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

Frank C. Schroeder*†‡, Andrew E. Taggi†§, Matthew Gronquist¶, Rabia U. Malik†, Jacqualine B. Grantʈ, Thomas Eisner**, and Jerrold Meinwald†‡

*Boyce Thompson Institute, Cornell University, Tower Road, Ithaca, NY 14853; Departments of †Chemistry and Chemical Biology and **Neurobiology and Behavior, Cornell University, Ithaca, NY 14853; ¶Department of Chemistry, SUNY Fredonia, Fredonia, NY 14063; and ʈSchool of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI 49931

Contributed by Jerrold Meinwald, July 17, 2008 (sent for review May 12, 2008) Extensive chemical analyses of spider venoms from many species aimed at enrichment or isolation of the compounds of interest. have revealed complex mixtures of biologically active compounds, In the case of the hobo spider, Tegenaria agrestis, direct NMR of which several have provided important leads for drug devel- spectroscopic analysis facilitated detection and characterization opment. We have recently shown that NMR spectroscopy can be of an entire family of natural products, the sulfated nucleosides, used advantageously for a direct structural characterization of the including the sulfated guanosine derivatives 1 and 2 (Fig. 1) (5). small-molecule content of such complex mixtures. Here, we report Except for a single example (6), sulfated nucleosides had com- the application of this strategy to a larger-scale analysis of a pletely escaped detection by conventional isolation and charac- collection of spider venoms representing >70 species, which, in terization protocols, although spider venoms had been subject to combination with mass spectrometric analyses, allowed the iden- intensive chemical scrutiny that had led to identification of tification of a wide range of known, and several previously hundreds of proteins, peptides, acylated polyamines, and various undescribed, small molecules. These include polyamines, common small-molecule neurotransmitters (7, 8). neurotransmitters, and amino acid derivatives as well as two The promising results with T. agrestis prompted us to initiate ECOLOGY additional members of a recently discovered family of natural NMR-spectroscopic screening of venom from a large selection products, the sulfated nucleosides. In the case of the well studied of spider species, both to determine the extent to which sulfated brown recluse spider, Loxosceles reclusa, sulfated guanosine de- nucleosides might occur among the various spider taxa and to rivatives were found to comprise the major small-molecule com- obtain an expanded and unbiased view of spider venom com- ponents of the venom. position in general.

