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VSFG spectra of the oil/water interface with varying bulk concentrations of SDS.
with special attention given to spectral features that are sensitive to the local hydrogenbonding environment.
Mismatch marker It's one of those problems that evolution has figured out and science is still struggling to comprehend: How do cells recognize and repair DNA-base mismatches, such as thymine (T)cytosine (C), that arise during the course of genetic recombination and replication. To unravel the cellular mechanisms involved, molecular probes that recognize mismatch sites are needed. Jacqueline K. Barton and Brian A. Jackson of the California Institute of Technology introduce a novel rhodium intercalator that preferentially binds to these mismatch sites and identifies the bases involved. The Caltech scientists designed an intercalator with ligands too large to fit into a correct base-matched DNA site but able to attach to the larger, perturbed site of a DNA-base mismatch. The result is [Rh(bpy).,(chrysi)]3+ (bpy=bipyridine; chrysi=5,6-chrysenequinone diimine), which is synthe-
Structure of the A-[Rh(bpy)2(chrysi)f3*.
174 A
The sum-frequency (SF) signal was greatly enhanced as the bulk surfactant concentration increased, because the interfacial concentration and the associated surface-charge density and electrostatic field are a function of the bulk concentration. As the charged surfactant is added to the aqueous phase, the dominant spectral feature remains the "icelike" (more ordered) O-H stretching mode, whereas little intensity comes from the "waterlike" (less ordered) O-H stretching mode. The authors infer that the prevailing structure of the interfacial water molecules in the presence of the charged surfactant is a tetrahedral arrangement similar to the structure of ice. (J Phys Chem B .998 102 569-76)
sized with A- and A-enanttomers. Once intercalated, photoactivation of this rhodium complex promotes strand scission and can yield distinctive products with differing mobility on a separation gel. The new rhodium intercalator was tested with a set of 17-mer oligonucleotides, each containing a DNA-base mismatch. The most intense cleavage pattern involves the A-rhodium complex and a CC mismatch; strong cleavage is also seen with TT and TC mismatches. Distinctive gel patterns are also recorded following cleavage of CA (A=adenine) and AA mismatches. How the rhodium complex functioned with differing orientations and sequences was also investigated with a series of DNA hairpins containing central CA mismatches. Irrespective of which bases flanked the CA mismatch, the rhodium complex binds to the perturbed site. Different cleavage patterns arose, however, depending on which bases were nearest neighbors For example, the cleavage intensity was higher when cytosines rather than nines flanked the cytosine in the mismatch site. As a general rule, the authors observed the most significant "cutting" at the most helix-destabilizing mismatches. In addition to unraveling repair mechanisms, this strategy could be a useful probe for the development of molecular diagnostics or chemotherapeutic agents. (/. Am. Chem. Soc. 1997, 119,12986-87)
Analytical Chemistry News & Features, March 1, 1998
SCIENCE
Peeling oligosaccharides Sequencing oligosaccharides can be tricky. If the reaction that cleaves the monosaccharides is too fast, information regarding sequence can be lost. If the reaction doesn't cause cross-ring cleavage of the monosaccharides, the linkage information, which is vital to determining the branching pattern, is lost. Carlito Lebrilla and his co-workers at the University of California-Davis had been using MALDI (matrix-assisted laser desorption/ionization) and Fourier transform ion cyclotron resonance (FT-ICR) MS to sequence oligosaccharides, but this approach alone failed to give them the new reducing ends they needed to elucidate the linkages. To solve their problem, they reached into the history of polysaccharide chemistry and revisited alkaline degradation, a more than 80-year-old reaction also known as the "peeling reaction", which they are combining with MALDI FTICRMS (Anal. Chem. 1199 70,663-72). Alkaline degradation cleaves the glycoside bond at the reducing end by (3-elimination to yield a new reducing end. Because of its inefficiency, it had been essentially abandoned as a method for degrading oligosaccharides to their individual components. Lebrilla's group turned the inefficiency to an advantage. Because the reaction cleaves the monosaccharides one at a time and leaves a significant part of the oligosaccharide intact, a ladder can be constructed from which the sequence can be determined. The group uses MALDI, collisioninduced dissociation (CID), and FT-ICRMS to analyze the products of the alkaline degradation. Sustained off-resonance irradiation CID causes the cross-ring cleavage of the
The structure of lacto-N-hexaose, a diantennary oligosaccharide sequenced by MALDI FT-ICRMS.
