Hey there! I've been following this site for around 6 years now, and started performing DMT extractions around the same time. Haven't felt the need to make an account before, but recently I've been seeing more and more discussions where I think my input could be valuable.

I'm currently a senior undergraduate in a top-10 chemistry program. My research experience is primarily in nucleic acid chemistry, but I hope to move into neurochem and drug design for my PhD work. I have a fair bit of organic chem experience from my classes, but more from my "private" research--primarily, but not exclusively on DMT extraction techniques and chemistry.

As for my experience as a psychonaut,I've chilled out significantly in recent years, but have fairly extensive experience with most classes of recreational and entheogenic psychoactives. I dislike picking a favorite compound, but if I had to it would have to be psilocin--if you haven't tried hydrolyzing your psilocybin exogenously I'd give it a shot. The compounds are quite similar, but psilocin definitely gives me a cleaner and calmer feeling trip. Other loves of mine include N,N-DMT, of course, ketamine, MDA and 2C-T-7.

I look forward to getting more involved on the site and helping to give back after all the information the nexus has blessed me with

Welcome Eleusis

Welcome to the Nexus!

Always good to have chemists join our forum

What is your method for hydrolyzing psilocybin ?

Have you ever tried DMT smoked during a psilo trip?

Regarding your DNA chemistry knowledge, what's your take on harmalas binding to DNA, do you think there's any significance to that?

Be well!

Glad for having professionals here

Thanks for the welcome

To hydrolyze psilocybin, just take your alkaloid extract, which should be primarily psilocybin and psilocin already (I'm not responsible for whatever strange tryptamines you might make by modifying other alkaloids!) and hold it at 70°C in aqueous condition around pH 3.0-5.0 for 8 hours to overnight. The lower the pH, the faster the reaction will take place, but your fraction of psilocin/psilocybin will suffer. After the reaction, a significant portion will still be in the form of psilocybin. To separate them,add the solution to a sep funnel with DCM. Carefully and slowly raise the pH--psilocin should start to move to the organic layer around pH 9. You have to be careful not to raise it too high, or you'll end up deprotonating the hydroxyl group and it'll stay in the water. I use DCM since it's heavier than water, so I can see when the precipitate starts to form. After getting all your psilocin into the organic layer, you can either precipitate out the remaining psilocybin from your water (the isoelectric point is around 4.0), or, if you had the foresight to use food safe chemicals like lemon juice and baking soda, just keep it around as some mushroom tea for microdosing purposes.

EDIT: Just want to add, psilocin is highly unstable in solutions, especially basic ones. You'll want to get it out of the DCM ASAP and consume it within a few days.

Some of the most powerful mental experiences of my life have come from smoking DMT during mushroom trips. I find it simultaneously makes the DMT trip both much more powerful than it would be otherwise as well as much calmer and more controllable. It's sort of like instead of your rocket blasting you off into outer-space, you're in a space-plane that just casually ascends to the stars.

My research is in RNA structure and catalysis, and I haven't heard of this before. I'll read some papers and get back to you on that. Though I can definitely say there is at least some significance--DNA and beta-carbolines all came from the same tree of life, there's no reason to be surprised they can interact!

Welcome Eleusis <3

Welcome!!

Great to have you join us and share!
Welcome!

Hello and welcome to the forum. How do you precipitate psilocin from DCM and psylocybin from water (sounds like you are doing that if I understand your post). Also, even if you used food safe stuff to do the warm acid dephosphorylation and to subsequently adjust the pH to increase the water/solvent partition, the water with remaining Psilocybin would not make a suitable tea because some DCM will move into the water (giving water with ~2% DCM), right?

In the literature the reason given to not go above pH9 is not because of depronation and lower solvent solubility (that won't happen until after pH9.4, but because of rapid oxidation in a basic environment, as mentioned in Cassales' paper where they are 1 full unit below the isolectric point and minimizing oxydation before optimizing solvent solubility). If my understanding is off let me know (thanks).

Also, in the literature it is generally accepted that a one hour warm acid bath essentially converts all the psilocybin into psiclocin (typically vinegar at 70C is used). Are you sure you have Psilocybin remaining?

I've attached a paper that goes over some of these points and/or has references to papers that do.