chemical prospecting ͉ Loxosceles ͉ metabolomics ͉ Results CHEMISTRY natural products ͉ neurotoxins Spider venom samples representing Ͼ70 species from 27 families (9, 10) were subjected to direct NMR spectroscopic analysis. For iological samples commonly contain complex mixtures of each species, one or more lyophilized samples corresponding to Bsmall molecules with diverse properties and functions. Their 5–10 ␮l of crude venom were dissolved, without purification,†† 1 1 characterization remains a challenge, because many analytical in D2O, and H NMR spectra were recorded. These H spectra approaches are primarily aimed at identifying specific classes of were used to assess the overall composition of nonvolatiles in known compounds. Several recent studies have demonstrated each venom sample. In a number of instances, the 1H NMR the feasibility of NMR-spectroscopic analyses of complex small- spectrum immediately suggested that simple amino acids and molecule mixtures, including the use of diffusion-ordered spec- amino acid derivatives typically found in spider venom repre- troscopy (DOSY) (1) or principal component analysis (PCA) in sented the major components. For example, in the 1H spectrum metabolomics (2) as well as the characterization of crude of Pisaura mirabilis venom, signals representing Ͼ90% of the unfractionated natural products extracts using routine 2D NMR small-molecule content of the venom could be assigned to spectra (3–5). Compared with MS analyses, 2D NMR spectro- glutamic acid, aspartic acid, 4-aminobutyric acid, and spermi- scopic investigations of small-molecule mixtures offer the benefit dine (Fig. 2). For venoms of this type, structural assignments of more detailed structural information, which is of particular were confirmed through NMR-spectroscopic mixing experi- relevance for the detection of unanticipated chemotypes. We ments using authentic samples of the identified compounds. The have recently reported on the utility of direct NMR spectro- scopic analysis of unpurified biological extracts as a powerful method for the discovery of natural products (5). Such analyses Author contributions: F.C.S., A.E.T., M.G., T.E., and J.M. designed research; F.C.S., A.E.T., provide a means for the rapid evaluation of biological samples in M.G., R.U.M., and J.B.G. performed research; F.C.S., A.E.T., M.G., R.U.M., and J.B.G. analyzed an unbiased and nondestructive manner and are particularly data; and F.C.S., M.G., and J.M. wrote the paper. useful for the characterization of glandular secretions with The authors declare no conflict of interest. specific biological functions. A standard set of spectra [1H, ‡To whom correspondence may be addressed. E-mail: [email protected] or fs31@ dqfCOSY, heteronuclear multiple quantum correlation cornell.edu. (HMQC), heteronuclear multiple bond correlation (HMBC), §Present address: DuPont Crop Protection, Stine–Haskell Research Center, Newark, DE and NOESY] collected for a crude biological sample will usually 19711. permit partial or complete structural characterization of its ††In some instances, centrifugation was necessary to remove insoluble material. In addition, major components as well as of many minor components. If these it cannot be excluded that in some cases lyophilization caused loss of volatile venom components; however, for the present study, limited availability of nonlyophilized ‘‘direct’’ NMR-spectroscopic analyses suggest the presence of venom samples precluded additional analyses aimed at volatile components, for exam- compounds of interest, final identification of individual compo- ple, via GC-MS. nents can be accomplished in a subsequent step, for example by This article contains supporting information online at www.pnas.org/cgi/content/full/ using additional MS analyses, through synthesis of proposed 0806840105/DCSupplemental. structures, or via NMR-monitored fractionation of the sample © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806840105 PNAS ͉ September 23, 2008 ͉ vol. 105 ͉ no. 38 ͉ 14283–14287 Downloaded by guest on September 23, 2021 O Like many of the venoms investigated, C. pastoralis venom was N O NH found to contain significant amounts of acylated polyamines, a N large family of natural products that has been extensively studied NH N N NH HO SO 2 (7). The presence of acyl polyamines in our unpurified venoms 3 O N N NH HO SO 2 was easily recognized based on the characteristic spin systems of 3 O 2 O OH their aromatic head groups, whose connections to the polyamine 1 chains were evident from the HMBC spectra. Polyamines fre- O OSO H 3 O H3C OH quently occurred as mixtures of several structurally very similar O compounds, and as a result, overlap of the corresponding H3C OH O HO NMR-spectroscopic signals in both 1D and 2D spectra did not O O HO H3C OH always permit complete characterization of individual com- OH HO pounds. Nonetheless, NMR-spectroscopic analyses enabled elu- cidation of the general structural features of the polyamines Fig. 1. Sulfated nucleosides from the hobo spider, Tegenaria agrestis. present in a sample, which, in cases where the spectra suggested the presence of unusual structural features, were investigated further. In the case of C. pastoralis, two major as well as