THH isomers - Synthetic and Natural THH differences?

[Mindlusion]

...then 2 isomers would form....these would be not distinguishable by gc/ms.

If that is true, why would that happen? We don't understand in vitro here...e.g. why would it not be partly natural 'in vivo' THH and partly racemic THH due to perhaps presence of reducing agents found in river water and/or another component matter (aluminum vessel...etc)?

I am not sure how the reduction to THH would occur during the boiling of the ayahuasca, perhaps it is being reduced by something in the plant itself, a protein or enzyme (reductases).

Indoles can oxidize molecules through a similar mechanism I posted above. In particular the beta-carbolines, the methoxy group pyrrole and amine nitrogens can donate electrons from their lone pairs and distribute the density by resonance. There must be something that can pick up those readily available electrons to break the pi-bond, but it looks like it would be energetically favored.

Formates are known reducing agents in vivo, catalyzed by an enzyme they are decarboxylated to form CO2 and hydride which acts as a reducing agent. Interestingly enough to reduce iminium ions! Of course, boiling solution obviously denatures proteins and enzymes, but I suspect something of this nature is happening.

Here is a link: Formic acid

To answer your question, THH becomes racemic in vitro because when it is in aqueuous solution such as in ayahuasca brew, it is protonated. The epimerization mechanism can occur to give you both enantiomers. The plant (in vivo) seems to produce the d-isomer selectively.

 

[downwardsfromzero]

Seeing as recent links have pointed me here, this seems like as good a place as any to post my thoughts on the process of THH and harmine formation during prolonged boiling of a harmaline-containing brew. The keyword here is disproportionation.

So, my thinking is that two molecules of harmaline will simultaneously oxidise and reduce each other to produce a molecule each of THH and harmine. Whether this requires the catalytic presence of harmine or THH we don't know. What this does suggest is that subjecting chromatographically pure harmaline to appropriate conditions (whatever they may be) would be instructive.


Inasmuch as the epimerisation of THH is concerned, is there really a decyclisation involved? Would isotopic labelling be of use in distinguishing this from other mechanisms? Attached is an image showing epimerisation mechanisms without decyclisation. Under acidic conditions the pathway on the left might apply, under basic conditions the slightly less plausible pathway on the right could apply. It's worth knowing that indoles are fairly readily protonated at the 3-position. This will undoubtedly contribute to the ease of epimerisation we see with THH.


EDIT: it seems that there's a "-H+" missing half way down on the left side of the diagram.

 

[ikra]

Hey , hope u doing well , please there's a reference of blue fluorescence of harmine ? i need that référence please .

 

[Brennendes Wasser]

Hello

You want to know if Harmin indeed has a blue fluorescence to verify if a THH conversion mostly gave pure THH?

So in general you will see the following:

THH = no fluorescence
Harmalin = green fluorescence (medium strength)
Harmin = blue fluorescence (very high strength)

But sadly if you cannot separate your sample into these 3 substances like with a TLC, then all 3 together will mostly just give you a blue fluorescence.

And as you would mostly carry over traces of Harmin, that means everything I checked in the Black Light was just either strongly blue or light blue ... not easy to determine if you have pure THH (if that is what you are searching for).

Even Harmalin itself is more looking green-blue to blue.

As I just made this today here is a picture of everything.

1 = THH + Harmalin + Harmin (but still mostly THH)
2 = THH (recrystallized 2x from Ethanol, only traces of other 2)

Now if you dont separate them then you would get this below the blacklight. 3 different wavelengths from high energy (254 nm) to low energy (360 nm). Probably black light LEDs you can buy online will be even 405 nm only to not cause any potential damage to eye.

As you can see there is never a big difference. At 254 nm everything is black. At 320 nm and 360 nm the mix with all 3 (#1) just glows a little stronger blue, but the MOSTLY THH (#2) is also quite a little blue due to traces even though it was recrystallized 2x.

