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Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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February 5, 2013

Vibrational Scent Wins A Round?

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Posted by Derek

I wrote here and here about Luca Turin's theory that our perception of smell is partly formed by sensing vibrational modes. (Turin is the author of an entertaining book on the subject of olfaction, The Secret of Scent, and also co-author of Perfumes: The A-Z Guide). His theory is still controversial, to say the least, but Turin and co-workers have a new paper out trying to shore it up.

A previous report from Vosshall and Keller at Rockfeller University had shown that human subjects were unable to distinguish acetophenone from its deuterated analog, which is not what you'd expect if we were sensing bond vibrations. Interestingly, this paper confirms this result. (References to all these studies are in the original paper, which is open-access, being in PLoSONE):

In principle, odorant isotopomers provide a possible test of shape vs. vibration mechanisms: replacing, for example, hydrogen with deuterium in an odorant leaves the ground-state conformation of the molecule unaltered while doubling atomic mass and so altering the frequency of all its vibrational modes to a greater or lesser extent. To first order, deuteration should therefore have little or no effect on the smell character of an odorant recognized by shape, whereas deuterated isotopomers should smell different if a vibrational mechanism is involved.

The experimental evidence on this question to date is contradictory. Drosophila appears able to recognize the presence of deuterium in odorant isotopomers by a vibrational mechanism. Partial deuteration of insect pheromones reduces electroantennogram response amplitudes. Fish have been reported to be able to distinguish isotopomers of glycine by smell. However, human trials using commercially available deuterated odorants [benzaldehyde and acetophenone] have yielded conflicting results, both positive and negative. Here, using GC-pure samples and a different experimental technique, we fully confirm Keller and Vosshall’s finding that humans, both naive and trained subjects, are unable to discriminate between acetophenone isotopomers.

But the paper goes on to show that humans apparently are able to discriminate deuterated musk compounds from their H-analogs. Cyclopentadecanone, for example, was deuterated to >95% next to the carbonyl, and to 90% at the other methylenes. It and three other commercial musks were purified and checked versus their native forms:

After silica gel purification, aliquots of the deuterated musks were diluted in ethanol and their odor character assessed on smelling strips. The parent compounds have classic powerful musk odor characters, with secondary perfumer descriptors as follows: animalic [Exaltone], sweet [Exaltolide], oily [Astrotone] and sweet [Tonalid]. In all the deuterated musks, the musk character, though still present was much reduced, and a new character appeared, variously described by the trained evaluators [NR, DG, LT and Christina Koutsoudaki, Vioryl SA] as “burnt,” “roasted,” “toasted,” or “nutty.” Naive subjects most commonly described the additional common character as “burnt.”

They found, by stopping the deuterium exchange early, that this smell showed up even at around 50% D-exchange or less. For more rigorous tests, they went to a "smelling GC", and double-blinded the tests. This gave clean compound peaks, and they were able to diminish the need to keep a memory of the previous smell in mind by capturing the eluted peak vapors in Eppendorf tube for side-by-side comparison.

This protocol showed that people are indeed unable to discriminate deuterated acetophenone from undeuterated - the Keller and Vosshall paper stands up, which will come as a relief to the author of the unusually celebratory editorial in Nature Neuroscience that accompanied it. To be sure, it also makes moot Turin's own objections to their work at the time, which questioned its experimental design and rigor.

But the deuterated musk experiment done this way are quite interesting. I'm going to just quote the entire section here:

All trials were performed with GC-pure catalytically deuterated [D fraction >90%] cyclopentadecanone [See Methods]. Each trial consisted of the assessment of 4 pairs of odorants, one deuterated and one sham-deuterated. The subjects were presented with a deuterated sample and their attention was drawn to the “burnt, nutty, roasted” character of the deuterated compound. Several other sample pairs were presented until the subjects were sure they could tell the difference between the two sample types.

