingly,
the prevalent macrolactone pattern in frog volatiles is also fol-
lowed in the structure of this sesquiterpene. Macrolactones
identified earlier, e.g. 2 and 4–6 are most likely derived from the
fatty acid pathway.[17] In contrast, 14 is the first sesquiterpene
macrolactone identified in frogs. The compound is closely re-
lated to niaviolide that additionally contains a C-2–C-3 double-
bond. Niaviolide was isolated from the androconial organs of
the African butterfly Amauris niavius.[16] Other sesquiterpenoid
macrolactones used in chemical communication are the
tris(norsesquiterpene)s cucujolide I [ferrulactone I, (4E,8E)-4,8-
dimethyldeca-4,8-dien-10-olide], employed as pheromone in
both cucujolid beetles[18] and Pieris butterflies,[9] and its isomer
The formation of the characteristic peaks m/z 109 and 168 suspensolide (3E,8E)-4,8-dimethyldeca-3,8-dien-10-olide.[19]
in the mass spectrum of 14 can be explained as shown in
Scheme 2. Initial cleavage led to ion 16 that loses a stable iso-
prene molecule, the 68 amu fragment mentioned above. The
distonic species 17 is formed with high intensity, stabilized by
the allylic cation. An acetyl radical loss leads to the base peak
m/z 109 (18) after H-transfer and double bond formation. The
loss of isoprene can also be observed in mass spectra of other
macrolides with the typical 1,5-dimethyl-1,5-diene structural
motif such as brassicalactone[9] and niaviolide.[16]
Frogolide is another example of the lactone ring as a pre-
ferred structural motif of volatile signals.[17] Some macrolides
are used by both insects and frogs, e.g. 2 and 4.[1,4,20] This might
implicate that frogs potentially obtain the macrolides from their
insect diet, which synthesize macrolides de novo[18,21] but can
also take them up from plants.[22] Experiments with a laboratory
colony of Mantidactylus betsileanus exclusively fed with fruit
flies showed de novo synthesis of 4, which was absent in the
food (unpublished). It seems likely that also the frogs investi-
With the two enantiomers in hand, the absolute configura- gated can synthesize 14 de novo, effectively using the terpene
tion of the natural compound was determined. Both enantio- biosynthetic pathway besides fatty acid metabolism to synthe-
mers were separated by GC on a chiral ꢀ-TBDMS-hydrodex size macrolides.
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Conclusions
We herein report a novel, unprecedented sesquiterpene macro-
lide, a rare class of natural products. The structure of frogolide
was determined to be (3S,6E,10E)-3,7,11-trimethyldodeca-6,10-
dien-12-olide (14). This compound is found in gular glands of
male African hyperoliid frogs as well as femoral glands of male
mantellid frogs endemic to Madagascar. We hypothesize that
frogolide is involved in chemical communication of these frogs,
although its exact function is yet unknown.
Experimental Section
General Remarks: Chemicals were obtained from commercial sup-
pliers and used without further purification unless otherwise noted.
Reactions were carried out in flame-dried vessels under a nitrogen
atmosphere. Conventionally dried solvents were distilled before use.
Purification of synthetic compounds was performed by column
chromatography with silica (silica gel 60, particle size 0.040–
0.063 mm, mesh 230–440 ASTM, Fluka) using ethyl acetate, pent-
ane, and diethyl ether as solvents. Thin layer chromatography was
performed using silica coated plates Polygram SIL G/UV254 (Ma-
cherey & Nagel) with molybdatophosphoric acid (10 % in ethanol)
for detection. 1H NMR- and 13C NMR spectra were acquired with
the following instruments (Bruker): AV II-300 (300 MHz for 1H and
75 MHz for 13C), DRX-400 (400 MHz for 1H and 100 MHz for 13C),
AV III-400 (400 MHz for 1H and 100 MHz for 13C) and AV II-600
1
(600 MHz for H and 150 MHz for 13C). Tetramethylsilane was used
as an internal standard (TMS, δ = 0 ppm). Multiplicities of the pro-
tons are described as singlets (s), doublets (d), triplets (t), quartets
(q), quintets (quint), sextets (sext), septets (sept), or multiplets (m).
