Carrots to reduce harmaline to THH?

[modern]

So I spent week testing this idea I had since I read about carrots enzymes reducing acetone to secondary alcohol and read up more and saw zinc/magnesium is used to preform the same thing. So in my mind I thought it would work to convert harmaline to THH. On a whim I tossed some carrot shaving into a acidic solution with what I thought was harmaline and left it two days and after removing the carrots and filtering well the color was BLUE and not the green from harmaline.
So then I decided to repeat the experiment with a carrot water to test black light, a magnesium control and a carrot test. No black light shine on carrot water.
I separated the harmine at 7-8 ph and the rest at 12ish. Then proceed to split the harmaline in two equal volumes and started the experiment.
Left everything for two days and before proceeding with filtering and precipitating the colors of the two did match with a blue color.
So to precipitate the thh from magnesium I did masake and got a highlighter yellow powder. and the carrot experiment I just precipitated as normal... it didn't precipitate at 10 but only at 12-13. I thought this was a good sign. I redissolved the hcl in acidic water and precipitated the same way... Also only precipitated at 12-13 range.

The crystals from the magnesium reduction were the nicest I've had yet. (pics)

Now to test I was originally going to do melting point test but since I was working with such small amounts I decided to do black light test after all the filtering. After making an equally acidic solution with citric acid rather than vinegar I placed the filter papers into separate beakers side by side. The magnesium control was a nice blue tone but the carrot was still green. There may have been some conversion however I don't have the ability to test. Visually I'd say it didn't convert however I'm not sure how strong the harmaline color is over THH. Would a 70% conversion of the harmaline still shine green? It wasn't the most noticeable difference but was still greener.

If someone with TLC paper would like to verify or someone with better just explain it doesn't work.

Carrots (Daucus carota)

 

[Transform]

So I spent week testing this idea I had since I read about carrots enzymes reducing acetone to secondary alcohol and read up more and saw zinc/magnesium is used to preform the same thing. So in my mind I thought it would work to convert harmaline to THH. On a whim I tossed some carrot shaving into a acidic solution with what I thought was harmaline and left it two days and after removing the carrots and filtering well the color was BLUE and not the green from harmaline.
So then I decided to repeat the experiment with a carrot water to test black light, a magnesium control and a carrot test. No black light shine on carrot water.
I separated the harmine at 7-8 ph and the rest at 12ish. Then proceed to split the harmaline in two equal volumes and started the experiment.
Left everything for two days and before proceeding with filtering and precipitating the colors of the two did match with a blue color.
So to precipitate the thh from magnesium I did masake and got a highlighter yellow powder. and the carrot experiment I just precipitated as normal... it didn't precipitate at 10 but only at 12-13. I thought this was a good sign. I redissolved the hcl in acidic water and precipitated the same way... Also only precipitated at 12-13 range.

The crystals from the magnesium reduction were the nicest I've had yet. (pics)

Now to test I was originally going to do melting point test but since I was working with such small amounts I decided to do black light test after all the filtering. After making an equally acidic solution with citric acid rather than vinegar I placed the filter papers into separate beakers side by side. The magnesium control was a nice blue tone but the carrot was still green. There may have been some conversion however I don't have the ability to test. Visually I'd say it didn't convert however I'm not sure how strong the harmaline color is over THH. Would a 70% conversion of the harmaline still shine green? It wasn't the most noticeable difference but was still greener.

If someone with TLC paper would like to verify or someone with better just explain it doesn't work.

Carrots (Daucus carota)

Well, this could be revolutionary if you've uncovered the secret of biochemical conversion of harmaline to THH. I'd hesitate to get over-excited just yet. It's still necessary to eliminate dilution factors and/or the presence of confounding factors like certain metal ions that may change the emission colour of harmala fluorescence, although the lengths that you've gone to in separating the alkaloids, along with the pH where precipitation occurred are rather encouraging.

Perhaps it would be worth trying the experiment again, maybe with a little extra ascorbic acid added, or one of the other cellular reducing agents. This could be fine-tuned by carefully exploring the exact chemical pathway and supplementing the necessary reductants and co-factors, like NAPDH, reduced biotin, and the like. Maybe it would be possible to speed up the reaction or increase the yield; the 70/30 ratio that you estimate would be the logical outcome of an equilibrium situation. Without removing the THH as soon as it was formed (which is essentially impossible in these circumstances) the reaction would have to be driven by stacking extra reducing agents on the input side of the equation.

If you really can't do any chromatography (check out enhanced paper chromatography before you write off the idea entirely), you can still attempt a pH-based separation as per the Van der Sypt (VDS) protocol.

Would a 70% conversion of the harmaline still shine green? It wasn't the most noticeable difference but was still greener.

Addressing this particular question, harmaline fluorescence is much stronger than that of THH and would tend to dominate even as a relatively low proportion of the mixture.
Anyhow, great bit of work, very intriguing. This trick will be shoehorned into the top of my to-do-list!

