Biosynthesis of Novel Tryptamines Aspirations.

[SKA]

Hai Nexians,

I was reading about Biosynthesis in the "DMT is everywhere" -Chapter of A.Shulgin's TIHKAL and this bit completely ignited my imagination.

"Eloquent examples of this(Manipulation of Biosynthesis in organisms) are the delightfull reports of indole syntheses
that were explored in Germany over the last few years. Visualise a vigorous growing mycelium culture that is synthesising psilocin
by bringing together the natural DMT in it's diet with a vital but non-too discriminating enzyme that has the capacity for
putting a hydroxyl group onto a Tryptamine. The mycelium has the knowledge of how to 4-hydroxy-late something, but doesn't
really care what that something is. The available target for this skill, in the natural mushroom, just happens to be DMT.
The product is 4-hydroxy-DMT, or psilocin and this is what the organism makes, because this is all that can be made.
There is 1 starting material, there is 1 synthetic capability, and there is 1 product.

Now let's manipulate things a bit. Let's keep this little fungal factory going, but wash out the DMT and replace it with
something like DET. The hydroxylating compulsion appears to be still there and is quite intact. What happens?
The mycelium produces a bunch of 4-hydroxy-DET, an interresting compound and an active psychedelic, which was first
reported by Sandoz laboratories, but a compound that is unknown in nature. I will wager that if the mycelia were to
be sprinkled with MIPT, the enzyme system would produce 4-HO-MIPT which is fully as potent as psilocin and unrecognised
in any legal system in any country that I am aware of. You are exploiting a natural process by providing it with an
unnatural starting material, and allowing it to generate a product that is intrinsically unnatural."

Now for me this raises some questions.

1) If mushrooms need DMT in their diet to produce 4-HO-DMT, does that mean the most commonly used grow-materials for
psilocin mushrooms, like straw, riceflower & Cowdung, contain DMT?
(would explain why I find DMT's smell strangely similair to cowdung-smell to a degree )

2) What if I were to crush a handfull of Anadenanthera Colubrina seeds(very rich in 5-HO-DMT) to powder and sprinkle this
powder onto a growing mycelium and/or mix it into the straw/riceflower/cowdung/vermiculite-cake before innoculation...
What would the Magic mushroom make out of the given 5-HO-DMT?

3)Now what compound would a Psilocybe mushroom make if I fed it dried, powdered plant material rich in 5-MeO-DMT?


Has anyone here ever experimented with this? I really feel this subject needs more research.

 

[joedirt]

Hai Nexians,

I was reading about Biosynthesis in the "DMT is everywhere" -Chapter of A.Shulgin's TIHKAL and this bit completely ignited my imagination.

"Eloquent examples of this(Manipulation of Biosynthesis in organisms) are the delightfull reports of indole syntheses
that were explored in Germany over the last few years. Visualise a vigorous growing mycelium culture that is synthesising psilocin
by bringing together the natural DMT in it's diet with a vital but non-too discriminating enzyme that has the capacity for
putting a hydroxyl group onto a Tryptamine. The mycelium has the knowledge of how to 4-hydroxy-late something, but doesn't
really care what that something is. The available target for this skill, in the natural mushroom, just happens to be DMT.
The product is 4-hydroxy-DMT, or psilocin and this is what the organism makes, because this is all that can be made.
There is 1 starting material, there is 1 synthetic capability, and there is 1 product.

Now let's manipulate things a bit. Let's keep this little fungal factory going, but wash out the DMT and replace it with
something like DET. The hydroxylating compulsion appears to be still there and is quite intact. What happens?
The mycelium produces a bunch of 4-hydroxy-DET, an interresting compound and an active psychedelic, which was first
reported by Sandoz laboratories, but a compound that is unknown in nature. I will wager that if the mycelia were to
be sprinkled with MIPT, the enzyme system would produce 4-HO-MIPT which is fully as potent as psilocin and unrecognised
in any legal system in any country that I am aware of. You are exploiting a natural process by providing it with an
unnatural starting material, and allowing it to generate a product that is intrinsically unnatural."

Now for me this raises some questions.

1) If mushrooms need DMT in their diet to produce 4-HO-DMT, does that mean the most commonly used grow-materials for
psilocin mushrooms, like straw, riceflower & Cowdung, contain DMT?
(would explain why I find DMT's smell strangely similair to cowdung-smell to a degree )

2) What if I were to crush a handfull of Anadenanthera Colubrina seeds(very rich in 5-HO-DMT) to powder and sprinkle this
powder onto a growing mycelium and/or mix it into the straw/riceflower/cowdung/vermiculite-cake before innoculation...
What would the Magic mushroom make out of the given 5-HO-DMT?

