I’ve always had a soft spot for detective novels. I even fancy myself a detective. Not like the ones you read about in pulp fiction made famous by Daly, Hamlet, and Spillane (to name a few), but a sleuth no less. I have spent the last couple of weeks playing detective in lab, you see. I am out on a hunt for molecules produced by these microbes I am studying. This type of investigative work is what attracted me to chemistry and biochemistry. At hand I have a set of known microbial metabolic pathways, a pretty good idea of what some of the intermediate and products are, a lot of chemistry knowledge, and a whole slew of really cool high tech tools.
The method is pretty straightforward. I feed my favorite microbe a known carbon source; it helps if I can radio-label a carbon or another atom on the substrate. I wait for a defined period of time, collect the media, and start the analysis. Step one: spin down cells, take off the supernatant, extract with dichloromethane, …
Yes, it is quite tedious, but many steps later, I have a small sample with metabolites extracted from the experimental microbial growth sample. Now the fun begins. First, what technique do I start with? The simplest is ultraviolet/visible (UV/vis) light spectra. What does the absorption spectra of your samples look like? Can you see something that resembles a conjugated backbone? How about an aromatic ring? Hints of structure here and there. But then you ask, does the sample contain one or many kinds of molecules? Can I somehow separate these molecules?
In this next step I might choose liquid chromatography (LC) or gas chromatography (GC). Which technique I use depends on certain properties of the molecules I am looking for, are they water-soluble? Are they volatile? For water-soluble molecules I select LC and for volatile molecules I select GC. Both of these separation techniques have a mass spectrometry instrument (MS) attached, allowing me to get a sense of the molecular mass of each separated compound. I inject and watch the molecules fly. Every peak I see reveals a wealth of information about the compounds ‘vitals’. What is the mass of the molecule? Does it fractionate into smaller defined compounds with a known mass? Do the fractionation patters I see match a compound previously characterized?
Next, I isolate and purify the compound using LC, collecting the fractions that have my compound of interest and use Nuclear Magnetic Resonance (NMR) spectroscopy to try to resolve ambiguities due to configuration around enantiomeric carbon atoms. I use proton NMR and carbon NMR and study the chemical shifts I see in my purified sample. From these set of data, I can deduce whether I have a ‘R’ or ‘S’ configuration around my enantiomeric carbon and a host of other structural relationships.
In the end, I have a larges set of ‘clues’ from which I will build a chemical structure of the compounds isolated from the microbial growth media. But that is not enough. In order to stake a claim to a new molecule and possibly a whole new molecular pathway, I have to present my findings to other chemists and microbiologists, convincing them that I have done my sleuthing well. It is very tedious work and most of the time you do not find a new molecule, but every once in a while, you isolate something, a molecule never seen before. The thrill of finding what has not been observed before, drives me to explore the secret metabolic pathways hidden in these microbial species.
Stay tuned to see how this tale turns out. HHH