News
SCIENCE
Aglow for antibiotics Antibacterial compounds used for veterinary therapeutic purposes have improved the health and productivity of farm animals. However, the use of these compounds is not without its downside. Overuse can lead to increased bacterial resistance to antibiotics. One way to reduce the negative effects is to monitor drug residues and make sure that only the needed amount of drug is used. In this issue (p. 4457), Matti T. Karp and colleagues at the University of Turku (Finland) describe a biosensor for the detection of one class of such antibacterial drugs the tetracyclines. The biosensor uses a genetically engineered E. coli strain that contains bacterial luciferase genes from P. luminescens and d regulatory unit that represses or promotes the production of luciferase, depending on the presence of tetracycline. The repressor protein (tetR) has a recognition site for tetracycline. When the tetracycline binds to the tetR, the protein changes conformation and dissociates from the promoter. The luciferase gene is turned on, and the cell luminesces. The sensor response depends on the pH and the external Mg2+ concentration. Increases in the Mg2+ concentration shifts the luminescence response curve toward higher amounts of tetracycline. The sensitivity of the sensor can be manipulated by adjusting the Mg2+ concentration. At sufficiently high concentrations of tetracycline, the bioluminescence begins to decrease, which the au-
Binding with mass spec As combinatorial libraries grow, so does the pressure on analytical scientists to find quick and effective ways to assay the many compounds for useful biological activity as drug candidates. Albert Heck working at the University of Warwick (United Kingdom) (now at Utrecht University, The Netherlands) has collaborated with Thomas Jorgensen and Peter Roepstorff, two molecular biologists at Odense University (Denmark), to find a new way of coping wiih such libraries. The team developed an electrospray ionization (ESI) MS technique for evaluating
704 A
thors attribute to the toxicity of tetracycline to the E. coli cells. "The tetracycline starts ts inhibit the protein synthesis machinery of the sensor bacteria," Karp says. Although more than 1000 tetracycline derivatives are known, only seven have been used extensively for clinical or veterinary applications. The sensor responds effectively to all of the clinically relevant tetracyclines, which actually showed a higher affinity (about 1000-fold) for the repressor protein than for ribosomes (their usual targets). Although 90 min were required for optimal sensitivity of luminescence, a detectable signal was produced within a few minutes. The detection limit was 2 ng/sample, which is in the picomole range. "We have focused on rapid diagnostics of antibiotic residues.
The diffusion rate of tetracycline in the cells controls the speed of the assay," Karp says. 'With very long incubation times of 10-20 h, sensitivity would be even better." The sensor is specific to tetracyclines. Other antibiotics, such as rifampicin, kanamycin, nalidixic acid, chloramphenicol, streptomycin, and erythromycin, do not induce luciferase production in the sensor cells—even at high concentrations over a wide range. Karp says, "No antibiotics other than tetracyclines will cause an increase in luminescence over the background signal." Potential areas of application for this sensor include the detection of tetracyclines in different food matrixes and serum. According to Karp, the next step is to develop an assay that is suitable for routine work. Celia Henry
Mode of action of the sensor on the molecular level.
