Chemical Education Today
Reports from Other Journals
Research Advances by Angela G. King
The quest for novel biologically active molecules routinely engages chemists in the purification and identification of natural products. Typically, chemical prospecting entails the collection of large numbers of specimens, homogenization, extraction, and lyophilization. The sample is then subjected to chromatographic separation techniques in order to obtain a pure compound. Recent work by Jerry Meinwald and his colleagues Frank Schroeder and Andrew Taggi has raised questions concerning the impact of this regimen on the natural products under investigation, and has demonstrated the ability to identify compounds in a biological mixture. Hololena curta is a species of spider whose venom had been previously studied by the Meinwald group. Among other compounds, it contains HF-7, a bis-sulfated glyconucleoside and reversible blocker of glutamate-sensitive calcium channels. At the time of its identification, HF-7 was a novel spider venom component, structurally differing from the other 100+ small-molecule toxins identified in spider venom, which are mostly acylpolyamines. Meinwald and his collaborators questioned why no compounds related to HF-7 had been identified in other spider venoms and began a search for them. Based on his earlier research, Schroeder initiated a novel NMR-based approach to spider venom analysis to identify additional toxins present in spider venom, including compounds related to HF-7, using spectral data as a guide. The first step of the new method is the acquisition of NMR spectra (1H and (1H,1H)-dqf-COSY) of the entire crude venom! These spectra are obtained before any fractionation or purification and serve as a fingerprint of the natural mixture. The small molecules present in the venom tend to give well-resolved signals that are easily distinguished from those of proteins and polypeptides. Phase-sensitive dqf-COSY spectra offer the advantages of predictable antisymmetric crosspeak shape and embedded multiplicity patterns. For some venoms, these initial NMR experiments have allowed structure determination of all prominent compounds. For venoms of complex composition, some fractionation is required. This approach was first tested on venom milked from the hobo spider, Tegenaria agrestis. The venom from this species had been previously studied but no sulfated nucleoside components had been identified. Using their new approach, the research team identified a family of sulfated nucleoside derivatives, completely characterizing four compounds and tentatively identifying three additional components, and trace amounts of HF-7. About 50% of the dry mass of T. agrestis venom is due to nucleoside-containing compounds. The rest of the sample consists of acylpolyamines, peptides, proteins, citrate, and inorganic salts. Most interesting is that when the venom was fractionated and the individual fractions analyzed by NMR spectroscopy, the bis-sulfated nucleosides indicated in the initial 10
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An Australian spider ( Delena sp.) before (top) and during (bottom) milking. With a device developed by biologist Jacqualine B. Grant at Cornell University, a mild electric shock is applied to the anesthetized spider, inducing it to squir t venom into a capillary tube.
NMR spectra of intact venom were not present! Instead researchers found predominately monosulfated guanosine and fucose. Without the data obtained on the crude venom, these natural products, representing over 50% of the venom sample, would have been overlooked since they decomposed during fractionation by reversed-phase HPLC. Once the problem was identified, it was overcome by adding a small amount of d5-pyridine to stabilize the bis-sulfated ribonucleosides as salts. Armed with this newfound knowledge of the stability of bis-sulfated ribonucleosides, Meinwald’s team returned to the venom of H. curta, the source of the kainite inhibitor HF-7. Using the crude venom NMR approach, at least five additional sulfated ribonucleosides were detected in H. curta venom samples. Researchers have moved forward and identified sulfated ribonucleosides in the venom of 12 more spider species. Sulfated nucleosides are likely to have potent biological activity. This clearly indicates that bioprospecting utilizing NMR analysis of unfractionated samples as a fingerprint may uncover more hidden treasures than an approach that rigidly requires pure compounds. What biological role do the sulfated nucleosides play? Schroeder has reported, “Their presence in spider venom suggests that they act in some fashion as neurotoxins, possibly in conjunction with other compounds present in the secretion. Strangely, the biological properties of sulfated nucleosides are largely unexplored. This is particularly surprising considering the large number of nucleoside derivatives and analogs which have been synthesized and tested in conjunction with efforts to interfere with viral replication or cancer progression.” His research team is currently developing syn-
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Photos courtesy of Frank Schroeder.
