Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Identification and Synthesis of Luteolide, a Highly Branched Macrolide Semiochemical from the Mantellid Frog Gephyromantis luteus Kristina Melnik,† Markus Menke,† Andolalao Rakotoarison,‡,§ Miguel Vences,‡ and Stefan Schulz*,† †
Technische Universität Braunschweig, Institute of Organic Chemistry, Hagenring 30, 38106 Braunschweig, Germany Technische Universität Braunschweig, Zoological Institute, Mendelssohnstraße 4, 38106 Braunschweig, Germany § Zoologie et Biodiversité Animale, Université d‘Antananarivo, BP 906, Antananarivo 101, Madagascar ‡
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S Supporting Information *
ABSTRACT: Luteolide is a 10-membered aliphatic macrolactone, (4R,8S,9S)-4,8dimethylundecan-9-olide ((−)-17), released by the femoral gland of males of the mantellid frog Gephyromantis luteus. Its structure was established using NMR, MS, and chiral GC and confirmed by stereoselective synthesis of different stereoisomers. Among the approximately 20 current macrolides known from the Mantellidae, luteolide is the first example of a volatile macrolide furnishing three stereogenic centers and an ethyl side chain.
A
These macrolides are structurally diverse and vary in size including 10- to 14-membered rings, the number of methyl branches, and the degree of unsaturation. Prominent examples are mantidactolide A (3),3 (S)-cucujolide III (4),4 (S)phoracantholide I (5),1 and gephyromantolide A (6)1 (Figure 1). Herein, we report the identification, synthesis, and stereochemical characterization of an unprecedented macrolide from the mantellid Gephyromantis luteus characterized by three stereogenic centers and an ethyl side chain, likely obtained by an ω-2-oxidation, in contrast to the ω-1-oxidation usually observed in frog macrolactones. This structural motif is unique among anuran volatile macrolides The extract of the femoral gland of two male specimens was analyzed by GC/MS showing the presence of only two compounds (Figure 2). The first compound was readily identified as the macrolactone frogolide (7), known from other
coustic, visual, and tactile signals are the prevalent channels of communication among anuran amphibians. Nevertheless, frogs of the Madagascar-endemic subfamily Mantellinae (Mantellidae) use the chemical channel as well.1 The males possess femoral glands on the ventral side of their hind legs,2 disseminating volatiles to induce activity and attraction in males and females. The first reported volatile frog pheromones were (R)-8-methylnonan-2-ol (1) and (S)phoracantholide J (2) produced by the mantelline Mantidactylus multiplicatus (Figure 1).1 The femoral glands of the mantelline frogs often contain a bouquet of volatiles, dominated by secondary alcohols and macrocyclic lactones.3−6
Figure 2. Total ion chromatogram of a single femoral gland extract of Gephyromantis luteus and structure of frogolide (7). Figure 1. Representative volatiles identified in various mantelline frogs. © XXXX American Chemical Society
Received: March 8, 2019
A
DOI: 10.1021/acs.orglett.9b00852 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters mantellids.7 The mass spectrum of the other, compound A (Figure 3), revealed a molecular mass of m/z 212.17745 (calcd 212.17763) obtained by HR-MS, consistent with the formula C13H24O2.
Then a stereoselective synthesis of 17 was performed to verify the proposal and to allow determination of the stereochemistry of A (Scheme 1). We proposed a synconfiguration for the ring substituents at C-4, C-8, and C-9, the latter also likely in the S-configuration, because other frog macrolides having similar configurations have been observed.1,3,4 A key step of the synthesis was a ring-closing metathesis (RCM) of ester 15 obtained from acid 12 and alcohol 14. The synthesis started with commercially available (S)-(−)-citronellal (S-8) that was converted by Wittig olefination into alkene 9.9 Regioselective epoxidation with m-CPBA gave epoxide 10.10 Oxidative cleavage with periodic acid and subsequent oxidation of the aldehyde 11 with silver nitrate and sodium hydroxide led to acid 12.11 The two adjacent chiral centers in alcohol 14 were obtained by asymmetric crotylation of propionaldehyde (13) with (+)-allyldiisopinocampheylborane (Ipc2BOMe) and cis-2-butene according to Brown et al.12 Steglich esterification of acid 12 and (S,S)-alcohol 14 with diisopropylcarbodiimide (DIC)13 gave ester 15 that was used in the following RCM with Grubbs’ second-generation catalyst.6 The obtained macrolactone 16 was hydrogenated with Rh/C to yield target compound (4R,8S,9S)-4,8dimethylundecan-9-olide ((−)-17). The use of Rh/C is crucial because hydrogenation with Pd/C led to epimerization of the methyl group in an allylic position, as reported previously for allylic stereogenic centers.14 Fortunately, comparison of NMR and MS spectra and the gas chromatographic retention index (RI) of A and the synthetic compound revealed the relative configuration to be as proposed. In parallel, another diastereomer, 4S,8S,9S-17, was synthesized in an identical sequence starting from (R)-citronellal (R-8) instead of S-8 and 3S,4S-14. The product verified differences of mass spectra and RI data among diastereomers of 17. For the determination of the absolute configuration, the (+)-17 enantiomer was also synthesized, starting from R-8 and using (−)-Ipc2BOMe, according to the established reaction sequence (Scheme 1). Next, the gas chromatographic enantiomer separation was performed