 

[modern]

I found it a very interesting idea however I am unable to test much further. I've always been interested in TLC papers but it is outrageously expensive here due to import taxes and such. I've also tried seperating thh and harmaline via ph but being so close to each other I've had difficulty? Maybe just lack of enough experience.

I've also used caapi as the starting material which tend to be on the lower end of harmaline? The best method would be using syrian rue which outside of brasil is the cheapest option.

The 70/30 conversion I guessed is based on the results of the paper being 50-90% depending on a few conditions. In any case this is a vary easy experiment to test so if someone with the ability to use tlc plates or machine to see end result components would very interesting.

The paper was very interesting to me and had me thinking many 'additives' that we've read about being added to brews being a form of alchemy to convert or remove certain compounds even when the plant is inactive.

 

[Woolmer]

This is cool! Here is a nice video that repeats the experiment from the paper.

If this works for imine reduction could it be used instead of NaBH4 in reductive amination of tryptamine?

 

[Transform]

This is cool! Here is a nice video that repeats the experiment from the paper.

I saw that video when it first came out - now that you remind me, this was nagging away in the background as sounding familiar. All the more props to @modern for actually trying it! It raises the question of what other plant material will aid desired transformations in a reaction/brew mixture, making @modern's comment all the more relevant:

The paper was very interesting to me and had me thinking many 'additives' that we've read about being added to brews being a form of alchemy to convert or remove certain compounds even when the plant is inactive.

This could lead to a huge leap forward in ayahuasca brewing science, and with plant brewing in general.

If this works for imine reduction could it be used instead of NaBH4 in reductive amination of tryptamine?

@Woolmer - the obvious thing to try would be NMT with acetone and carrot peel (and I think this synth talk is allowed since we're not using dangerous or watched materials). If your hypothesis turns out to be valid, the product would be MIPT... However, I do note that the two examples given consisted of reductions of a ketone group to a secondary alcohol.

The putative reduction of the imine group harmaline could be easier because of the proximity of the aromatic indole moiety. A standalone imine such as the NMT/acetone adduct might be less amenable to reduction via this method. That's no reason not to try the experiment, of course - but it should not be too much of a surprise to see the formation of a considerable amount of IPA as a byproduct. It would be a case of optimising the reaction conditions, perhaps.

I've always been interested in TLC papers but it is outrageously expensive here

Check that thread again, all you really need is blotting paper, a couple of sheets of glass, a solvent-resistant container, and the correct solvent blend.

 

[Woolmer]

The obvious thing to try would be NMT with acetone and carrot peel (and I think this synth talk is allowed since we're not using dangerous or watched materials). If your hypothesis turns out to be valid, the product would be MIPT... However, I do note that the two examples given consisted of reductions of a ketone group to a secondary alcohol.

The putative reduction of the imine group harmaline could be easier because of the proximity of the aromatic indole moiety. A standalone imine such as the NMT/acetone adduct might be less amenable to reduction via this method. That's no reason not to try the experiment, of course - but it should not be too much of a surprise to see the formation of a considerable amount of IPA as a byproduct. It would be a case of optimising the reaction conditions, perhaps.

I found this honors thesis that tried to reduce imines using pea enzymes. They say the pea mixture caused the solution to become too acidic thereby favoring hydrolysis.

Only using 0.2 M sodium phosphate buffer did they get some reduction but hydrolysis was still the major product. How could one further optimise these conditions? Lower temperatures? Addition of co-factors as you mentioned @Transform? Add acetone in stoichiometric amounts in an aprotic solvent with the amine? Monitoring pH to stay around 5 (where imines are more stable)? Sorry if these are silly questions but also just musing to myself while learning the chemistry.

They also cite papers that successfully used other vegetables to reduce ketones -- Arracacha, beetroot, coriander, yam's had high conversion %. Could be worth trying out.

I will try the harmaline reduction when I get time. Nice find @modern !

 

[Transform]

I found this honors thesis that tried to reduce imines using pea enzymes. They say the pea mixture caused the solution to become too acidic thereby favoring hydrolysis.

Only using 0.2 M sodium phosphate buffer did they get some reduction but hydrolysis was still the major product. How could one further optimise these conditions? Lower temperatures? Addition of co-factors as you mentioned @Transform? Add acetone in stoichiometric amounts in an aprotic solvent with the amine? Monitoring pH to stay around 5 (where imines are more stable)? Sorry if these are silly questions but also just musing to myself while learning the chemistry.

They also cite papers that successfully used other vegetables to reduce ketones -- Arracacha, beetroot, coriander, yam's had high conversion %. Could be worth trying out.

I will try the harmaline reduction when I get time. Nice find @modern !