3)Now what compound would a Psilocybe mushroom make if I fed it dried, powdered plant material rich in 5-MeO-DMT?


Has anyone here ever experimented with this? I really feel this subject needs more research.

1) No. They get DMT from the decarboxylation of Tryptophan. They do not have DMT directly in their diet. What Shulgin's blurb was missing is that there are also n-methylating enzymes present that convert tyrptamine to N,N-DMT.


2) & 3). My guess is that the 5' OH would interfer with the hydroxylation at the 4 position. I'm guessing their would be an electrostatic repulsion from the current 5' OH wit the enzyme trying to place another hydroxyl in such close proximity. This is just my best guess. Obviously if it worked you would have 4, 5-hydroxy-n,n-dimethyltryptamine. I'd be willing to bet this would have to be synthesized in a lab though!

peace

 

[SKA]

Yes indeed Shulgin continues on the next page about how DMT is made in plants/fungi and the role of decarboxylases & N-methyl-transferases.
He mentions how Tryptophane is decarboxylated to produce Tryptamine, which in turn is converted by methylation into DMT.

I assume mushrooms produce DMT in the same way, except that they proceed to hydroxylate the DMT at the 4 position, creating psilocin.
So for psilocin-rich magic mushrooms you'd need a Tryptophan-rich substrate. Correct?

Does anyone have more specific information on the biosynthetic enzymatic steps/processes Psilocybe mushrooms use to produce Psilocin?

 

[joedirt]

Yes indeed Shulgin continues on the next page about how DMT is made in plants/fungi and the role of decarboxylases & N-methyl-transferases.
He mentions how Tryptophane is decarboxylated to produce Tryptamine, which in turn is converted by methylation into DMT.

I assume mushrooms produce DMT in the same way, except that they proceed to hydroxylate the DMT at the 4 position, creating psilocin.
So for psilocin-rich magic mushrooms you'd need a Tryptophan-rich substrate. Correct?

Does anyone have more specific information on the biosynthetic enzymatic steps/processes Psilocybe mushrooms use to produce Psilocin?

Actually a substrate rich in tryptophan would likely not produce significantly stronger mushrooms as the mushroom would only use the tryptophan that it needed. Essentially I believe the decarboxylation is the rate limiting step....

However.... I have read reports of people spiking the substrate with tryptamine and dramatically increasing the potency...sorry don't have a link right now. This should allow the mushroom to produce Psilocin/Psilocybin at a rate equal to the next rate limiting enzyme catalytic step.


I believe the biosynthetic process for psilocybin production is similar to the following were the enzyme Name/Class is show in ()..


Tryptophan --> (aromatic amino acid decarboxylase) ---> Tryptamine --> ( Tryptamine 4' Hydroxylase ) --> 4-OH-Tryptamine --> (N-Methyl Transferase) (N-Methyl-Transferase) --> Psilocin --> (Phosphorylation) --> psilocybin

I believe there could be minor routes were tryptamine is actually N-meythylated first to form DMT and then 4- hydroxylated, but I don't recall anyone confirming that DMT was present in psilocybe mushrooms...thus I believe the above pathway is the predominant one.

 

[joedirt]

Of course this web site has a graphic showing the N-Methylation step first.

File:Psilocybin biosynthesis.png - Wikipedia

en.wikipedia.org

Now I ned to go back and double check some references because I really don't think mushrooms have significant quantities of DMT present and I'm pretty sure if the production was similar to the above link there would be DMT present.

Anyone else confirm or deny DMT in psilocybe mushrooms?

 

[Dr_Sister]

Sister has also read about mushroom growers adding either tryptamine or DMT to their growing medium to make shrooms more potent, but has no first hand experience with it. Pretty sure it is discussed at length over at the Shroomery though.

Facinating topic!

 

[Infundibulum]

Of course this web site has a graphic showing the N-Methylation step first.

File:Psilocybin biosynthesis.png - Wikipedia

en.wikipedia.org

Now I ned to go back and double check some references because I really don't think mushrooms have significant quantities of DMT present and I'm pretty sure if the production was similar to the above link there would be DMT present.

Don't believe everything wikipedia says...

The presence of both norbaeocystin and baeocystin in mushrooms argue that the methylation steps occur after the hydroxylation and phosphorylation steps.

If not this, then you'll have to assume that baeocystin and norbaeocystin are degradation by-products, which is difficult to envisage. You may also want to entertain the idea that the biosynthetic pathway of psilocybin is different from psilocin, i.e. whereas psilocybin is formed by the methylation of norbaeocystin and baeocystin, psilocin is formed by a dmt intermediate. But there's little if any evidence for any of the above.