The connectivities of the carbon atoms are described as primary
(CH3), secondary (CH2), tertiary (CH), or quaternary (Cq). GC/MS anal-
yses of synthetic products were performed with a GC HP6890/MSD
HP5973 combination (Hewlett Packard) and natural samples were
analyzed with a GC 7890A/MSD 5975C combination (Agilent Tech-
nologies). Mass spectrometry was performed in electron ionization
mode (EI) with 70 eV. Fused-silica capillary columns HP-5MS (Agilent
Technologies, 30 m, 0.25 mm i.d., 0.25 μm film thickness) were used
with helium as the carrier gas. High resolution mass spectrometry
data were obtained with a GC 6890 gas chromatograph (Agilent)
equipped with a Phenomenex ZB5-MS column (30 m, 0.25 mm i.d.,
0.25 μm film thickness) coupled with a time-of-flight mass spec-
trometer JMS-T100GC, GCAccuTOF (JEOL, Japan), operated in EI
mode (70 eV). JEOL MassCenter Workstation Software was used.
The instrument was calibrated with PFK to reach a resolution of
5000 (fwhm) at m/z 292.9824. Chiral gas chromatography was per-
formed with a Hydrodex-ꢀ-6-TBDMS-column (25 m, 0.25 mm i.d.,
Macherey–Nagel). IR spectra were acquired with a Tensor 27
(Bruker) by using the diamond-ATR-technique, and GC/IR analysis
was performed using a GC 7890B (Agilent Technologies) gas chro-
matograph coupled to a DiscovIR instrument (Dani Instruments).
The samples eluting from the GC column were deposited on a
cooled ZnSe disc at – 40 °C using a disc speed of 4 mm/min. The
gas chromatograph was equipped with an Agilent HP-5 column
(30 m, 0.25 mm i.d., 0.25 μm film thickness) with helium as the
carrier gas. The resulting infrared spectra had a resolution of 4 wa-
venumbers and were normalized and processed using GRAMS/AI
9.2 software by Thermo Fisher Scientific Inc. modified with work-
books provided by Dani Instruments. The peaks are listed with wave
numbers in cm–1. Intensities are described with s (strong), m (me-
dium), w (weak) and br (broad). Optical rotation was measured with
Figure 5. Total ion chromatogram of the gas chromatographic enantiomer
separation of 14 on a chiral ꢀ-TBDMS-hydrodex phase. Temperature program:
isothermal for 60 min at 110 °C, then with 2 °C/min to 160 °C, followed by a
sharp ramp of 25 °C/min to 220 °C. (A) racemic mixture; (B) S-14; (C) R-14;
(D) natural extract of a gland extract of H. cinnamomeoventris; (E) coinjection
of the natural extract and S-14. Peak identities were confirmed by GC/MS.
Table 1. Frog species containing frogolide (14) in their femoral or gular gland
extracts obtained from males.
Species
Frog family
Hyperolius cinnamomeoventris
Hyperolius viridiflavus
Gephyromantis granulatus
Gephyromantis leucomaculatus
Gephyromantis luteus
Guibemantis liber
Guibemantis pulcher
Guibemantis tornieri
Mantella aurantiaca
Spinomantis aglavei
Hyperoliidae
Hyperoliidae
Mantellidae
Mantellidae
Mantellidae
Mantellidae
Mantellidae
Mantellidae
Mantellidae
Mantellidae
Mantellidae
Spinomantis taravatra
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a Dr. Kernchen Propol Digital Automatic polarimeter with 1 cm cu-
vettes at a wavelength of 589 nm. Free-living adult male specimens
of the target amphibian species were caught in the field at night
in 2013 and 2014, during the respective breeding season; males
emitting advertisement calls and thus being in the breeding condi-
tion (30 mL) was added. The aqueous phase was extracted with
ethyl acetate (3 × 75 mL). The organic phase was washed with brine
(50 mL) and dried with MgSO4. After removal of the solvents, col-
umn chromatography (pentane/Et2O, 20:1) yielded the desired
methyl ester 10 in 73 % yield over three steps (819 mg, 3.25 mmol).