several 1 H NMR spectra of several other spider venom samples, for minor acylated polyamines were detected in the 1H NMR example, Dysdera sp., Phyxioshema sp., and Scytodes fusca, spectrum. Analysis of the 2D NMR spectra yielded partial showed primarily broad, unresolved peaks characteristic of large structures for the two major polyamines (Tables S2 and S3). Both peptides and proteins but were virtually devoid of any significant compounds appeared to possess a 2,5-dihydroxybenzoylamin- signals representing small molecules. A summary of the small opropylamine head group and terminate with a 4-guanidinobu- molecules we identified in our venom library is given in Table 1. tylamine (Agmatine) moiety. Complete characterization of the Venom samples of more interesting composition that could intervening diaminoalkane segments was not possible, however, not be characterized based solely on their 1H NMR spectra were because of multiple signal overlap. Subsequent analysis of the analyzed further by using 2D NMR spectroscopy, including sample by ESI-MS-MS nevertheless allowed the remainder of dqfCOSY, NOESY, HMQC, and HMBC spectra. For the venom both structures, 5 and 6, to be determined (Figs. S2 and S3). For samples analyzed in this way, the resulting sets of spectra these analyses, the molecular [M ϩ 1]ϩ peaks corresponding to provided sufficient signal dispersion and connectivity informa- polyamines 5 and 6 were identified based on the structural tion to assign all major signals to discrete structural fragments, characteristics previously identified in the NMR spectra. For which, in many cases, allowed immediate identification of com- example, collisionally induced fragmentation of a terminal ag- plete structures. For example, the highly convoluted 1H NMR matine moiety produces a characteristic fragment ion having a spectrum of Coelotes pastoralis venom (11) clearly indicates that mass-to-charge (m/z) ratio of 114 (12). A tandem experiment this venom represents a complex mixture (Fig. 3). However, scanning for those precursor ions giving rise to a fragment of based on the corresponding dqfCOSY spectrum, structures m/z ϭ 114 revealed two major components in the venom sample, could be easily proposed for the venom’s major components as whose abundance, elemental composition, and molecular mass well as for many minor components. Among the minor compo- indicated that they correspond to the two major polyamines nents, we identified a sulfated nucleoside, whose presence was observed in the NMR spectra. Complete structures could then suggested by cross-peaks at 5.35 ppm/6.13 ppm, 5.35 ppm/4.74 be confirmed based on the predictable fragmentation patterns ppm, and 4.36 ppm/4.42 ppm that appeared to belong to the (12) observed in daughter scans acquired for the selected 2Јand 5Ј protons of a ribose spin system, which are character- molecular [M ϩ 1]ϩ peaks. Aside from these two compounds istically shifted downfield in sulfated derivatives (5). Further from C. pastoralis, the major acylated polyamines present in our analysis using HMQC and HMBC spectra, as well as negative-ion venom samples appeared to belong to known types and were not electrospray-ionization mass spectrometry (ESI-MS) and UV characterized in detail. spectroscopy, confirmed the presence of the previously unde- Among the most interesting investigated in the course scribed disulfated guanosine derivative 3 [Fig. 4 and supporting of this survey were three species from the Loxosceles: information (SI) Fig. S1 and Table S1]. In addition, several Loxosceles arizonica (13), Loxosceles deserta (14), and the well compounds commonly found in spider venom were identified, known brown recluse, Loxosceles reclusa (13). Loxosceles spiders including citric acid, isocitric acid, cytosine, ␥-amino butyric are notable for the severe necrotic lesions that may result from acid, and octopamine. their bites to humans (15). Surprisingly, we found sulfated nucleosides to comprise by far the most abundant components in the venoms of all three species. Our NMR-spectroscopic analyses revealed that all three Loxosceles species contained B 2,5-disulfated guanosine (3) as the major component, in addition B S G S to small amounts of what appeared to be 2-sulfated guanosine (4) A A G (Fig. 5 and Table S4). Because the concentrations of the B monosulfated derivative 4 in Loxosceles spp. venom were low, G S and the amounts of venom we had available were limited, we A G were unable to characterize 4 fully via HSQC and HMBC spectra. Therefore, a sample of 4 was synthesized via nonselec- tive, partial sulfation of a large excess of unprotected guanosine. NMR-spectroscopic analysis of the resulting mixture of mono- sulfated guanosine derivatives confirmed our structural assign- 5.0 4.0 3.0 2.0 ment of 4 as the 2-sulfated derivative. The presence of the chemical shift [ppm] guanine moiety in 3 and 4 was further confirmed by UV Fig. 2. The 1H NMR spectrum of P. mirabilis venom. Signals corresponding to spectroscopy (Fig. S1) and via negative-ion ESI-MS (Tables S1 Ϫ glutamic acid, aspartic acid, 4-aminobutyric acid, and spermidine are labeled and S4), which showed peaks corresponding to [M-1] at m/z G, A, B, and S, respectively. 442.0 and 362.0, respectively.