on a chiral Hydrodex β-6TBDM phase (see Supporting Information, Figure S4). Coinjection experiments established the absolute configuration of natural A to be (4R,8S,9S)-4,8-dimethylundecan-9-olide ((−)-17). Macrolide 17, for which we propose the name luteolide, also occurs in low concentrations in other mantellids, such as Mantidactylus betsileanus. The unknown compound C in G. moseri6 is also identical to 17. Luteolide exhibits several structural features that differentiate it from other frog macrolides.1,3,4,6,7 It is the first macrolide with an ω-2 oxidation pattern instead of the usually occurring ω or ω-1 oxidation.15 Furthermore, it exhibits three stereogenic centers and an odd-numbered carbon chain. Related macrolides such as decanolides are most likely derived from the fatty acid biosynthetic pathway15,16 that leads to methyl branches at even numbered positions by incorporation of methyl malonate in the biosynthesis instead of malonate.3 Interestingly, the stereochemistry of methyl branches and macrolide ring seems to be conserved in the Mantellidae. The biosynthesis of luteolide potentially involves the incorporation of two methylmalonate and two malonate units and a propionate starter unit leading to the observed substitution pattern (see Supporting Information, Figure S6). The high structural diversity of semiochemicals derived from the femoral glands of the species-rich mantelline frogs is
Figure 3. EI-mass spectrum of natural compound A, identified as luteolide.
The mass spectrum of A resembled those of other frog macrolactones, although no match was found.8 Microhydrogenation of the natural sample with Pd/C led to no change in the mass spectrum, indicating a saturated cyclic structure. The ions m/z 183 (M − 29) and 154 (M − 58) indicated an ethyl group next to the C−O single bond.8 Gratifyingly, direct NMR analysis of the natural sample was possible with the material obtained from only two frogs, about 500 μg per frog. The signals of lactone 7 were assigned by comparison with reported data,7 confirming its identity with frogolide (7) (see Supporting Information Figures S1 and S2 and Table S1).7 The remaining signals were used to elucidate the structure of compound A, which was proposed to be 9ethyl-4,8-dimethylundecan-9-olide (17). Key 1H,1H COSY and HMBC correlations are shown in Figure 4 (for detailed
Figure 4. Key 1H,1H COSY (blue line) and HMBC (red arrows) correlations of the proposed structure for unknown compound A.
analysis, see Supporting Information Figure S3 and Table S2). The shift of C-1 (172.3 ppm) indicated an ester carbonyl group that correlated with H-2 and H-9 in the HMBC spectrum. The shift of C-2 (35.1 ppm) corresponded to an αCH2 group next to a carbonyl group. Carbon 9 (81.5 ppm) showed a downfield shift compared to methyl substituted analogues,3 and COSY correlations between H-9, H-12, and H-13 indicated an ethyl group at this position, explaining the shift difference. Two additional methyl groups were found at C-4 and C-8, confirmed by HMBC correlations of H-10 with C-3 and C-5 as well as H-11 with C-7, C-8, and C-9. B
DOI: 10.1021/acs.orglett.9b00852 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Scheme 1. Stereoselective Synthesis of (4R,8S,9S)-4,8-Dimethylundecan-9-olide ((−)-17) and its Diastereomer 4S,8S,9S-17
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striking, especially in Gephyromantis. The eight species of this genus tested so far contained high numbers of up to 14 macrolides in their femoral glands.17,18 This is even more surprising because these frogs are active vocal communicators with loud and species-specific advertisement calls. In mantelline frogs the main olfactory organ (MOO) is not connected to the vomeronasal organ (VNO). In Mantidactylus betsileanus, the pheromonal macrolides are detected in the MOO and not in the VNO, suggesting an evolutionary connection of olfactory anatomy with chemical communication.18 Despite being volatile, most likely the macrolide pheromones play a role in short-range communication, or are even directly applied when males rub their femoral glands on the dorsum of females during mating. Odorous communication is also observed in other frog families.19 Macrolides found in mantellid frogs include saturated and unsaturated compounds ranging in size between C10 and C16, varying in the degree and position of methyl groups, and are derived from the fatty acid or terpene biosynthetic pathways. Some compounds have been detected across unrelated mantellid species, varying in relative amounts. This suggests the macrolide biosynthetic genes might be conserved in this group of amphibians. We suggest identifying differential expression of genes potentially related to macrolide biosynthesis as a promising future approach to understand the genomic basis of the astonishing variation of these compounds in mantellids. In summary, we identified the new macrolactone luteolide (17) and established a robust synthetic route that in the future will allow exploration of the biological function of macrolide 17.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Stefan Schulz: 0000-0002-4810-324X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Deutsche Forschungsgemeinschaft for funding with Grant No. SCHU 984/10-1/2 and the Madagascan authorities for research and export permits. Work in Madagascar was carried out in the framework of a collaboration accord between TU Braunschweig and the Université d’Antananarivo, Département de Biologie Animale.