tion were specifically selected. The frogs were sedated by the appli- The (R)-enantiomer was obtained using (R)-dihydrofarnesol in 65 %
cation of a small quantity of benzocaine, which is absorbed through yield over 3 steps under identical conditions. TLC (pentane/Et2O,
the frog's skin, and euthanized by an overdose of the same sub- 20:1): Rf 0.43. H NMR (300 MHz, CDCl3): δ = 0.94 (d, JH,H = 6.6 Hz,
stance in the field laboratory. Tissue from the vocal sac (hyperoliids) 3 H, CH3), 1.26–1.42 (m, 2 H, CH2), 1.60 (s, 6 H, 2 × CH3), 1.68 (d,
or the femoral gland (mantellids) was removed with sterilized scis- 3JH,H = 1.1 Hz, 3 H, CH3), 1.90–2.07 (m, 7 H, 3 × CH2, CH), 2.08–2.36
1
3
sors and tweezers, and immediately fixed in dichloromethane for
chemical analysis. Samples were stored in 1 mL GC vials sealed
with Teflon-lined caps to prevent modification or evaporation of
compounds.
(m, 2 H, CH2), 3.67 (s, 3 H, CH3), 5.07–5.13 (m, 2 H, 2 × CH) ppm. 13
C
NMR (75 MHz, CDCl3): δ = 15.9, 17.7, 19.6, 25.3, 25.7, 26.7, 30.0, 36.7,
39.7, 41.6, 51.3, 124.1, 124.3, 131.3, 135.7, 179.7 ppm. EI-MS (70 eV):
m/z (%) 252 (2) [M]+, 237 (2), 209 (55), 177 (18), 163 (3), 151 (6), 135
(7), 123 (41), 109 (100), 95 (26), 85 (13), 81 (24), 73 (8), 69 (99), 59
(S,E)-2,3-Dihydrofarnesol (8): The catalyst [Ru{(R)-tol-binap}](OAc)2
(324 mg, 0.36 mmol, 0.02 equiv.) was added to a solution of (E,E)-
farnesol (7, 4.00 g, 17.99 mmol, 1.0 equiv.) in degassed MeOH
(35 mL) in a 40 mL cylindrical glass vial equipped with a magnetic
stirrer bar that was transferred into a Teflon-lined high-pressure
hydrogenation autoclave. The autoclave was purged with nitrogen,
filled with hydrogen gas (35 bar), and the solution was stirred for
5 h at room temperature. The crude reaction mixture was then
stirred for 10 min with added silica (3.24 g, 1000 wt.-% of catalyst)
and then filtered. The filtrate was stirred again with activated char-
coal (16 g, 400 wt.-% of starting material) for 15 min and filtered
through a short silica plug. (S,E)-2,3-dihydrofarnesol (8) was ob-
tained as a light yellow liquid ready to use without further purifica-
tion in the next step (yield: 3.89 g, 17.34 mmol, 96 %). (R,E)-2,3-
dihydrofarnesol was obtained in 89 % yield by using [Ru{(S)-tol-
binap}](OAc)2 as the catalyst.[11] 1H NMR (400 MHz, CDCl3): δ = 0.91
(15), 55 (18), 41 (51). IR (GC-IR): ν = 2961 (s), 2921 (s), 2853 (s), 2729
˜
(w), 1738 (s), 1702 (w), 1672 (w), 1437 (s), 1377 (s), 1289 (s), 1261
(m), 1197 (s), 1153 (s), 1108 (m), 1009 (m), 838 (m) cm–1. (S)-10:
[α]2D1.6 = –4.6 (c = 1.01 in CH2Cl2). (R)-10: [α]2D5.0 = +4.1 (c = 0.78 in
CH2Cl2).