The only way to really make a difference between them is to do a TLC. That one used the solvent mix by Mindlusion (4:1:0,1 Methanol:Ethyl Acetate:NH3). You can now take a look at the substances separately. THH is at the bottom, but Harmin just above, Harmalin will be completely on the top.

Now you can see the following:

THH @ 254 nm = (weak) black
Harmalin @ 254 nm = black
Harmin @ 254 nm = dark blue

THH @ 320 nm = (nearly) invisible
Harmalin @ 320 nm = light blue
Harmin @ 254 nm = (strong) medium blue

THH @ 360 nm = (nearly) invisible
Harmalin @ 360 nm = green-blue
Harmin @ 360 nm = (strong) light blue

So you see if you have a mix of all 3 it is hard to get any ideas. Even traces of the other 2 will make it look blue under the blacklight. Harmalin is a little green compared to Harmin ... but when you have ONLY Harmalin I would also call it blue. Just with Harmalin compared to Harmin it has a slight more green colour.

The only thing which might be feasible (to verify purity of a THH conversion) is to weigh the SAME AMOUNT of Harmalas vs. your THH reaction product. Like 10 mg on 1 ml is good. Then put a paper into each of these and compare the fluorescence in the blacklight. If fluorescence has become WAY less than probably there is not much of Harmin/Harmalin left. But even if you still have 1 % of each it will give you that blue fluorescence. :|

 

[endlessness]

It's the inverse, it's harmaline just above THH, and harmine is toward the top.

 

[Brennendes Wasser]

Oh man screwed that whole stuff already twice in a row :roll: :roll:

Thanks I probably will never get that difference. So now it's corrected ...

Also makes more sense chemically = closer structural resemblance of Harmalin + THH should give them more similar properties.

And Harmin has the biggest Chromophor with 3 aromatic rings, so it should also have the strongest absorption / fluorescence at the highest wavelength. :thumb_dow


PS:

I was writing this only to give a visual explanation about fluorescence for Ikra. But now I see that the first post is even also exactly about that topic and even has some much better-looking pictures But then this is a much better reference, but then there is a real proof of the fluorescence anyways?

So maybe Ikra can explain again what she/he means if that is not the answer, if she/he might come online again ...

 

[An1cca]

I would suggest microscopy for the identification of the different alkaloids. Pure and mixed samples can easily be evaluated following in-situ recrystallization with ammonia. And they're beautiful to look at as well.

 

[downwardsfromzero]

Looking back through this thread, two questions still deserve definite answers:

- what is the yellow-fluorescing stuff in Endlessness' caapi tlc at the same rf as THH? Oleamide seems like a good candidate but a definitive analysis seems desirable. 2-d tlc would be a good place to start - rotate the plates by 90° and run them again with a different solvent system. The yellow stuff and the THH should resolve separately given the right solvents.

- we still don't know if the two enantiomers of THH have different pharmacology - or if THH gets racemised under physiological conditions. Also, would a cold-extracted caapi THH sample be different from brewed caapi THH extracted after prolonged simmering? Is Callaway's claim based on experimental evidence?

 

[endlessness]

Looking back through this thread, two questions still deserve definite answers:

- what is the yellow-fluorescing stuff in Endlessness' caapi tlc at the same rf as THH? Oleamide seems like a good candidate but a definitive analysis seems desirable. 2-d tlc would be a good place to start - rotate the plates by 90° and run them again with a different solvent system. The yellow stuff and the THH should resolve separately given the right solvents.

- we still don't know if the two enantiomers of THH have different pharmacology - or if THH gets racemised under physiological conditions. Also, would a cold-extracted caapi THH sample be different from brewed caapi THH extracted after prolonged simmering? Is Callaway's claim based on experimental evidence?

So why do you think the yellow stuff is not simply racemic THH afecting the fluorescence?

 

[downwardsfromzero]

So why do you think the yellow stuff is not simply racemic THH afecting the fluorescence?

