NMR-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species

Abstract

Extensive chemical analyses of spider venoms from many species have revealed complex mixtures of biologically active compounds, of which several have provided important leads for drug development. We have recently shown that NMR spectroscopy can be used advantageously for a direct structural characterization of the small-molecule content of such complex mixtures. Here, we report the application of this strategy to a larger-scale analysis of a collection of spider venoms representing >70 species, which, in combination with mass spectrometric analyses, allowed the identification of a wide range of known, and several previously undescribed, small molecules. These include polyamines, common neurotransmitters, and amino acid derivatives as well as two additional members of a recently discovered family of natural products, the sulfated nucleosides. In the case of the well studied brown recluse spider, Loxosceles reclusa, sulfated guanosine derivatives were found to comprise the major small-molecule components of the venom.

Fig. 1.

Sulfated nucleosides from the hobo spider, Tegenaria agrestis.

Fig. 2.

The ¹H NMR spectrum of P. mirabilis venom. Signals corresponding to glutamic acid, aspartic acid, 4-aminobutyric acid, and spermidine are labeled G, A, B, and S, respectively.

Fig. 3.

Detailed NMR-spectroscopic analysis of C. pastoralis venom. (A) The ¹H NMR spectrum revealing a highly complex composition. (B–D) Excerpts of the corresponding dqfCOSY spectrum with color-coded annotation of cross-peaks in yellow, citric acid (CIT); orange, γ-aminobutyric acid (GAB); green, acylated polyamine 5 (APA-5); red, acylated polyamine 6 (APA-6) and disulfated guanosine 3 (SNU-3); light blue, other polyamines; black, isocitric acid (ICA) and octopamine (OCT); pink, 1,3-diaminopropylene section of polyamines; and dark purple, 4-hydroxybenzoic acid (4-HB). In addition, D shows evidence for two minor 2,5-dihydroxybenzoic acid derivatives (purple and green, 2,5-DHB).

Fig. 4.

New natural products 3-6 identified from C. pastoralis and Loxosceles spp. venom.

Fig. 5.

The ¹H NMR and dqfCOSY spectra obtained for L. reclusa venom. Cross-peaks corresponding to 2,5-disulfated guanosine (3) and 2-sulfated guanosine (4) are marked blue and red, respectively.