14284 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806840105 Schroeder et al. Downloaded by guest on September 23, 2021 Table 1. Small molecules detected in spider venoms organized by family Small Family Species Small molecules Family Species molecules

Agelenidae Agelena gracilens striatus e Agelena labyrinthica i Pisauridae Dolomedes tenebrosus b,d,e, Agelena orientalis i Dolomedes gertschi b,d,e Agelenopsis aperta (21, 22) d,i Pisaura mirabilis b,e Agelenopsis pennsylvania tristis (23) e Benoitia tadzhika Salticidae Phidippus ardenus Calilena sp. Phidippus johnsoni curta (6, 21) d,h Phidippus sp. d,f Loxosceles arizonica h Scytodidae Scytodes fusca Loxosceles deserta h Segestriidae Ariadna sp. c,d Loxosceles reclusa h Sparassidae Delena cancerides d,e,g,i Tegenaria agrestis (5) d,h Eusparassus oculatus a,b,e,f,g,i Tegenaria domestica b,d,g,i Holconia flindersi b,d,e Amaurobiidae Coelotes pastoralis d, h Isopedella canberrensus d,e Paracoelotes birulai (21) d,i Isopedella leai b,d Araneidae Aculepeira sp. d,i Micrommata virescens virvirescens a,ed diadematus c,d,e Neosparassus diana b,d,e Araneus sp. e Olios sp. e Araneus tartaricus d,e Theraphosidae Aphonopelma seemani a,b,d,i Argiope lobata Aphonopelma sp. b,d,i d.e Avicularia avicularis b,e,i Larinioides cornutus d,e,i Ceratogyrus marshalli (cornuatus) b,d,g

Larinioides sp. e,i Chilobrachys sp. a,d,i ECOLOGY Clubionidae Cheiracanthium punctorium Citharischius crawshayi d,e punctorium Cyrtaucheniidae Anemesia incana e Haplopelma albostriatum a,d,e,i Dipluridae Phyxioshema sp. Haplopelma lividum a,d,e,f,i Dysderidae Dysdera sp. Heteroscodra maculata B Eresidae Eresus sp. e Ornithoctonus huwena a,b,d,e Stegodyphus sp. e Phormictopus cancerides b,d Filistatidae Kukulcania sp. b Psalmopoeus cambridgei b,d,g CHEMISTRY Gnaphosidae Drassodes sp. e Selenocosmia hainana a,b,d,e,i Hexathelidae Macrothele sp. (24) b,i Theridiidae Latrodectus tadzhicus i Lycosidae Rabidosa sp. d,e Steatoda giorra Hogna sp. d,e,i Steatoda paykulliana b,e,i Lycosa proegrandis d,e,f Thomisidae Misumena vatia b,d,i Nemesiidae Raveniola micrura b,d,e,g,i Thomisus onustsus b,i Oecobiidae Urotea sp. Titanoecidae Titanoeca sp. d,e Oxyopidae Peucetia viridans e,i Zodariidae Lashesana sp. b,d,e Palpimanidae Palpimanus sp. d

a, acetyl choline; b, ␥-aminobutyric acid; c, choline; d, citric acid; e, glutamic acid; f, histamine; g, octopamine; h, sulfated nucleosides; I, other nucleosides.

Discussion Our results show that direct NMR-spectroscopic analysis can A well known limitation of NMR spectroscopy is its relatively be used to screen complex biological samples such as spider poor sensitivity. Despite continuing advances in probe design venom for the presence of small molecules. In combination with and available magnet field strength, NMR spectroscopic sensi- mass-spectrometric analyses and synthesis, this methodology tivity is often orders of magnitude lower than that of MS (16). enabled us to detect and identify compounds without purifica- tion. In the case of the sulfated guanosine derivatives that we Nonetheless, our analyses, which in most cases had to rely on identified in venoms obtained from C. pastoralis and Loxosceles samples of only 5–25 ␮l of crude venom, allowed detailed spp., complete structures could be proposed based on the NMR NMR-spectroscopic characterization of most components ac- Ϸ data obtained for the entire venom, with subsequent confirma- counting for more than 1–5% of the small-molecule content of tion via independent synthesis. A different situation was pre- the venom samples. For example, as shown in Fig. 3, most of the sented by the acylated polyamines detected in C. pastoralis, which signals visible in the dqfCOSY spectrum of C. pastoralis could be occur as part of a larger family of structurally similar compounds. assigned to distinct spin systems. The threshold for detection Here, signal overlap in the NMR spectra prevented full NMR- necessarily varied for different compound classes and depended spectroscopic characterization of the individual components. on the size and complexity of the venom samples. For example, However, the NMR spectroscopic analyses revealed important whereas 1% of a sulfated ribonucleoside derivative could have structural features which provided guidance in the rational been detected even in the most dilute venom samples investi- pursuit of further analyses via MS leading to full identification. gated, the presence of as much as 5% of an unusually function- Complementing MS analyses, NMR-spectroscopic screening alized polyamine might not have been detected in venoms seems particularly well suited for the survey of arrays of related containing large amounts of other polyamines. samples, because the efficiency of analysis inevitably increases as