Methyl (3S,6E,10E)-12-Hydroxy-2,3-dihydrofarnesenoate (11): A
solution of SeO2 (44 mg, 0.396 mmol, 0.2 equiv.) and tBuOOH
(3.96 mmol, 2.0 equiv., 0.72 mL of a 5.5
M solution in nonane) in
CH2Cl2 (10 mL) was stirred for 30 min at room temperature.[13] Ester
10 (0.5 g, 1.98 mmol, 1 equiv.) was added at 0 °C. After stirring for
5 h at room temperature Na2SO3 solution (10 mL) was added, and
the aqueous phase was extracted three times with ethyl acetate
(3 × 15 mL). The combined organic phases were washed with brine
(10 mL), dried with MgSO4, and the solvents were removed. Column
chromatography (pentane/ethyl acetate, 2:1) yielded 25 % of the
desired product (133 mg, 0.495 mmol) 11 and 13 % of its regioiso-
3
[d, J(H,H) = 6.6 Hz, 3 H], 1.14–1.43 (m, 5 H), 1.60 (s, 6 H), 1.68 (s, 3
H, CH3), 1.96–2.09 (m, 6 H), 3.63–3.74 (m, 2 H), 5.07–5.13 (m, 2 H)
mer 12 (69 mg, 0.257 mmol). The (R)-enantiomer of 11 was ob-
1
ppm. 13C NMR (100 MHz, CDCl3): δ = 15.9, 17.7, 19.5, 25.4, 25.7, tained similarly in 22 % yield. TLC (pentane/EtOAc, 2:1): Rf 0.54. H
3
26.7, 29.2, 37.2, 39.7, 39.9, 61.2, 124.3, 124.6, 131.3, 134.9 ppm. EI- NMR (400 MHz, CDCl3): δ = 0.94 (d, JH,H = 6.6 Hz, 3 H, CH3), 1.18–
MS (70 eV): m/z (%) 224 (1) [M]+, 209 (1), 181 (33), 163 (19), 149 (2),
1.40 (m, 2 H, CH2), 1.60 (s, 3 H, CH3), 1.66 (s, 3 H, CH3), 1.94–2.34
(m, 9 H, 4 × CH2, CH), 3.67 (s, 3 H, CH3), 3.98 (s, 2 H, CH2), 5.08–5.12
(m, 1 H, CH), 5.36–5.40 (m, 1 H, CH) ppm. 13C NMR (100 MHz, CDCl3):
δ = 13.6, 15.9, 19.5, 25.2, 25.9, 29.8, 36.6, 39.2, 41.5, 51.3, 68.7, 124.4,
125.6, 134.7, 134.8, 173.8 ppm. EI-MS (70 eV): m/z (%) 250 (14) [M
– H2O]+, 235 (2), 219 (1), 207 (3), 199 (51), 194 (4), 181 (3), 175 (6),
167 (100), 161 (4), 149 (16), 139 (28), 121 (47), 107 (36), 93 (30), 81
137 (5), 123 (54), 109 (21), 95 (47), 81 (71), 69 (100), 55 (26), 41 (56).
IR (GC/IR): ν [cm–1] 3274 (br.s), 2965 (s), 2926 (s), 2863 (s), 2853 (s),
˜
1676 (w), 1451 (s), 1377 (s), 1107 (m), 1060 (s), 1013 (m). (S)-8:
[α]2D1.6 = +24.2 (c = 0.96 in CH2Cl2). (R)-8: [α]2D5.0 = –26.4 (c = 1.10 in
CH2Cl2).