The Eppendorf tubes were heated in a solid heating block to 50C. The samples were arranged in rows according to their type. The experimenter randomized the order of the tubes within the rows by means of two flips of a coin (first flip: first or second two positions, second flip: first or second spot within those). The rows were then mixed randomly by a further coin flip per d/H pair (heads: swap positions, tails leave in situ).

Subjects were first given a training pair and told which was deuterated and which sham-deuterated. The experimenter then left to watch the experiment through a window. Subjects were then presented with the unlabeled, position-randomized pairs of deuterated and sham-deuterated GC-pure samples and asked to say which was which.

The subject, wearing nitrile gloves to avoid contamination, smelled first one and then the other sample. Multiple sniffs at each sample were allowed. The subject was asked to identify the deuterated sample and to place it to one side. After four trials the tubes were placed under the UV light source and identified. The subject was not informed of the outcome. To avoid habituation, the subject then rested for 15 minutes before attempting the next trial.

The results are shown in table 2. Eleven subjects were used. Two subjects tired before reaching the desired number of 12 trials. Two were able to go beyond to 13 and 17 trials respectively. The binomial p values range between 0.109 [6/8 correct] to 7.62×10−6 [17/17 trials]. These are independent trials, and an aggregate probability for all trials [119/132 correct] can be calculated: it is equal to 5.9×10−23.

As it happens, musks are at nearly the top of the molecular weight range for odorant compounds. The paper mentions a rule of thumb among fragrance chemists that compounds with more than 18 carbons rarely have any perceptible odor, even when heated (and different people's noses can top out even before that). Musks tend to smell quite similar even with rather different structures, which suggests that a small number of receptors are involved in their perception. Here's Turin's unified theory of musk:

We suggest therefore that a musk odor is achieved when three conditions are simultaneously fulfilled: First, the molecule is so large that only one or a very few receptors are activated. Second, one or more of these receptors detects vibrations in the 1380–1550 cm-1 range. Third, the molecule has intense bands in that region, caused either by a few nitro groups or, equivalently, many CH2 groups. A properly quantitative account of musk odor will require better understanding of the shape selectivity of the receptors at the upper end of the molecular weight scale, and of the selection rules of a biological IETS spectrometer to calculate the intensity of vibrational modes.

It's safe to say that this controversy is very much alive, no matter what the explanation might be. Leslie Vosshall of Rockefeller has already commented on this latest paper, wondering if compounds might be enzymatically altered in the nose (which would also be expected to show a large difference with deuterated compounds). I'll await the next round with interest!

Comments (27) + TrackBacks (0) | Category: Chemical News


COMMENTS

1. Curious Wavefunction on February 5, 2013 10:28 AM writes...

Interesting. I wonder if the truth lies somewhere in between, with vibration being important for some compounds and shape being important for others. I also wonder how this squares with the combinatorial smell sensing mechanism which got its discoverers a Nobel Prize a few years ago.

For what it's worth, I gave a seminar on Turin's theory a few years ago, and 9 out of 10 of my test subjects could distinguish between the smell of dimethyl sulfide and dimethyl sulfide-d6.

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2. rogi on February 5, 2013 10:31 AM writes...

Derek and other lab rats. I submit to you that there is something to be said for this. I lay down a challenge: Crack open a vial of deuterated methanol (D3COD) and regular methanol and place a drop or two on the lab bench. The deuterated version smells subtly different than regular methanol.We did this experiment with a bunch of former colleagues (n=15) and all but 3 (we did a blinded experiment) could tell the difference.

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3. Young Padawan on February 5, 2013 10:40 AM writes...

Reminds me of something related: t-butyl vs. TMS in an odorant
Angew. Chem. Int. Ed. 2007, 46, 3367 –3371

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4. Eskimo on February 5, 2013 10:42 AM writes...

As CWf points out, there are so many olfactory receptors. I imagine that deuteration might make a difference in binding/signaling for some, but not for others.

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5. lt on February 5, 2013 11:00 AM writes...