A New Approach to Natural Product Identification
Chemical Education Today
Reports from Other Journals theses for the preparation of several of the sulfated nucleosides identified in spider venom, as well as of a number of analogs. This is necessary, because the amounts of venom available are clearly too small to provide quantities of nucleosides large enough for biological testing. Looking forward, Schroeder states, “With the synthetic samples in hand and with the collaboration of biologists and molecular pharmacologists, we will be able to subject sulfated nucleosides to a broad range of biological screening.”
More Information 1. Taggi, A.; Meinwald, J.; Schroeder, F. A New Approach to Natural Products Discovery Exemplified by the Identification of Sulfated Nucleosides in Spider Venom. J. Am. Chem. Soc. 2004, 126, 10364–10369. 2. A fuller description of this research is available at http:// pubs.acs.org/cen/news/8232/8232notw4.html; a description of Meinwald’s research in chemical ecology is available at http:// www.chem.cornell.edu/fs31/mission_statement.html (both sites accessed Oct 2004). 3. SpiderPharm specializes in the production of venoms from spiders and other arthropods. The company maintains a Web site with links to research on spider venom at http:// www.spiderpharm.com/ (accessed Oct 2004).
Acrylamide in French Fries Acrylamide is a human neurotoxin, known rodent carcinogen and probable human carcinogen. In the four years since its identification in food, a desire to reduce its levels in food has developed due to associated health risks. Several reports indicate that asparagines and sugars are precursors of the acrylamide in food, which forms through the Maillard reaction. Eliminating acrylamide formation in foods by stopping the Maillard reaction is not completely desirable, since it produces desirable taste and aroma in foods. A Canadian research team led by Adam Becalski recently identified the relationship between possible acrylamide precursors, namely sugars and amino acids, present in food and the level of acrylamide produced. The investigators used french fries as the food item for the investigation. Commercial french fries were found to have levels of acrylamide between 60 and 1800 ng/g. Different levels of sugars and amino acids in the raw potatoes and different processing methods were suggested as the source of the variance in acrylamide levels. Researchers selected 66 potato samples with a large range in sugar (glucose, fructose, and sucrose make up more than 90% of all sugars in a potato) and amino acid composition. The levels of sugars and amino acids in the raw potatoes were measured using an enzyme-based assay and a modified commercially available EZ:fast amino acid kit, respectively. Each potato sample was then cut into french fries and cooked in an identical two-step process. The resulting fries were then subjected to an isotope dilution (13C3acrylamide) LC–MS/MS method. Asparagine levels in raw potato alone were not strong predictors of resulting acrylamide. However the levels of sugars correlate strongly with the acrylamide levels observed in 12
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french fries, with formation of acrylamide favored by an excess of sugars. Additional research suggests that cold storage temperatures enhance acrylamide levels. While commercial production of french fries might be limited by the variety of potato cultivars available on a commercial scale, this work clearly demonstrates that if a variety low in sugar is selected, acrylamide levels can be significantly reduced when using a standard industry cooking method.
More Information 1. Becalski, A.; Lau, B.; Lewis, D.; Seaman, S.; Hayward, S.; Sahagian, M.; Ramesh, M.; Leclerc, Y. Acrylamide in French Fries: Influence of Free Amino Acids and Sugars, J. Agric. Food Chem. 2004, 52, 3801–3806. 2. This research is complementary to a gas chromatography class research project investigating french fries. Crowley, J.; DeBoise, K.; Marshall, M.; Shaffer, H.; Zafar, S.; Jones, K.; Palko, N.; Mitsch, S.; Sutton, L.; Chang, M.; Fromer, I.; Kraft, J.; Meister, J.; Shah, A.; Tan, P.; Whitchurch, J. Classroom Research: GC Studies of Linoleic and Linolenic Fatty Acids Found in French Fries, J. Chem. Educ. 2002, 79, 824. 3. A detailed description of the Maillard reaction, which results in non-enzymatic browning, can be found at http:// www.agsci.ubc.ca/courses/fnh/410/colour/3_82.htm. A lower level explanation and hands-on activity is also available: http:// www.exploratorium.edu/cooking/meat/INT-what-makes-flavor.html (both sites accessed Oct 2004).