Schroeder et al. PNAS ͉ September 23, 2008 ͉ vol. 105 ͉ no. 38 ͉ 14285 Downloaded by guest on September 23, 2021 A B

2.0

1,3-PDA GAB 2.5

ICA CIT 3.0 APA-6

APA-5 3.5 ppm

3.5 3.0 2.5 2.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 ppm

2,5-DHB C 4.0 D APA-5+6 4-HB 2,5-DHB 4.5 7.0

5.0 OCT OCT

5.5 7.5 ppm CYT SNU-3 6.0 CYT

6.0 5.5 5.0 4.5 4.0 ppm 7.5 7.0 ppm

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, ␥-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).

known chemotypes are encountered repeatedly. Quick recogni- Amaurobioidea (C. pastoralis) and Scytodoidea (Loxosceles tion of the NMR-spectroscopic signals representing such known spp.). However, given the variability of spider venom composi- chemotypes then facilitates detection of unexpected or previ- tion even among spiders from individual families, it seems likely ously uncharacterized compounds (17). Particularly noteworthy that similar compounds occur in other superfamilies as well. in the present study is the fact that sulfated nucleosides were The physiological properties of the sulfated nucleosides re- found to comprise the major components in the venoms of the main largely unexplored, primarily because their discovery is still three Loxosceles species, including the brown recluse spider, L.

reclusa. That sulfated nucleosides should comprise the major HDO small-molecule components of this previously well studied venom and yet have remained undetected until now, provides powerful testimony to the effectiveness of primary NMR- spectroscopic screening in natural-products research. Based on 4.0 the results of this study, it appears that sulfated nucleosides occur in the spider superfamilies Agelenoidea (A. aperta, H. curta),

4.5 [ppm] chemical shift

O O N N NH NH 5.0 N N NH N N NH HO SO 2 HO 2 3 O O 3 4 5.5 OH OSO3H OH OSO3H

OH O 6.0 H N NH N N N N 2 p H H H R NH 6.0 5.5 5.0 4.5 4.0 chemical shift [ppm] OH 5: R = OH 6: R = H Fig. 5. The 1H NMR and dqfCOSY spectra obtained for L. reclusa venom. Fig. 4. New natural products 3-6 identified from C. pastoralis and Loxosceles Cross-peaks corresponding to 2,5-disulfated guanosine (3) and 2-sulfated spp. venom. guanosine (4) are marked blue and red, respectively.