I'm often surprised when people who work with proteins are themselves surprised when the molecules they study act like molecules. As if it were unimaginable that concepts like lock and key or induced fit are but the most crude of generalizations and that things should really be that simple and nature is somehow wrong when it does not behave like our models suggest. Or that shape and vibrations are somehow independent of each other and that "good" molecules have to make a choice about which of their properties are allowed to manifest in any given interaction.

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6. HI on February 5, 2013 11:02 AM writes...

To Curious Wavefunction. Buck and Axel won the Nobel Prize for discovering the large family of genes that encode olfactory receptors. Their Nobel Prize is secure, regardless of whether Turin is right (which I am skeptical of).

Olfactory receptors are just GPCRs after all. If Turin is right, why shouldn't this mechanism apply to other GPCRs as well?

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7. istvan on February 5, 2013 11:29 AM writes...

Such speculations make me wonder whether anyone has done the conventional logP measurements in octanol/deuterated water. (And I am lazy to look up 'deuterium bonding' vs 'hydrogen bonding'.)

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8. hi, hi on February 5, 2013 12:35 PM writes...

@6,

I dunno if Turin claims that this is a universal mechanism or not, but is there a fundamental reason why it should be? Is it not possible that some GPCRs recognize shapes of litle rocks of a ligand, while for some others vibrational effects are just as important? As #5 points out, we just deal with mental models of reality. I can see a continuum where some GPCR-ligand interactions are described to within experimental uncertainty by simply considering shapes and H-bonds, while for some others (say peptides) ligand conformations due to rotations around sigma bonds are important; and for others even vibrational modes need to be modelled to fully recapitulate experiment.

Any reason why not?

BTW, speaking of ligands as rocks - wikipedia tells me that adamantane smells of camphor. Fascinating!

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9. RM on February 5, 2013 12:52 PM writes...

Does the "The subjects were presented with a deuterated sample and their attention was drawn to the “burnt, nutty, roasted” character of the deuterated compound. Several other sample pairs were presented until the subjects were sure they could tell the difference between the two sample types." raise warning flags for anyone else? In a test that purports to determine if people can determine a difference between the two compounds, first telling them that there *is* a difference between the two compounds and then conditioning them until they themselves believed there was a difference between the compounds seems a little like begging the question. I'd be interested to hear how long it took for the subjects to be sure they could tell the difference, whether there were any subjects that just didn't think they could tell the difference, and what then happened to those subjects.

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10. Derek Lowe on February 5, 2013 1:20 PM writes...

#7 Istvan, here's a reference: http://www.sciencedirect.com/science/article/pii/0378517384900577

"Isotopic effects are demonstrated in the lipophilicity, measured by shake-flask and HPLC methods, of a series of deuterated aromatic compounds. The results indicate that deuterated compounds are less lipophilic than their protium isomers by about −0.006 per deuterium atom on the log Poct scale. This isotopic effect is satisfactorily accounted for by differences in molar volumes of isotopomers. The partition coefficient of benzene and toluene is critically evaluated in view of the volatility of these compounds."

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11. Henning Makholm on February 5, 2013 1:27 PM writes...

@7 RM: As far as I understand the quotes, the test was not just whether they could get the subjects to agree/believe that there is a difference (all sorts of problems with that), but whether subjects to learn the difference well enough to reliably tell the deuterated samples from the standard ones under double-blind conditions. If that protocol was followed as described, there doesn't seem to be much room for self-deception or other placebo effects.

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12. Mike W on February 5, 2013 1:40 PM writes...

What I find interesting about this controversy is that Vosshall and the other defenders of the default (null) hypothesis have essentially zero basis for their hypothesis - it is just an assumption that smell receptors are like other GPCRs in recognizing shape. Yet they often vociferously go after Turin. I don't find this stance scientific. Science consists in gathering evidence and testing hypotheses; in the absence of such the correct stance is "I don't know," not "the null hypothesis is true, dammit, nah nah nah I don't hear you."

The opsins work by translating photon-triggered isomerizations of (essentially) retinal into conformational changes of the proteins and release of retinal, and yes, shape is important here. It doesn't seem too ridiculous to think that a probably metal-large-ligand trigger could induce a conformational change of a protein, possibly through a high-low spin transition, with VAT involved or not. Ultimately, shape would again be important. The issue is translating another type of input into a shape change.