Methyl (S,E)-2,3-Dihydrofarnesenoate (10): A solution of pyrid-
ine·SO3 (2.12 g, 13.32 mmol, 3.0 equiv.) in DMSO (50 mL) was stirred
for 15 min at room temperature. This mixture was added to a solu-
tion of (S)-8 (1.00 g, 4.45 mmol, 1.0 equiv.) and NEt3 (3.08 mL,
44.50 mmol, 10.0 equiv.) in CH2Cl2 (150 mL) at 0 °C. After stirring
for 3 h at room temperature water was added (100 mL). The aque-
ous phase was extracted three times with CH2Cl2 (3 × 50 mL), and
the combined organic phases were dried with MgSO4. The crude
aldehyde 9 was used directly in the next reaction step after removal
of the solvents.[12]
(22), 77 (8), 69 (43), 65 (4), 59 (17), 55 (29), 41 (36). IR (GC-IR): ν =
˜
3279 (br.s), 2956 (s), 2918 (s), 2853 (s), 1736 (s), 1672 (w), 1438 (s),
1377 (s), 1290 (s), 1198 (s), 1154 (s), 1105 (m), 1072 (s), 1011 (s), 958
(w), 879 (w), 844 (m) cm–1. S-11: [α]D21.6 = –2.4 (c = 0.98 in CH2Cl2).
R-11: [α]2D5.0 = +3.4 (c = 1.80 in CH2Cl2).
(3S,6E,10E)-3,7,11-Dodec-6,10-dien-12-olide, Frogolide (14):
Lithium hydroxide (97 mg, 4.06 mmol, 20 equiv.) was added to the
ester 11 (55 mg, 0.203 mmol, 1 equiv.) dissolved in a mixture of
THF (2 mL), methanol (2 mL), and water (1 mL).[15] The resulting
mixture was heated to reflux at 80 °C for 14 h. After addition of sat.
Aldehyde 9 (989 mg, 4.45 mmol, 1.0 equiv.) was added to a solution
of NaH2PO4 (5.34 g, 44.5 mmol, 10.0 equiv.), 2-methyl-2-butene
(20 mL), tert-butanol (15 mL) in THF (50 mL) and water (50 mL).
Sodium chlorite (2.01 g, 22.25 mmol, 5.0 equiv.) was added partially
in three equal portions every 45 min. After addition of the last
portion, the mixture was stirred for an additional hour. Water was
added (50 mL), and the aqueous phase was extracted three times
with ethyl acetate (3 × 75 mL). The organic phase was washed with
brine (50 mL), dried with MgSO4, and the solvents were removed.
The residue was taken up in DMF (15 mL) and methyl iodide
NH4Cl solution (3 mL), the mixture was acidified using 2
M HCl (ca.
1 mL), and the aqueous phase was extracted three times with ethyl
acetate (3 × 5 mL). After removal of the solvent, the resulting crude
acid 13 was used directly in the next step without further purifica-
tion.
Triethylamine (144 mg, 200 μL, 1.42 mmol, 7 equiv.) and bis(2-oxo-
3-oxazolidinyl)phosphinic chloride (BOP-Cl, 155 mg, 0.609 mmol,
3 equiv.) were added to a solution of the hydroxyacid 13 (52 mg,
0.203 mmol, 1 equiv.) in CH2Cl2 (5 mL). The resulting mixture was
(1.264 g, 8.9 mmol, 2.0 equiv.) and K2CO3 (0.922 g, 6.68 mmol, stirred for 6 h at room temperature, and sat. NH4Cl solution (5 mL)
1.5 equiv.) were added. After stirring for 30 min, sat. NaHCO3 solu-
was added. The aqueous phase was extracted three times with
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1558.