EDIT: To be clear, I am not precipitating psilocin from DCM. I evaporate off the DCM and do a recrystallization from water (water recrystallization--so unusual you do this!). The psilocybin should precipitate out of water at its isolectric point around pH 4.

You're right, DCM is soluble in water at about 25g/L. If you intended on keeping the aqueous layer to drink you'd want to use ether or something else nontoxic as the organic solvent. I should've been specific that DCM doesn't qualify as food safe In my own use I've kept around the solution,dissolved more extract if necessary, and put it through the acid-cat hydrolysis again to maximize yield.

If you are relatively quick, you shouldn't have to be too concerned about base-catalyzed destruction of your molecules. Just be sure to neutralize it again after getting the psilocin out. Though you can certainly keep pH lower if you'd like, that paper had a goal I didn't--they wanted to quantitatively determine the concentrations of alkaloids, so were avoiding destroying any at all. For me, that wasn't as much of a concern. I was more concerned about obtaining a quality separation of the PSOP and PSOH.

The reason I spend so much time on hydrolysis is simple: I'm not sure how long it takes!Better safe than sorry. And there is definitely still psilocybin present. Reaction rate is independent of ratio of products/reactants at equilibrium (K). I've used GC to check on the composition of my extracts. Could you point me to the source on the hydrolysis going to completion? Maybe I'm doing something wrong.

Thanks for your answers, now it makes more sense. However, I still don't get some parts of your answer:

I have not seen psylocybin precipitate cleanly from water from a simple mushroom extract. Sure, solubility in water is minimized at pH4, but you if you start evaporating/cooling the solution and other gunk precipitates first or with it. Same if you try to salt it out with ammonium sulfate or acetone. I've also tried to crash the gunk out at a pH a few units away from 4, then go back to 4 and get psilocybin to precipitate without success. If you have a way to precipitate it out of water it would be great if you could share the details.

Regarding the pH not wanting to be above 9, if you wanted a good separation without concern for destroying product wouldn't you want to get to pH 9.37 (above 9)? You said don't go above 9, but I think you mean stay within 0.5 pH units of 9.37 (or something along those lines). The only reason to not go above 9 is to avoid oxydation since 9.37 is the ideal target for solvent extraction. Or am I missing something? Maybe you are just rounding that could make logical sense (although 9.5 is closer to the pI than 9).

Regarding dephosphorylation effective completion time, See text below (from "An aqueous–organic extraction method for the isolation and
identification of psilocin from hallucinogenic mushrooms." ) for a claim that all psylocybin is converted with an hour soak in dilute acetic acid followed by heat (the hour soak is described in the main text). Full paper is here. The mushroom enzymes may play a role, what species where you using when you found Psilocybin still present?

Thanks so much for that paper! I've been looking for something like that forever.

You can definitely get psilocybin to drop out of water. Isoionic precipitation can require extreme precision, but with dilute NaOH from a burette dropwise you should be able to do it, even with relatively low concentrations of PSOP.

And yeah, I think the point of minimum solubility on PSOH is about 9.4, I more meant to be careful with it. My post wasn't intended as a full recipe, just a quick sum-up of my method from memory.

Due to security reasons, I'd rather not share the mushroom used. It was a regional species that would narrow down my location quite a bit.

Alright, that clears other things up, thank you. You can see we can be pretty rigorous here, but it's all in the interest of having valid information with unknowns and assumptions spelled out.

As far as psylocybin dropping out, I was wondering/asking if it can be done without anything else crashing out with it. I guess there could be a window if one knew the conditions at which psylocybin starts to precipitate, Do you have more details on that? I've played around with mushroom extracts at 50% and 30% ammonium sulfate and varied the pH, unfortunately nothing new seem to crash out near pH4. It could have fallen out at a different pH along with other proteins because I overdid the ionic strength though.

I can imagine that in the pH vs ionic strength plane there may be a path that one can take to end up at a point were only psylocybin precipitates. Knowing the temperature effect on both gunk and psylocybin would also help.

Do you have any details on the isolectric precipitation of psylocybin (e.g. ionic strength and temperature precipitation boundary phase curve while at pH4)? Or better yet, have you made psylocybin from a mushroom extract precipitate from water (or methanol), and if so how did you do it? Thanks.

I haven't done anything quantitative on mushroom alkaloids, but maybe I should Once I'm finished with undergrad, I should have a bit more financial freedom to play around with.

