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Alkaloids play an important role in the defense strategies of insects and other arthropods. Especially from ladybird beetles and from certain ants an amazing variety of defensive alkaloids have been identified [1,2]. In most of these compounds, an usually unbranched carbon chain is attached at one or more sites to a nitrogen atom, forming linear as well as mono-, di-, and tricyclic structures. Recent analyses of the hemolymph from Chilocorus and Exochomus ladybird beetles led to the identification of a new series of "dimeric" alkaloids (1, 2]. The carbon skeletons of these compounds are mostly quite complex, and unusual in that they consist of two unbranched chains of, most likely, acetogenin origin [3]. Other dimeric and trimeric alkaloids have been identified from the poison gland secretion of Myrmicaria ants [4]. In most of the above cases, NMR-spectroscopy played a major role in the process of structure determination. Recently, we identified a new type of dimeric alkaloid, psylloborine A (3), from the hemolymph of the 22-pointed ladybird beetle, Psyllobora vigintiduopunctata (L.). Exclusive correlation spectroscopy (E.COSY) [5] was used as the key tool in assigning this structure.

Fig. 1: Structures of three recently identified "dimeric" alkaloids.

The alkaloid could easily be isolated from whole-body extracts of P. vigintiduopunctata. After determining its molecular composition using positive ion electrospray mass spectroscopy as C₂₆H₄₀N₂, we continued with a standard set of proton and carbon NMR experiments.

As shown in the top part of Figure 2, the proton NMR spectrum of psylloborine is extremely congested: Of the 40 protons, 36 have chemical shift values within a range of less than 2.5 ppm. In this situation it seemed impossible to analyze the proton spin systems on the basis of phase-sensitive DQF-COSY spectra alone. The part of the DQF-COSY shown in Figure 2 clearly shows the enormous amount of overlap especially near the diagonal. We therefore conducted a series of E.COSY experiments, which so far have mostly been used for the exact determination of proton-proton coupling constants in peptides and other compounds of known constitution [5]. As shown in Figure 3, coupling constants that are difficult to extract from the DQF-COSY spectrum due to overlap or insufficient resolution, can be determined very neatly from the E.COSY spectrum, using crosspeaks at the less crowded periphery of the spectrum. With this approach, most of the vicinal H,H-coupling constants in the two spin systems of psylloborine A (3) could be determined. Thus, in conjunction with heteronuclear NMR spectra, the constitution of psylloborine could be determined as shown in Figure 1.

Fig. 2: 0.75-1.64 ppm region of the ¹H-NMR and the DQF-COSY spectrum of psylloborine A (3) (C₆D₆, 500 MHz). The vicinal coupling constants of the proton 1'-H_eq (1.37 ppm) can not be directly extracted, due to poor resolution in F2 and overlap with other crosspeaks, e. g. of the proton 8-H_eq at 1.36 ppm. The E.COSY signals corresponding to the crosspeaks (1-H_eq/1-H_ax) and (1'-H_eq/1'-H_ax) are shown in Fig. 3.

Fig. 3: (A): Crosspeak of the geminal pair 1-H_eq/1-H_ax in the E.COSY spectrum of psylloborine A (3) (CD₂Cl₂, 500 MHz). The passive vicinal couplings J(1eq,2) and J(1eq,9a) as well as the active coupling J(1eq,1ax) can be determined without interference from components of opposite phase. (B): E.COSY crosspeak of the geminal pair 1'-H_eq/1'-H_ax. Although, in comparison to the corresponding DQF-COSY spectrum (Fig. 2), the crosspeak is greatly simplified, the passive vicinal coupling constants J(1'eq,9a') and J(1'eq,2') cannot be directly extracted. Due to partial overlap, only the sum J(1'eq,9a')+J(1'eq,2') can be determined. Since J(1'eq,9a') is accessible from the E.COSY crosspeak of 9a'-H/1'-Hax (not shown), J(1'eq,2') can be calculated. The E.COSY crosspeak 1'-H_eq/1'-H_ax also allows one to determine the active geminal coupling J(1'eq,1'ax). In addition, a small four-bond coupling J(1'eq,3'eq) is revealed.

For a more detailed description of our investigations and experimental details, please see our recent publication in Tetrahedron:

Psylloborine A, a New Dimeric Alkaloid from Ladybird Beetles. F. C. Schröder, T. Tolasch, Tetrahedron 1998, 54, 12243-12248.

References

1. King, A. G.; Meinwald, J. Chem. Rev. 1996, 96, 1105-1122; Daloze, D.; Braekman, J.-C.; Pasteels, J. M. Chemoecology 1995, 5/6, 173-183.

2. Braekman, J. C.; Daloze, D. in Studies in Natural Products, Vol. 6; Atta-ur-Rahman Ed.; Elsevier: Amsterdam, 1990; pp. 421-466.

3. Timmermanns, M.; Braekman, J.-C.; Daloze, D.; Pasteels, J. M.; Merlin, J.; Declerq, J.-P. Tetrahedron Lett. 1992, 33, 1281-1284; McCormick, K. D.; Attygalle, A. B.; Xu, S.-C.; Svatoë, A.; Meinwald, J. Tetrahedron 1994, 50, 2365-2372; Shi, X.; Attygalle, A. B.; Meinwald, J.; Houck, M. A.; Eisner, T. Tetrahedron 1995, 51, 8711-8718.

4. Schröder, F.; Franke, S.; Francke, W.; Baumann, H.; Kaib, M.; Pasteels, J. M.; Daloze, D. Tetrahedron 1996, 52, 13539; Schröder, F.; Sinnwell, V.; Baumann, H.; Kaib, M. Chem. Commun. 1996, 2139; Schröder, F.; Sinnwell, V.; Baumann, H.; Kaib, M.; Francke, W. Angew. Chem. Int. Ed. Engl. 1997, 36, 77-80.

5. Griesinger, C.; Sörensen, O. W.; Ernst, R. R. J. Magn. Reson. 1987, 75, 474; Biamonti, C., Rios, C. B., Lyons, B. A., Montelione, G. T. Multidimensional NMR Experiments and Analysis Techniques for Determining Homo- and Heteronuclear Scalar Coupling Constants in Proteins and Nucleic Acids. In Advances in Biophysical Chemistry; Bush, C. A. Ed.; JAI Press Inc.: Greenwich, Connecticut, USA, 1994; pp. 51-120.