That’s the culprit! Separating enantiomers with bacteria

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  • Published: Jul 16, 2012
  • Author: Jon Evans
  • Channels: Electrophoresis
thumbnail image: That’s the culprit! Separating enantiomers with bacteria

Question the victim

That’s the culprit! Separating enantiomers with bacteria

The best person to identify the perpetrator of a crime in a line-up is usually the victim, and it turns out that the same is true when identifying biologically-active enantiomers. According to Chinese scientists, when looking to distinguish between the two enantiomers of the antibacterial compound ofloxacin, it’s best to ask bacteria.

Like many drug molecules, ofloxacin is naturally chiral, meaning that it exists in two mirror-image structural forms known as enantiomers. As is also often the case, one of these enantiomers is much more biologically active than the other, with the antibacterial activity of (S)-ofloxacin many times higher than that of (R)-ofloxacin.

To separate different enantiomers, scientists often employ compounds known as chiral selectors, which interact with one enantiomer more than the other. By incorporating chiral selectors such as cyclodextrins, chiral crown ethers, polysaccharides or proteins in an electrolyte, enantiomers can be effectively separated by capillary electrophoresis (CE).


Call in the bacteria

For antibacterial compounds, bacteria should work just as well, as clearly the biologically-active enantiomer will interact with the bacteria more than the non-active enantiomer, because that is how it produces its antibacterial effect. What is more, by using bacteria as the chiral selector, scientists will not only be able to separate the different enantiomers, but they should also gain an insight into the way the active enantiomer interacts with the bacteria.

So a team of Chinese chemists led by Zhining Xia at Chongqing University decided to try this out with the two ofloxacin enantiomers. They tested three different bacteria as chiral selectors: Escherichia coli and Pseudomonas aeruginosa, both of which are Gram-negative; and Staphylococcus aureus, which is Gram-positive.

To ensure that the bacteria wouldn’t interfere with the detection of the two ofloxacin enantiomers by UV absorption, as bacteria also absorb UV light, the scientists only inserted a narrow plug of bacteria into the capillary. Because the enantiomers migrate faster than the bacteria, they travel through the plug, where (S)-ofloxacin should interact more than (R)-ofloxacin, and both reach the UV detector before the bacteria, preventing any interference.


Case solved

This is indeed exactly what happened. For all three bacteria, (S)-ofloxacin took longer to migrate through the plug than (R)-ofloxacin, causing the enantiomers to separate and produce two clear peaks in the subsequent electropherogram. The separation process was also quick, taking less than seven minutes.

As hoped, the process revealed useful information about the interaction between the bacteria and enantiomers, because the enantiomers were better separated by the Gram-negative bacteria than by the Gram-positive bacteria. This supports an earlier finding indicating that ofloxacin, which produces its antibacterial effect by inhibiting an enzyme involved in DNA replication, is more effective against Gram-negative bacteria than Gram-positive bacteria. According to Xia, this process could also be used to probe the mechanism of action and toxicity of antibacterial compounds, as well as the development of antibacterial resistance

What is more, bacteria might be just the start. By incorporating other target cells in a CE electrolyte, this process offers a way to separate and study the enantiomers of many other drugs. If that does turn out to be the case, bacteria and other cells could find themselves regularly called upon to help scientists with their enquiries.

Related Links

Journal of Separation Science (Article in Press): "Enantioselective analysis of ofloxacin enantiomers by partial-filling capillary electrophoresis with bacteria as chiral selectors"

Article by Jon Evans

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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