Iron Phenyl Hydrazide: A Novel Class of Web-Toxin From the Spider Nephilaclavipes.

Marques, M.R.^1; Ribeiro, S. P¹.; Mendes, M. A¹.; Souza, B.M¹.; Tormena, C. F².; Rittner, R³.; Palma, M.S¹.

1 Dept. Biology - CEIS / IBRC - UNESP - Rio Claro, SP, CEP: 13506-900 - Brazil; 2 Dept. Chemistry, F.F.C.L.R.P - São Paulo University, Ribeirão Preto, SP, Brazil; 3 Physical Organic Chemistry Laboratory - Chemistry Institute - UNICAMP - Campinas - CEP: 13083-970 – Brazil.

The orb-web spiders are polyphagous animals in which the web plays a very important role in prey capture. The viscous droplets covering the capture threads of the spider webs constitute part of the strategy for prey paralysis/killing. It is necessary to know the chemical composition of these droplets to understand its function in web mechanics and prey capture. GABA, N-acetyltaurine, choline, betaine and isethionic acid have been found in the Argiope sp webs. During field observations, we have noted that immediately after capture by the webs, the preys already presented toxicity signs, prior venom inoculation by the spider. The fact suggests the existence of toxins in the web of Nephila clavipes. This research has as objectives to isolate and to assign the chemical structures  of the most abundant low molecular weight toxins in the viscous droplets from the webs of Nephila clavipes and characterize their mechanisms of action. The crude extract of Nephila clavipes web was chromatographed under RP-HPLC in a C-18 semi preparative column (SHISEIDO 10 x 250 mm). Elution was carried out in a linear gradient concentration from 5% to 60% (v/v) MeCN (containing 0.1% TFA) and monitored at 215 nm. The chromatographic profile of the crude extract presented six fractions. Fraction 5 was among the most abundant ones and caused paralysis in the assayed insects (Apis mellifera). This fraction was rechromatographed under isocratic conditions by using 18% (v/v) MeCN (containing 0.1% TFA), resulting in more 3 subfractions. The subfraction 5.3 was submitted to mass spectrometry (ESI-MS and ESI-MS/MS), ¹H-NMR and ICP elementary analysis for metals detection and quantitation. We found out a compound of low molecular mass: 205 Da. The new compound was designated by Iron Phenyl Hydrazil Ethyl Alcohol. This type of compound is not so common among natural products, mainly in spider venom toxins. Apparently, this compound seems to be a potent insectide, even when topically used on prey insects; in addition to this, the aromatic azides were also described as strong antibiotics. The structural and mechanistic characterization of this type of compound may be used in a forthcoming future, as references for the development of novel pharmaceuticals / agrichemicals.

Supported by FAPESP; CAT/CEPID/FAPESP

Correspondence to: mmarques@rc.unesp.br