14286 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806840105 Schroeder et al. Downloaded by guest on September 23, 2021 very recent and amounts available from their natural sources are sweep width in F1 were acquired. For further characterization of selected insufficient for broad biological screening. We have recently samples, nongradient HMQC (phase-sensitive), gradient HSQC (phase- completed the synthesis of a small library of sulfated nucleosides, sensitive), gradient or phase-cycled HMBC, phase-cycled phase-sensitive which is currently being evaluated through the National Insti- NOESY spectra (using a mixing time of 600 ms), or phase-sensitive ROESY spectra (mixing time 300 ms) were acquired. tutes of Health’s Molecular Libraries Screening Network (MLSCN). MS. MS analyses were carried out as HPLC-MS or via direct injection of the Materials and Methods sample via syringe pump by using a Micromass Quattro I tandem MS operated in positive- or negative-ion electrospray mode and an Agilent Series 1100 Spider Venom. Venom samples were purchased commercially from Fauna liquid chromatograph (10 ϫ 250 mm; Supelco 5-␮m ODS preparative column Laboratories, Ltd. and Spider Pharm or were acquired directly from live eluted at a flow rate of 3.4 ml/min) equipped with a diode array detector. A specimens in our laboratory. In all cases, the venom was milked by using gradient elution was used, which started with a solvent composition of 5% electric stimulation of the venom gland and subsequently lyophilized (18, 19). methanol and 95% water for 3 minutes and then progressed to 100% meth- anol by 40 min. After UV-detection and before ESI-MS detection, the HPLC Sample Preparation. Venom was obtained frozen in lyophilized form, gener- effluent was split 40:1. Before injection onto the HPLC, spider venom samples ally as a white powder. To each sample was added 0.75 ml (for use of normal were redissolved in water and filtered over glass wool. 5-mm NMR tubes) or 0.3 ml (for use of Shigemi tubes) of D2O (100 atom % D). The resulting suspension was subjected to sonication for 1 min and then Synthesis. A stirred suspension of guanosine in dry dimethylformamide (95 centrifuged for 5 min. The supernatant was removed and analyzed via NMR mg, 0.36 mmol) under argon was cooled to Ϫ10°C, and sulfur trioxide- spectroscopy. After completion of NMR-spectroscopic experiments, select dimethylformamide complex (75 mg, 0.49 mmol) was added in one portion. samples were lyophilized and redissolved in water for MS analyses. Authentic The mixture turned clear within 2 min and was allowed to warm to 22°C. After samples of common small molecule neurotransmitters, which were used to stirring for 2 h, the reaction mixture was cooled to 0°C, and 1 ml of saturated confirm identities, as well as all NMR solvents, were purchased from Aldrich aqueous potassium bicarbonate solution was added with stirring. Subse- and Acros Chemicals. quently, the mixture was evaporated to dryness, and a portion (10 mg) was analyzed by NMR spectroscopy. The (1H,1H)-dqfCOSY (1H,13C)-HSQC, and NMR Spectroscopy. All analyses were carried out by using a Varian INOVA (1H,13C)-HMBC spectra revealed a mixture containing 2Ј,3Ј,5Ј-trisulfated, 3Ј,5Ј- 600-MHz NMR spectrometer, which was equipped with a 5-mm inverse- disulfated, 2Ј,5Ј-disulfated, and 5Ј-sulfated guanosine as the major products, detection HCN probe. Shigemi tubes were used as needed. NMR spectra were along with smaller amounts of 2Ј- and 3Ј-sulfated guanosine. Chemical shift acquired at 25°C by using the standard pulse sequences provided by Varian. values of the 2Ј-sulfated and the 2Ј,5Ј-disulfated isomers were in agreement

Venom samples of complex composition were characterized via a phase-cycled with those determined for the natural product. See Tables S1 and S4–S7 for ECOLOGY phase-sensitive dqfCOSY spectrum by using the following parameters: 600-ms NMR-spectroscopic data. acquisition time, 250–600 complex increments in F1, and 8, 16, 32, or 64 scans per increment, depending on the concentration of the sample. The numbers ACKNOWLEDGMENTS. We thank Prof. Andrey Feodorov of Fauna Laborato- of increments (ni) were chosen so that the digital resolution in F1 was roughly ries, Ltd., for providing spider venoms. This work was supported in part by the same for all extracts. Generally, 40–80 complex increments per 1 ppm of National Institutes of Health Grant GM 53830.

