Part 2: Tryptophan discovery: How do we know what we know?

[mike_malaska@deepspace.social]

That's a beautifully circular definition. It means if I can come up with another molecule that fits your observations, then you don't have a unique ID.

(thus invalidating your discovery.)

It puts the onus on the inventor (or discoverer) to have a solid defensible characterization from alternate possibilities.

(Key question: "You claim your identification uniquely fits the data, how did you verify that? Didja check ALL possible molecules????")

[mike_malaska@deepspace.social]

Any actual astrobiological detection is going to get A LOT of scrutiny since “extraordinary claims require extraordinary evidence”.

So yeah, go hardcore, satisfy the skeptics. If the underlying molecular detections are not solid, you are building your evidence on sand.

(And.....fun fact – MS-only analysis has NEVER been able to identify a complex organic chemical structure de novo.)

[mike_malaska@deepspace.social]

So how do natural product chemists* perform de novo structure elucidation?

Firstly, is you start with something you know is pure. You check with GC, HPLC under a few different column conditions to make sure that there is only one peak and not something else hiding under it. You should only see one retention time peak and no other bumps or shoulders.

Then you know that you are only dealing with one thing and you don’t have to worry if Signal A is coming from Compound B.

[mike_malaska@deepspace.social]

*(in my past career, I was not a natural product chemist, but was second in the drug discovery chain.

You know those stories where they identify a deep sea sponge alkaloid compound that kills cancer cells? Yeah, my old job was to figure how to modify that natural product to make it a better drug candidate: increase activity, remove toxicity, improve solubility, etc.

"Here's 20 mg of a funky paspalinine derivative, chemically modify it to make it work better. Go."
((it was actually very fun.))

[mike_malaska@deepspace.social]

For de novo structural elucidation, the workhorse is NMR (nuclear magnetic resonance). With NMR you can run a bunch of different techniques. My favorite minimal set for an unknown is 1H NMR, 13C NMR, DEPT-135, HSQC (1H-13C correlation), HMBC (multiple bond coupling experiments) and maybe ROSY (coupling) or NOSY (through-space interaction) experiments if you need it.

[mike_malaska@deepspace.social]

The beauty about NMR is that it is completely non-destructive and (unlike MS) does not lie. Unless you have a weird relaxation delay (you can compensate for that) you will see all the protons (and usually the carbons) under standard instrument settings.

There are a lot of other weird funky pulse sequences you can run too if you need to try to figure stuff out.

And, the best part, you can recover all the sample when you are done.

[mike_malaska@deepspace.social]

The two downsides of NMR is it can fool you with symmetry. But a simple MS run can help sort that out. The other bummer is that you can’t extract really hardcore purity information from it.

NMR is only quantitative to about 95%. So you need another technique to check for high purity. (such as: GC, LC, elemental analysis)

[mike_malaska@deepspace.social]

The absolute best structural confirmation is single-crystal high resolution X-ray analysis. That gets you the positions and orientations of all the molecules. If it is a really highly resolved structure, you can see (or infer) the hydrogen atoms.

But X-ray crystallography is really hard (and expensive) to do right off the bat. NMR will always be the tool of choice.

[mike_malaska@deepspace.social]

(and in a funny twist, protein structure is usually figured out by X-ray crystallography. But proteins don’t normally exist in crystal form, they are fluidy-loosey-goosey flexible. Crystallization can introduce new conformational artifacts.

To get it right and determine the protein bonding interactions in native solution you use…..NMR!)

[mike_malaska@deepspace.social]

For MS, a HRMS (high resolution mass spectrometry) experiment is my first choice. That gives you the molecular ion to lots of decimal places and thus gives you the exact mass and thus empirical formula so you know what atoms and how many of them you are dealing with. It doesn’t give you structure, but it nails the empirical formula.

[mike_malaska@deepspace.social]

MS also has lower resolution options. That can give you a rough idea of the weight, maybe of the parent molecule, and maybe some pieces and parts as it breaks into chunks. You might combine it with a front-end separation (like GC or HPLC) so you can analyze mixtures of molecules by separating them first then sending little tiny subsamples of them into the MS chamber as they come off a column.