As for other things crashing out with psilocybin, that typically isn't a problem with isoelectric precipitation given it's specificity. You shouldn't have many proteins or free peptides in your extract after an A/B extraction + re-crystallization anyway. If you have an accurate way of measuring pH, you can always go back and forth past the IEP with NaOH and HCL--this is a convenient way of increasing the polarity of the aqueous layer slowly while maintaining appropriate pH. If that's too slow for you, nothing wrong with just adding NaCl, just be careful, you can salt out everything if you add too much!

Well after A/B and re-x precipitation, isoelectric precipitation is not as interesting since you would have done all the hard work already.

The breakthrough for a home extractor would be to do a water extract tea and then in the next step be able to get psylocybin to precipitate alone by adjusting the ionic strength and pH (and temperature). That would be a significant breakthrough.

I will try again sometime, this time being more carefull. I believe ammonium sulfate has more precipitation power vs NaCl because of the Amonium and Sulfate ions hydration layer (position in the Hofmeister series). So, the idea would be to start at say 10% ammonium sulfate at pH 4 and add ammonium sulphate slowly. The pH may go up while doing so, then use sulfuric acid (car battery supply) and/or ammonia (from household cleaning) to stay at the isolectric point. The concern I have is the psylocybin being very tangled up (or even in a bond) with proteins and enzymes. Another difficulty would be psylocybin converting to psiclocin at pH4 when enzymes are present before crashing out, but maybe those can be dentatured up-front while making the tea with a slightly alkaline boil.

In short, the mushroom extract is a very interesting system with a lot of challenges. Even when attempts at isolation fail (repeatedly ) it is fun to just try to figure it out.

Unless you're trying to do an extract without changing pH (which seems like an exercise in frustration) I don't think you should be as concerned about interactions with proteins as you are. Pretty much all proteins are going to be
a)Denatured above 40C
b)Hydrophilic (the portion of membrane and other hydrophobic proteins is quite small)

Additionally, the Hofmeister series is particular to proteins, and based on interactions with the negatively charged peptide backbone. The solubility effects are going to be quite different in tryptamines, which are typically (psilocybin is an exception here though--maybe we can find a way to exploit that) positively charged at neutral pH. On top of that,higher salt concentration has a stabilizing effect on protein folding, which consequently has a major effect on their solubility. You wouldn't see any of this in tryptamine molecules.

(After writing the above paragraph I did some research and found an excellent paper on this. It seems although they don't align perfectly, the Hofmeister series is still a pretty accurate scale for smaller organics. Though ammonium sulfate would still not be a great choice because of how reactive it is. Sodium sulfate perhaps? Paper:https://pubs.acs.org/doi/full/10.1021/acs.oprd.7b00197)


And yeah, you definitely couldn't use isoelectric precipitation as a first round method of extraction--far too fickle to apply in a chaotic biomaterial environment. I still think you can't really beat A/B pulls for this sort of stuff, at least not as a first round extraction.

I question the need for a easier method of getting concentrated alkaloids--if one is really interested in the chemical details, they need to put in the effort to understand it anyway, and if one's just looking for a good time, some lemon-mushroom tea should serve them just fine.

Trust me, an easy kitchen method of getting concentrated mushroom alkalodis would be welcomed and useful. Turning a bag of dry mushrooms in the freezer into a loose stable powder that can go in one's personal natural medicinal cabinet next to harmalas, DMT, salvinorin, 5-MEO, bufo, and mescaline would be pretty neat.

I think we are agreeing and aligned at this point. Thanks for the feedback and link to the paper, it looks interesting. I'm not an expert, but I think the Hofmeister series also applies to smaller molecules because of the hydration layer effect: some ions are better than others at changing its size.

As far a the biological mess that is the crude extract, we've found here at the nexus that adding acetone crashes out a lot of junk (from either water or methanol extracts). Based on a very crude UV flashlight fluorescent intensity visual check it looks like the actives stay in solution (currently this is an unproven assumption which needs to be tested).

So one path we are looking for final cleanups are attempts to crash out the alkaloids after the acetone crash. You pointed out going to pH4 and systematically increasing the ionic strength, that's something worth trying (previous attempt was a crude pH shift on al already high ionic strength solution). There are other paths mentioned on the Nexus currently being tested.