The point is, whatever we may think the possibilities are, we assign the null hypothesis to "shape, and shape alone" for no real reason. The "celebratory editorial" states that Turin's theory was assigned as much closer to unicorn status than goat. I'm not sure why. The prior evidence against his theory is what, exactly?

I do not have any opinion as to the validity of Turin's ideas, but I see nothing that would incline me to reject his ideas out of hand. As any organic chemist knows, there are plenty of very different compounds that smell similar, and structurally similar compounds that smell different. That alone makes me interested in seeing more tests of Turin's theory, as opposed to simply going on believing something else for which there is no data.

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13. Pete on February 5, 2013 1:48 PM writes...

Did they check the tritium levels in the deuteriated compounds?

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14. Henning Makholm on February 5, 2013 2:12 PM writes...

@Mike W: Perhaps any explanation that involves "vibrations" just smells too much like homeopathy for some people's tastes?

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15. leftscienceawhileago on February 5, 2013 2:18 PM writes...

A minor point, but I am pretty sure they meant "isotopologue" and not "isotopomer".

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16. Mike W on February 5, 2013 2:25 PM writes...

@Henning: I've never heard anyone around this issue mention homeopathy, thank goodness. Turin has no doubt GPCRs are involved, of course, the question is of their mechanism of triggering.

I assume "smells too much" was unintentional?

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17. Henning Makholm on February 5, 2013 2:40 PM writes...

@Mike W: Naturally. The idea that anyone posting comments on a blog would ever intentionally word his comment in a playful manner is preposterous.

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18. newnickname on February 5, 2013 3:59 PM writes...

@various: As a grad student, I was at first amazed to learn that you can separate C-deutero and C-protio isomers by HPLC, so there is some strong evidence for a difference in weak interactions with solid - liquid phases. And you can separate deuterio-diastereomers from each other.

The pK acidity of hetero-D and hetero-H is also different. Once bound to a receptor, if pK or H/D bonding is important for signalling, there you go again.

If people can detect an H/D odor difference (I'll be off to the lab to test that soon), does acknowledging a phys chem difference between H/D compounds as above mean that Turin is correct? I don't see weak forces (HPLC example) being vibrational. I guess that delta-het-(D/H) acidity could be treated that way, but that's more of a stretch than a vibration.

Specifically, to DMSO: Aren't there sometimes decomposition products in DMSO accounting for most of the odor? Then, 99% DMSO-d6 from an ampoule might be more pure and not smell the same as that 2 year old bottle coming out of the safety cabinet with all kinds of adsorbed vapors therein.

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19. David Borhani on February 5, 2013 4:42 PM writes...

@Curious Wavefunction: was it clearly demonstrated that the DMS and DMS-d6 were identically "pure" --- apropos to @newnickname's comment, DMSO smells "bad" due to minor impurities.

I think the PlosOne paper lacked a few possible alternative explanations, and they missed a serious opportunity to add weight to their argument:

1. They claim, but do not demonstrate, that the conformational ground states (and [though not mentioned] any easily accessible energetically excited conformations) of protio- and deutero-musk are the same. Yet, they also note that deuteration significantly alters the mobility of the musk compound in a gas chromatograph (GC). The altered mobility could be due (as they posit) to (quantum) dispersion effects (essentially, electron–electron correlations). But if that's true (i.e., altered affinity of the deuterium atoms for the GC substrate material, which seems certainly possible), then it's also true that the deuterium atoms in the musk compound must have altered affinity for themselves, i.e. the (ground state) conformation might change (or the proportion of ground & excited states might change, or which excited state conformation). Different conformation = different smell, long ago conclusively demonstrated (e.g., the carvone enantiomers: one smells of caraway, the other spearmint). And of course, the altered dispersion effects could also differentially affect the affinity to one olfactory receptor compared to another. Tickle different receptors differentially, and you've got a different smell.