CH2Cl2 (3 × 5 mL), and the combined organic phases were dried
with MgSO4. After removal of the solvents, a short silica filter col-
umn using pentane/diethyl ether (20:1) yielded frogolide (14) in
42 % yield over two steps (20 mg, 0.085 mmol). The (R)-enantiomer
was obtained in 63 % yield with upscaling by the factor of two. TLC
(pentane/Et2O, 20:1): Rf 0.62. 1H NMR (400 MHz, CDCl3): δ = 1.01 (d,
[3] M. Vences, G. Wahl-Boos, S. Hoegg, F. Glaw, E. Spinelli Oliveira, A. Meyer,
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12, 2731–2738.
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3JH,H = 6.0 Hz, 3 H, CH3), 1.18–1.43 (m, 2 H, CH2), 1.53, (s, 3 H, CH3),
2
1.64 (s, 3 H, CH3), 1.86–2.35 (m, 9 H, 4 × CH2, CH), 4.07 (d, JH,H
=
=
2
3
12.7 Hz, 1 H, CH2) 4.88 (d, JH,H = 12.7 Hz, 1 H, CH2), 5.14 (t, JH,H
7.9 Hz, 1 H, CH), 5.31–5.34 (m, 1 H, CH) ppm. 13C NMR (100 MHz,
CDCl3): δ = 14.7, 14.9, 20.8, 23.7, 24.4, 29.3, 36.0, 38.9, 43.6, 67.1,
126.4, 126.7, 130.4, 133.4, 172.4 ppm. EI-MS (70 eV): m/z (%) 236 (7)
[M]+, 221 (1), 208 (1), 175 (1), 168 (88), 153 (5), 135 (6), 121 (12),
109 (100), 105 (4), 93 (44), 81 (24), 77 (8), 67 (45), 55 (20), 41 (32).
IR (GC-IR): ν = 2957 (s), 2929 (s), 2862 (s), 1739 (s), 1690 (w), 1444
˜
(s), 1378 (m), 1362 (m), 1304 (m), 1288 (s), 1256 (m), 1236 (m), 1221
(s), 1181 (w), 1071 (s), 1028 (m), 984 (w), 857 (m) cm–1. S-14:
[α]2D1.6 = –46.3 (c = 0.99 in CH2Cl2) R-14: [α]2D5.0 = +36.1 (c = 0.61 in
CH2Cl2).
Acknowledgments
We thank the Deutsche Forschungsgemeinschaft through re-
search grant SCHU 984/10-1 and the Austrian Science Fund
(FWF): P25612 for financial support. Work in Madagascar was
carried out in the framework of a collaboration accord between
TU Braunschweig and the Université d'Antananarivo, Départe-
ment de Biologie Animale. We are grateful to the Malagasy au-
thorities for research and export permits. Specimen collection
in Rwanda was conducted within a collaboration between the
University of Vienna and the University of Koblenz-Landau, Ger-
many. We would like to particularly thank M. Dehling and P. M.
Maier for their support. Specimen collection and export was
authorized by the Rwanda Development Board.
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Keywords: Natural products · Terpenoids · Pheromones ·
Macrocycles · Lactones
[22] S. Schulz, Eur. J. Org. Chem. 1998, 13–20.
[1] D. Poth, K. C. Wollenberg, M. Vences, S. Schulz, Angew. Chem. Int. Ed.
2012, 51, 2187–2190; Angew. Chem. 2012, 124, 2229.
Received: February 6, 2018
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Full Paper
Volatile Macrolactones
Frogolide is a new sesquiterpene mac-
rolactone identified in gular and femo-
ral scent glands of the African frog
families Mantellidae and Hyperoliidae.
Frogolide is the first frog macrolide of
terpenoid origin. The identification,
synthesis, and determination of the
absolute configuration are presented.
M. Menke, K. Melnik, P. S. Peram,
I. Starnberger, W. Hödl, M. Vences,
S. Schulz* .............................................. 1–7
Frogolide – An Unprecedented Ses-
quiterpene
Macrolactone
from
Scent Glands of African Frogs
DOI: 10.1002/ejoc.201800199
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