Selectively Sensing Mercury with OligonucleotideBased Detectors Mercury is introduced into the environment through numerous processes: volcanic activity, mining, and the combustion of solid waste, for example. Due to environmental hazards associated with Hg2⫹, the need to rapidly and selectively detect its presence is critical. Chemosensors are molecules used to detect the presence of some species (substrate) through a change in their interaction with light upon substrate binding. Detectors need to be water-soluble for use in the environmental field, and most sensors employed today utilize small synthetic molecules that bind Hg2⫹. Researchers at Tokyo Metropolitan University have now developed an oligonucleotide (ODN)-based detector that is water soluble and offers greater selectivity for mercury over other metal ions that may be present in the sample. The new sensors consist of an ODN functionalized at the 3´ terminus with a fluorescein fluorophore (F) and at the 5´ terminus with a dabcyl quencher group (D). The ODN sequence contains a thymine-rich mercury binding region and a linker sequence. The sensors work by using mercury(II) ions to coordinate base pairs between thymine residues in two different binding sequences. This causes the formation of a hairpin turn and brings the 3´ and 5´ termini in close contact. Their close proximity allows an enhanced fluorescence resonance energy transfer (FRET) between F and D. The end result is significant quenching of fluor-escent emissions relative to a random coil conformation. As [Hg2⫹] increases, the observed intensity of the D-ODN-F fluorescence decreases
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Chemical Education Today
Reports from Other Journals Substances other than mercury ions found in environmental samples may also quench the sensor’s fluorescence. The antisensor described above can be used to determine whether observed quenching is from mercury binding or a sample artifact. If the quenching occurs in both the presence and absence of the antisensor, it is most likely due to contaminants in the sample and the presence of Hg2⫹ is questionable. If the quenching dissipates when the antisen-sor is present, the sample being studied contains mercury(II) ions. The new oligonucleotide-based sensor for mercury(II) offers several advantages: water solubility, selective detection of Hg2⫹, and control experiments. This utility may be further exploited by developing sensors with varying sensitivity by altering the sequences of binding sites and linkers, and using different donor–acceptor FRET combinations to detect different metal ions with different fluorescence emissions wavelengths. A schematic representation of quenching of fluorescence from F upon binding of Hg2⫹ ions and resulting hairpin structure formation. Reprinted with permission from Angew. Chem. Int. Ed. 2004, 43, 4301. Copyright 2004.
within a concentration range of 40–100 nM Hg2⫹. Researchers postulate that the sensor does not work at concentrations lower than 40 nM since several mercury ions must bind to the sensor at the same time to form the hairpin structure. An antisensor having the same sequence as D-ODN-F but lacking the F and D residues was used to check the reliability of the new sensor. When D-ODN-F and the antisensor were mixed in solution, with the antisensor in excess, addition of Hg2⫹ resulted in only a slight change to the fluorescence spectrum of D-ODN-F due to the antisensor sequence binding the mercury ions and substantially decreasing the concentration of mercury in solution.
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More Information 1. Ono, A.; Togashi, H. Highly Selective Oligonucleotide-Based Sensor for Mercury(II) in Aqueous Solutions. Angew. Chem. Int. Ed. 2004, 43, 4300–4302. 2. Young, J. A. Chemical Laboratory Information Profile: Mercury(II) Nitrate Monohydrate. J. Chem. Educ. 2003, 80, 1373. 3. For help in creating interest among students in environmental issues concerning mercury, see Judd, C. News from Online: Mercury and Our Environment. J. Chem. Educ. 2001, 78, 570. 4. A description of a new small synthetic mercury detector, for comparison, appeared in this column: J. Chem. Educ. 2004, 81, 1086. Angela G. King is Senior Lecturer in Chemistry at Wake Forest University, P.O. Box 7486, Winston-Salem, NC 27109;
[email protected].
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