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REFERENCES
(1) Poth, D.; Wollenberg, K. C.; Vences, M.; Schulz, S. Angew. Chem., Int. Ed. 2012, 51, 2187−2190. (2) Vences, M.; Wahl-Boos, G.; Hoegg, S.; Glaw, F.; Spinelli Oliveira, E.; Meyer, A.; Perry, S. Biol. J. Linn. Soc. 2007, 92, 529−539. (3) Poth, D.; Peram, P. S.; Vences, M.; Schulz, S. J. Nat. Prod. 2013, 76, 1548−1558. (4) Menke, M.; Peram, P. S.; Starnberger, I.; Hödl, W.; Jongsma, G. F. M.; Blackburn, D. C.; Rödel, M.-O.; Vences, M.; Schulz, S. Beilstein J. Org. Chem. 2016, 12, 2731−2738. (5) Hötling, S.; Haberlag, B.; Tamm, M.; Collatz, J.; Mack, P.; Steidle, J. L. M.; Vences, M.; Schulz, S. Chem. - Eur. J. 2014, 20, 3183−3191. (6) Peram, P. S.; Vences, M.; Schulz, S. Org. Biomol. Chem. 2017, 15, 6967−6977. (7) Menke, M.; Melnik, K.; Peram, P. S.; Starnberger, I.; Hödl, W.; Vences, M.; Schulz, S. Eur. J. Org. Chem. 2018, 2018, 2651−2656. (8) Schulz, S.; Peram, P. S.; Menke, M.; Hötling, S.; Röpke, R.; Melnik, K.; Poth, D.; Mann, F.; Henrichsen, S.; Dreyer, K. J. Nat. Prod. 2017, 80, 2572−2582. (9) Green, S. A.; Matos, J. L. M.; Yagi, A.; Shenvi, R. A. J. Am. Chem. Soc. 2016, 138, 12779−12782. (10) Schroeter, M.; Weyerstahl, P. Synth. Commun. 2004, 34, 1535− 1540.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00852. Experimental procedures, NMR spectra, enantiomer separation, mass spectral data analysis, proposed biosynthetic pathway (PDF) C
DOI: 10.1021/acs.orglett.9b00852 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters (11) Taber, D. F.; DeMatteo, P. W. J. Org. Chem. 2012, 77, 4235− 4241. (12) (a) Brown, H. C.; Bhat, K. S. J. Am. Chem. Soc. 1986, 108, 5919−5923. (b) Ghosh, A. K.; Anderson, D. D. Org. Lett. 2012, 14, 4730−4733. (13) Neises, B.; Steglich, W. Angew. Chem., Int. Ed. Engl. 1978, 17, 522−524. (14) Li, N.-S.; Piccirilli, J. A. Tetrahedron 2013, 69, 9633−9641. (15) Schulz, S.; Hötling, S. Nat. Prod. Rep. 2015, 32, 1042−1066. (16) Dräger, G.; Kirschning, A.; Thiericke, R.; Zerlin, M. Nat. Prod. Rep. 1996, 13, 365−375. (17) Junk, A.; Wenzel, S.; Vences, M.; Nowack, C. Zool. Anz. 2014, 253, 338−344. (18) Nowack, C.; Peram, P. S.; Wenzel, S.; Rakotoarison, A.; Glaw, F.; Poth, D.; Schulz, S.; Vences, M. J. Zool. 2017, 303, 72−81. (19) (a) Tyler, M. J. Pheromones and amphibian behavior. In Biological Toxins and Bioterrorism; Springer Netherlands, 2015; pp 559−569. (b) Starnberger, I.; Poth, D.; Peram, P. S.; Schulz, S.; Vences, M.; Knudsen, J.; Barej, M. F.; Rödel, M.-O.; Walzl, M.; Hödl, W. Biol. J. Linn. Soc. 2013, 110, 828−838. (c) Starnberger, I.; Preininger, D.; Hö dl, W. Anim. Behav. 2014, 97, 281−288. (d) Schulte, L. M.; Yeager, J.; Schulte, R.; Veith, M.; Werner, P.; Beck, L. A.; Lötters, S. Anim. Behav. 2011, 81, 1147−1154. (e) Schoeppner, N. M.; Relyea, R. A. Funct. Ecol. 2009, 23, 1114− 1121. (f) Hamer, R.; Lemckert, F. L.; Banks, P. B. Biol. Lett. 2011, 7, 361−363. (g) Brunetti, A. E.; Lyra, M. L.; Melo, W. G. P.; Andrade, L. E.; Palacios-Rodríguez, P.; Prado, B. M.; Haddad, C. F. B.; Pupo, M. T.; Lopes, N. P. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 2124−2129. (h) Brunetti, A. E.; Merib, J.; Carasek, E.; Caramão, E. B.; Barbará, J.; Zini, C. A.; Faivovich, J. J. Chem. Ecol. 2015, 41, 360−372.
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DOI: 10.1021/acs.orglett.9b00852 Org. Lett. XXXX, XXX, XXX−XXX