Excellent question. I thought the yellow stuff was naturally-sourced material. Synthesis using zinc reduction will definitely produce racemic material (presumably you didn't use a chiral auxiliary...) The natural material at least has a chance of being in enantiomeric excess. Running the 2-d tlc should help clear up whether the spots are THH and only THH.

Has anyone else replicated this yellow fluorescence with natural, optically pure THH? Is the weaker fluorescence of the synthed material due to some paradoxical self-quenching that only occurs through the two enantiomers 'fighting' each other?! Or is there something, some impurity, from the reduction process that is affecting the result? Traces of zinc? Dimers?

It would be fantastic to see results from specific tests on separated enantiomers compared with the racemic material, and also borohydride-produced material compared with the stuff made using zinc, maybe even THH synthesised from 6-MeO-T via Pictet-Spengler.

I still find your results very intriguing!

 

[endlessness]

So why do you think the yellow stuff is not simply racemic THH afecting the fluorescence?

Excellent question. I thought the yellow stuff was naturally-sourced material. Synthesis using zinc reduction will definitely produce racemic material (presumably you didn't use a chiral auxiliary...) The natural material at least has a chance of being in enantiomeric excess. Running the 2-d tlc should help clear up whether the spots are THH and only THH.

Has anyone else replicated this yellow fluorescence with natural, optically pure THH? Is the weaker fluorescence of the synthed material due to some paradoxical self-quenching that only occurs through the two enantiomers 'fighting' each other?! Or is there something, some impurity, from the reduction process that is affecting the result? Traces of zinc? Dimers?

It would be fantastic to see results from specific tests on separated enantiomers compared with the racemic material, and also borohydride-produced material compared with the stuff made using zinc, maybe even THH synthesised from 6-MeO-T via Pictet-Spengler.

I still find your results very intriguing!

Thanks for the thoughts!

actually I meant the other way around, the yellow fluorescence being the d-isomer, and the faint shine that later turns dark, being the racemic mixture.

but yeah, more tests needed (as always )

 

[downwardsfromzero]

One more thing, the right chiral-substituted borane should provide a route to enantiopure THH from harmaline. Otherwise it's possibly a matter of column chroma using a chiral substrate. (But not always: Demystifying racemic natural products in the homochiral world - Nature Reviews Chemistry )

In most cases, chiral HPLC is the preferred method for the resolution of mixtures of enantiomers. However, in racemic mixtures, some component enantiomers have been reported as non-separable on chiral HPLC. They are exemplified by selaginellin enantiomers, which can be interconverted in solution77. Chiral resolution of the constituents of the pyridocarbazole alkaloids, stritidas A–C (30–32), failed on a Phenomenex Lux cellulose-2 column20. The phenylpropanoid derivatives, xanthiifructins A/B (enantiomeric forms of 33), were separable on a Chiralpak AD-H column and a Phenomenex Lux cellulose-2 column, but not their analogue xanthiifructin C (34) which structurally differs by the lack of the propenyl moiety on its second phenyl group97. Similarly, the enantiomers of the phenylpropanoid derivative penicilliode A (26) were not distinguishable on many chiral columns (Phenomenex Lux celluloses-1, 2, 3, 4 and 5 and FLM Chiral ND(2)), whereas their analogues penicilliode B (27) and penicilliode C (35) were successfully resolved in their respective enantiomers44. In all, available chiral HPLC columns still fail to distinguish between physical properties of components of certain enantiomer mixtures.

(Stritida A (30) in particular resembles THH fairly closely so Phenomenex Lux Cellulose-2 would possibly be a less desirable choice of column substrate.)


It would be nice to know if there are any other instances of racemic mixtures displaying different fluorescence properties than the pure enantiomers. DL-histidine is less soluble than the enantiopure form, for example ( https://pubs.rsc.org/en/content/articlepdf/2021/ce/d1ce01199e [downloads pdf]) so it is possible to imagine that other physical properties could vary in a similar way - and the intermolecular forces involved in crystallisation could well lead to variations in fluorescence behaviour. In absence of an expert in this specific field, this will require some scouring of the literature.