vancomycin analogues as new antibiotics. forming between peptidoglycans and kills The approach correlates peaks in their the bacterium. spectra directly with the binding of antiWorryingly, resistance, even to vancomybiotic candidates to bacterial cell wall pepcin, is becomming more comon, as bacteria tides and so avoids the complex and timehave swapped the D-alanine in their growing consuming spectroscopic titration techbacterial overcoat for a D--actate or D--erine. niques typical in such studies. This change repels vancomycin rather than hooking it—removing its inhibitory effects Bacterial drug resistance is a major and allowing the bacterium to mature. problem, and natural products such as the glycopeptide vancomycin are now used by In searching for effective vancomycin hospitals as last-resort drugs against killer analogues to bring up the rearguard, medicibugs such as Staphylococcus aureus. Vanco-nal chemists have discovered that the solumycin, and its chemical cousin ristocetin, tion association binding constant (K^ bework by hooking on to the D-Ala-D-Ala setween the antibiotic and the bacterial peptide quence of peptidoglycan precursors cell wall is an accurate indicator of potential present in the growing bacterial cell wall. activity as a drug. Now, Heck and his colThis stops stabilizing cross-links from leagues have found that they can determine
Analytical Chemistry News & Features, November 1, 1998
Shifting masses 2(l values of 0.730 and 0.714 cm, respecenced by something other than the mass tively, whereas the dihydrobenzofuran exof the molecule." Shifts along the m/z axis can be a good perienced little or no chemical mass shift. The chemical mass shift is particularly thing, particularly if they allow the sepaThe isobars naphthalene and rfr-nitrobenzene pronounced for nitroaromatic compounds. ration of seemingly unseparable ions. In differ in molecular mass by only 0.003 m/z, Cooks' group wants to exploit these large tlit' October 15 issue of Analytical Chembut they were readily separated l)ecause, at a shifts to detect nitroaromatic compounds istry (p. 4448), Graham Cooks and his z,, of 0.707 cm, d-,-nitrobenzene had a chemica in complex mixtures. "The driving force is co-workers at Purdue University demass shift of 0.6 m/z. explosives detection," says Cooks. "If you scribed experiments in which they could have a [feature] like a 0.7 m/z shift that separate isobaric and isomeric comCooks emphasizes that their results do you can use on top of any other feature in a pounds in an ion trap mass spectrometer not mean that commercial ion trap mass complex mixture, it could be extremely simply by changing the distance between spectrometers produce mass spectra with valuable in detecting the presence of one the end-cap electrodes. Although this wrong m/z values. "You can set up the inclass of compounds or eliminating another." effect was first observed by George Stafstrument to run like any ion trap and give ford and John Syka of Finnigan in 1983, good mass-to-charge values. Or, you can If the term chemical mass shift seems Cooks and his co-workers are the first to set it up by adjusting the geometry so that vaguely familiar, it should. "The parallel examine these shifts systematically and the fields are unusual. In this experiment, with NMR is intentional," says Cooks. "We to demonstrate isomeric ion separation we didn't want the m/z to come out 'right'. couldn't call them chemical shifts because We wanted the apparent mass to be influthe name was reserved." Celia Henry Changing the distance between die end-cap electrodes (tiie z„ value) in an ion trap mass spectrometer introduces higher order electric fields. Certain compounds respond to these electric fields in ways that result in a "chemical mass shift". In this system, the end-cap electrodes were mounted on moving stages connected to a linear micrometer. The 2„ could be adjusted from 0.684 to 0.920 cm in 0.0076-cm or smaller increments. At values greater than or equal to the z value of commercial instruments (0.783 cm), the effects of the higher order fields cancel each other, and there is no chemical mass shift With this system, Cooks and his coworkers separated the isobaric pairs of (/-nitrobenzene/naphthalene and />-nitrotoluene/3-ethoxyaniline, as well as the isomers acetophenone and dihydrobenzofuran. For example, the chemical shift for Separating the structural isomers acetophenone and dihydrobenzofuran with chemical mass acetophenone was 0.5 m/z and 0.9 m/z at shifts.
Electrospray can measurr the eolution binding constants for noncovalent complexes of vancomycin.
Ka values using only the relative ion intensities in ESI-MS. This has allowed them to develop a fast, parallel method for high throughput screening of vancomycin-type compounds. In comparison, solution spectroscopic techniques only work on one receptorligand complex at a time. Heck and his colleagues tested their method on vancomycin itself and three peptide ligands, which represented the precursor peptides of the bacterial cell wall. They found the vancomycin-peptide complexes were transported intact from solution into the vacuum gas phase of the mass spectrometer. The equilibrium concentrations in solution were represented by the MS peak intensity of a particular complex
relative to the summed intensities of all complexes and the free antibiotic. In addition, the team investigated the pH stability of the ristocetin-peptide complex using ESI-MS and compared the results with the conventional circular dichroism approach. Very similar results were produced with both, so Heck believes ESI-MS could be used not only to assay potential drug candidates but also to provide an estimate of their likely efficacy at different pH values. Heck adds that the ESI method should be applicable to a variety of other systems, such as enzyme inhibitors, because the technique is based solely on ion intensities. David Bradley
Analytical Chemistry News & Features, November 1, 1998 705 A