Yeah, it all gets a bit tricky there - aprotic solvents risk denaturing the enzyme, so it's risky taking the aqueous 'potency' much below that of plant physiological conditions. Having an excess of the un-iminised starting amine in addition to the imine would possibly help drive the reaction, as well as helping with the pH. Harmaline, of course, has the advantage of being a stable imine. Lower temperatures might reduce the efficiency of the catalyst; it would be prudent to list all of the factors that might be involved in optimisiing the reaction, and looking at where trade-offs have to be made.

As far as co-factors go, having a brief scan of the literature shows that glucose appears to be the most readily available feedstock, although that would possibly compete in the imine formation. Carrots do come pre-packed with a certain amount of glucose anyhow.

I think we should all look at replicating the harmaline reduction for now, before trying things like optimising the reaction and branching out into different substrates and vegetables. I do wonder, though, if the intrinsic biochemistry of a given plant can be used to guide viable choices for particular types of reaction. Isolated enzymes have been used as reagents for quite some time now so to some extent it's a matter of trawling through the literature and joining up the dots - or making friends with the right enzymologist/biotechnologist.

The other thing I'm thinking is that a flow reactor would probably be useful for this type of work. I reckon this could be as simple as running an aquarium pump to circulate the reaction mixture through a carrot-packed column ( ) Continuous monitoring would enable pH correction as well as keeping an eye on reaction progress. Maybe an inert atmosphere would help prevent the reverse (oxidation) reaction too?

 

[Josh.lobbs]

The colour difference might be because the carrots reduce the harmaline to a specific enantiomer.

from my understanding the carrots have an enantiomeric excess of 99% so you might have a sample of 99% (S) or (R) Tetrahydroharmine

 

[Transform]

I tried this recently in an impromptu fashion what with rue tea and carrot peelings being ongoing features of my kitchen. The carrot peel was surprisingly happy about standing, unrefrigerated, for two days in the rue brew, although the liquid itself got turbid. The odour of the liquid was, if anything, improved. This could be considered fortunate since the carrot was scrubbed but not sterilised. [The follow-up experiment will involve carrot strips peeled after sanitising the vegetable with IPA.]
A small portion of untreated rue brew was reserved for comparison.

The yellow colour of the rue brew was greatly diminished after carrot treatment.

This is not altogether surprising, since the carrot strips also absorbed a significant amount of fluorescent material, which became apparent after inspection with a near-UV light.

The fluorescence of the treated brew was both diminished in intensity (probably in large part due to the adsorption of material by the carrot tissue) and shifted in a bluish direction, which was much more apparent to the naked eye. Future tests will be recorded using the camera with colour correction turned off in an attempt to capture this colour shift more effectively.
First picture is with the 'spy pen' light, second and third are with the Blufixx torch:

Use of a yellow filter highlights the diminished fluorescence over the colour shift:

The treated brew seemed to revert to a more greenish fluorescence after boiling, but this may be due to the increased concentration of harmaline upon evaporation.

More reports of further experiments to follow, as and when they occur.

Addendum: with more attention to hygiene, I may yet try consuming the carrot shavings after the soak, if the brew appears to be in good sanitary at that point. I'm in two minds whether to try any of the current "carrotised" brew, even though it smelled much better than a usual brew (which, admittedly, isn't saying much - wet dog is an easy aroma note to beat) but the dominant coffee and carrot notes were quite appealing and also without a single unpleasant undertone. The major off-putting factors of the raw, carrot-treated brew were the turbidity, the viscosity, and the fact that it was obviously alive with some unknown range of microorganisms; it's also kind of disappointing that the brew smells nowhere near as nice after a prolonged simmer.

 

[Transform]

it's also kind of disappointing that the brew smells nowhere near as nice after a prolonged simmer.

I forgot the pan on the stove while reducing it down a bit (well, more the intention was to bring it to a boil to knock out any bad microorganisms before having a taste). The carrot + harmala odour apparently makes a nice incense - my lady friend commented positively on the scent afterwards and was quite amused by my explanation.

Anyhow, I still hope to repeat this experiment as outliined in one or more of the posts above.

 

[Woolmer]

Enantioselective Bioreduction of Medicinally Relevant Nitrogen-Heteroaromatic Ketones

Purple carrots worked best for these guys!

 

[acacian]

@Transform Syrian Rue seed is actually used as an incense too so that makes sense.. but perhaps carrot is the missing ingredient. A least by burning it you can be sure you knocked out any bad microorganisms

 

[Transform]

Enantioselective Bioreduction of Medicinally Relevant Nitrogen-Heteroaromatic Ketones

Purple carrots worked best for these guys!

I shall be holding out for purple carrots - that will be the sign for action!

 

[Transform]

@Transform Syrian Rue seed is actually used as an incense too so that makes sense.. but perhaps carrot is the missing ingredient. A least by burning it you can be sure you knocked out any bad microorganisms

If anything the carrot added a sweet floral note to the mix so we could be onto something here. As far as eliminating microorganisms goes, this was probably overkill, however. You've now set me thinking whether it's worth trying a bit of microscopy to check for endospores in the carbonised residue, yet another project I can't afford to embark upon.