saccharide at the reducing end of the oligosaccharide. Because alkaline degradation produces numerous side reactions and products, the FT-ICRMS is necessary—particularly for the exact mass determinations. Lebrilla says, "An exact mass determination for each of the fragments is pretty important, so that we can fish out the real product of the reaction from the chemical noise." The size of oligosaccharides is generally not a problem for sequencing. The big problem is the branching. "If [the oligosaccharide] is too highly branched, you may get a lot of information near the reducing end, but you won't get much information at the non-reducing end." The alkaline degradation occurs at different rates, which depend on the linkages. Therefore, if a branched oligosaccharide has antennae with different linkages, it will be relatively simple to determine the linkage at the branch points. Another strength of the alkaline degradation/MALDI FT-ICRMS combination is that it can also pinpoint the location of sialic acid and fucose on oligosaccharides. In traditional oligosaccharide sequencing, these residues often fall prey to nonselective glycosidic bond cleavage. Although the method provides sequence and linkage information, it does not provide complete structural information. In particular, it does not provide information about either the stereochemistry or a and P linkages. "The bad side is that you may never get full structural information [with MS]," says Lebrilla. "For that, you still have to use something such as NMR, but those techniques require significantly more material." Lebrilla points out that obtaining sufficient quantities of material will always be a problem for oligosaccharide analysis. They have thus far used the method to sequence a range of known oligosaccharides. The next step is to use the method to sequence unknown samples. To that end, they are collaborating with Jerry Hedrick of the molecular and cell biology department at the University of California-Davis to sequence oligosaccharides involved in frog egg fertilization. "There's been some work done with NMR in which they've gotten a few full structures, but they've only looked at the more abundant components," says Lebrilla. "We want to go back and see what the less abundant components are that they couldn't do with NMR We want to do real samples and see if we can get results."
Simple, yet sophisticated How can one build a detector for CE that leaves the capillary undisturbed and therefore can be placed anywhere along its length? That does not need a window in the capillary coating that would weaken the device? That can be constructed with less than $100 worth of materials? A schematic of the contactless conductivity detector. Such a detector was prewhen an electric frequency is applied. sented in the Feb. 1 issue of Analytical Chemistry (p. 563)3 It was seveloped dnd Schnell works with frequencies in the 20- to 40-kHz range, thereby keeping tested by a team at the Institute of Analytical Chemistry and Radiochemistry at the capacitive reactance low. the Leopold-Franzens-University InnsIn an earlier version of the detector, bruck (Austria). The Institute has a the two electrodes were made by paintlong research tradition in isotachoing a conductive silver varnish directly phoresis, CE, HPLC, and coupled methonto the CE capillary coating, rather ods. As long ago as the 70s, Erhard than by using syringe needles. The silSchnell, a now-retired professor at Innsver varnish provided excellent sensitivbruck, had the idea of a contactless con- ity for the signals because no air gap ductivity detector with capacitive couexisted between the electrodes and the pling. However, it wasn't realized until capillary. The construction is more comhis retirement and the rise of CE. plicated than with cannulas, however, Giinther K Bonn the head of the Instiand it leaves the detector's position tute offered a small laboratory to fixed. In the newer cannula version, the Schnell that would allow him to bring detector can be placed anywhere along his ideas to fruition Because a retired the capillary. In fact, Zemann hopes that professor does not have an official reit will be possible to investigate the fate search group Andreas J Zemann also of CE bands during their migration a professor at Innsbruck and his group through the capillary would use and test the new device With the newer detector, conductivity The detector consists of two syringe cannulas that are mounted 2 mm apart so that the CE capillary can be pushed through both of them (see figure). The cannulas act as capacitors that, together with a homemade frequency generator and amplifying electronics, measure the electrolyte conductivity inside the gap between them. When an analyte zone passes, the conductivity increases, which will be registered as a peak. The conductivity is measured via the capacitive properties of the detection unit, which is in fact a resistor-capacitor unit
Up close with the new detector.
is measured axially along the capillary (over the length of the gap between electrodes) and not across the capillary diameter. Therefore, capillaries of a different diameter give similar signals. The length of the gap is of minor importance. The detector linearity is excellent (from less than 1 ppm to more than 1000 ppm ffo sodium and chloride), and the detection limits are comparable with instruments on the market. Schnell and Zemann hope to improve the detection limits even more. For this purpose the use of more sophisticated electronics will be sary to optimize the waveform of the frequency generator and to improve the grounding Zemann also plans to develop a suppressor device that could be coupled to Schnell's detector and would improve the baseline by bringing the background conductivity to almost zero Veronika R. Meyer
Celia Henry Analytical Chemistry News & Features, March 1, 1998 175 A