2. Acetophenone is conformationally rather boring compared to the musk macrocycle. Pretty much all acetophenone can do is twiddle its methyl group, or point the carbonyl group in or out of the benzene ring plane. By contrast, conformational analysis of macrocycles like the musk was (and is) a challenging area, and led to, for example, one of the programs that got Schrodinger started, Clark Still's Macromodel. So, no surprise there.

3. Missed opportunity: What they need to do is to demonstrate that a conformationally "rigid", i.e., an odorant with only one "conformation", changes its smell upon deuteration. Then I'll be more convinced (though affinity changes could also still play a role). Camphor would be a great test system: it is quite rigid, and it has a very distinctive smell. The trick would be perdeuterating it, I suppose, but there are likely similar systems that might be more amenable.

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20. Curious Wavefunction on February 5, 2013 6:01 PM writes...

@19: Good point, but are there *any* cases where deuteration caused a measurable difference in ground state conformations? I would be most interested in any such study.

In addition I am not sure how much difference even dissimilar conformations would make, as long as one molecule can access the other molecule's binding conformation; after all energy differences between conformations can easily be quite small and surmountable, 2-3 kcal/mol at most, and deuteration might simply alter the distribution of conformations rather than their identity. But I agree, a conformationally rigid molecule would take one confounding variable out of the picture.

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21. gippgig on February 5, 2013 9:38 PM writes...

Is it possible that changing the vibrational frequencies of a molecule would affect its preferred conformations? That would be consistent with an isotope effect in cyclopentadecanone (lots of possible conformations of these macroring compounds) but not acetophenone (hardly any) - but then there shouldn't be any effect for methanol (conformations - what conformations?) (#2).

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22. gippgig on February 5, 2013 9:42 PM writes...

Oops, I see #19 beat me to this. Should have hit refresh before commenting...

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23. ama on February 6, 2013 2:47 AM writes...

as #18 said shape is only one part of receptor recognition, electrostatics is another. On top of this affinity is further dependent on conformational freedom of the ligand and the protein, as entropic effects (desolvation ...). A change from H->D could (besides what #18 listed) have a subtle effect on affinity as kinetics (kon/koff). This could influence the signaling (pathway) of the GPCR. The vibrational mode would be in this case a descriptor (a measurable observable) for this difference.

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24. David Borhani on February 6, 2013 9:16 AM writes...

@20, @23: I suspect a few kcal/mol would be all it takes. If there is only one receptor for musk, 1.4 kcal/mol difference (say, biasing one conformation over another in the perdeuterated musk) could equate to a partial vs. full agonist. Same olfactory neuron gets stimulated, but how is that altered signal (different spike frequency, perhaps) interpreted by our brain? With more than one receptor --- i.e., several olfactory neurons --- the signal could easily become more complex, i.e. a different perceived odor.

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25. Mark_cresset on February 6, 2013 10:47 AM writes...

Whatever the deutero-effect turns out to be, it's not just limited to olfactory receptors. I worked on an ion channel project at a big pharma where we were aiming for a complicated profile of agonist/antagonist/inverse agonist against different subtypes. One of the lead compounds had a CD3, added to improve half life (kinetic isotope effects!). The biologists swore blind that the CD3 compound had a different profile to the CH3 one - tested over multiple different batches. This leads me to believe that if deuterated compounds really do behave differently then it is a more general feature of ligand-protein interactions, rather than some magic property of olfactory receptors.

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26. luca turin on February 6, 2013 6:41 PM writes...

Very interesting and informed debate, for a change. Thanks everyone !

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27. eugene on February 7, 2013 5:38 AM writes...

I agree with number 25. Does a protein detect a substrate via vibrations then, if a deuterium analogue has such a different activity profile? That natural systems process isotopologous compounds at different speeds from their non-labeled analogues has been known for a long time.

I can say to the same effect, that smell is caused by quantum tunneling and the wavefunction of an H atom. The D is less likely to tunnel over to some other conformation when it's inside the receptor, and therefore there is a different smell.

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