1. Cobas JC, Groves P, Martin-Pastor M, De Capua A (2005) New applications, processing 12. Chesnov S, Bigler L, Hesse M (2000) The spider Paracoelotes birulai: Detection and methods and pulse sequences using diffusion NMR. Curr Anal Chem 1:289–305. structure elucidation of new acylpolyamines by on-line coupled HPLC-APCI-MS and CHEMISTRY 2. Halouska S, Powers R (2006) Negative impact of noise on the principal component HPLC-APCI-MS/MS. Helv Chim Acta 83:3295–3305. analysis of NMR data. J Magn Res 178:88–95. 13. Gertsch WJ, Mulaik S (1940) The spiders of Texas. Bull Am Mus Nat Hist 77:307–340. 3. Dossey AT, Walse SS, Rocca JR, Edison AS (2006) Single-insect NMR: A new tool to probe 14. Gertsch WJ, Ennik F (1983) The spider genus Loxosceles in North America, and the West chemical biodiversity. Am Chem Soc Chem Biol 1:511–514. Indies (Araneae, Loxoscelidae). Bull Am Mus Nat Hist 175:264–360. 4. Zhang F, Dossey AT, Zachariah C, Edison AS, Bruschweiler R (2007) Strategy for 15. Vetter RS, Visscher PK (1998) Bites and stings of medically important venomous automated analysis of dynamic metabolic mixtures by NMR. Application to an insect . Int J Dermatol 37:481–496. venom. Anal Chem 79:7748–7752. 16. Lacey ME, Subramanian R, Olson DL, Webb AG, Sweedler JV (1999) High-resolution 5. Taggi AE, Meinwald J, Schroeder FC (2004) A new approach to natural products NMR spectroscopy of sample volumes from 1 nl to 10 ␮l. Chem Rev 99:3133–3152. discovery exemplified by the identification of sulfated nucleosides in spider venom. 17. Schroeder FC, et al. (2007) Differential analysis of two-dimensional NMR spectra: New J Am Chem Soc 126:10364–10369. natural products from a pilot-scale fungal extract library. Angew Chem Int Ed 46:901–904. 6. McCormick J, et al. (1999) Structure and total synthesis of HF-7, a neuroactive glyco- 18. Grant JB, Land B (2002) Transcutaneous amphibian stimulator (TAS): A device for the nucleoside disulfate from the funnel-web spider Hololena curta. J Am Chem Soc collection of amphibian skin secretions. Herpetol Rev 33:38–41. 121:5661–5665. 19. Norment BR, Smith OE (1968) Effect of Loxosceles reclusa Gertsch and Mulaik venom 7. McCormick KD, Meinwald J (1993) Neurotoxic acylpolyamines from spider venoms. against hemocytes of Acheta domesticus (Linnaeus). Toxicon 7:141–144. J Chem Ecol 19:2411–2451. 20. Tzouros M, Chesnov S, Bienz S, Hesse M, Bigler L (2005) New linear polyamine deriv- 8. Schulz S (1997) The chemistry of spider toxins and spider silk. Angew Chem Int Ed Engl atives in spider venoms. Toxicon 46:350–354. 36:314–326. 21. Jasys VJ, et al. (1990) Isolation, structure elucidation, and synthesis of novel hydroxy- 9. Platnick NI (1997) Advances in Spider 1992–1995 with Redescriptions lamine-containing polyamines from the venom of the Agelenopsis aperta spider. JAm 1940–1980 (Am Mus of Nat Hist, New York). Chem Soc 112:6696–6704. 10. Platnick NI (2007) The World Spider Catalog, ver 7.5 (Am Mus of Nat Hist, New York), 22. Quistad GB, Lam WW, Casida JE (1993) Identification of bis(agmatine)oxalamide in venom http://research.amnh.org/entomology/spiders/catalog/index.html. from the primitive hunting spider, Plectreurys tristis (Simon). Toxicon 31:920–924. 11. Ovtchinnikov SV (1841) The nominotypical spider subgenus Coelotes. Tethys. Entomol 23. Yamaji N, et al. (2004) Structure and enantioselective synthesis of polyamine toxin Res 2: 35–48. MG30 from the venom of the spider Macrothele gigas. Tetrahedron Lett 45:5371–5373.

Schroeder et al. PNAS ͉ September 23, 2008 ͉ vol. 105 ͉ no. 38 ͉ 14287 Downloaded by guest on September 23, 2021