It is far more likely that psilocin is a degradation product of psilocybin in mushrooms rather than a substrate for psilocybin, which makes the wiki diagram wrong. If methylation proceeds prior to hydroxylation, then you should be able to see DMT and NMT in mushrooms (which you don't) and if phosphorylation occurs in the end you should be able to see 4-HO NMT (which to my knowledge you don't).

 

[SnozzleBerry]

However.... I have read reports of people spiking the substrate with tryptamine and dramatically increasing the potency...sorry don't have a link right now. This should allow the mushroom to produce Psilocin/Psilocybin at a rate equal to the next rate limiting enzyme catalytic step.

The original papers can be found on Shroomery over here, they documented up to a 300% increase in alkaloid production, iirc. There was a member of the nexus who successfully added tryptamine to his substrate about a year or a year-and-a-half ago and reported, in addition to an increase in potency, that the fruits were greatly mutated and dwarfed. His results seemed to confirm the old experiment and sounded rather impressive to me.

 

[joedirt]

Of course this web site has a graphic showing the N-Methylation step first.

File:Psilocybin biosynthesis.png - Wikipedia

en.wikipedia.org

Now I ned to go back and double check some references because I really don't think mushrooms have significant quantities of DMT present and I'm pretty sure if the production was similar to the above link there would be DMT present.

Don't believe everything wikipedia says...

The presence of both norbaeocystin and baeocystin in mushrooms argue that the methylation steps occur after the hydroxylation and phosphorylation steps.

If not this, then you'll have to assume that baeocystin and norbaeocystin are degradation by-products, which is difficult to envisage. You may also want to entertain the idea that the biosynthetic pathway of psilocybin is different from psilocin, i.e. whereas psilocybin is formed by the methylation of norbaeocystin and baeocystin, psilocin is formed by a dmt intermediate. But there's little if any evidence for any of the above.

It is far more likely that psilocin is a degradation product of psilocybin in mushrooms rather than a substrate for psilocybin, which makes the wiki diagram wrong. If methylation proceeds prior to hydroxylation, then you should be able to see DMT and NMT in mushrooms (which you don't) and if phosphorylation occurs in the end you should be able to see 4-HO NMT (which to my knowledge you don't).

I concur and that is essentially what I was proposing, but you never know. In all likelihood there is some trace amount of DMT in shrooms as the laws of statistical mechanics pretty much state that every reaction that can happen will happen in some capacity... In any event unless someone can show a journal article or other experimental evidence of significant quantities of DMT I'll stand bye the original biosynthesis I laid out in the prior post... Always good to have a sanity check with the other member though!

Gracias Amigo!

 

[Infundibulum]

I concur and that is essentially what I was proposing, but you never know. In all likelihood there is some trace amount of DMT in shrooms as the laws of statistical mechanics pretty much state that every reaction that can happen will happen in some capacity... In any event unless someone can show a journal article or other experimental evidence of significant quantities of DMT I'll stand bye the original biosynthesis I laid out in the prior post... Always good to have a sanity check with the other member though!

Gracias Amigo!

shrug,

sure we may never know, buuuuuuut...... actually one could go as far as hypothesizing that mushrooms' methyltransferase only recognizes the 4-phosphate ester in norbaeocystin and methylates it to baeocystin and psilocybin. That is, their methyltransferase cannot recognize 4-HO-tryptamine to methylate it to 4-HO-NMT and subsequently 4-HO-dmt.

This is probably why you don't find 4-HO tryptamine and 4-HO-NMT in mushrooms. If the methyltransferase(s) from mushrooms do actually have such an exquisite 4-phosphorester indole substrate specificity, then they will be unable to methylate not only 4-HO-tryptamine and 4-HO-NMT, but tryptamine as well. Which quite throws the speculation about the presence of dmt in mushrooms out of the window....

 

[SKA]

But another possible explanation for why you don't find 4-HO-Tryptamine and 4-HO-NMT in Magic Mushrooms could be if they decarboxylate Tryptophan into Tryptamine first,
then Methylate that Tryptamine into DMT and then 4-hydroxylate that DMT into Psilocin.


Shulgin specificly mentions the Magic Mushrooms 4-Hydroxylase enzyme being not-too-discriminating on what it 4-hydroxylates.
He also sais that sprinkling DET into the mycelium, makes the mushrooms growing from it produce 4-HO-DET.
I suppose he didn't make that up. I wish he had gone in more detail about this.

Are we hypothising about how mushrooms make psilocin because none of us is a biochemist? Or because simply no one really knows?

 

[Infundibulum]

But another possible explanation for why you don't find 4-HO-Tryptamine and 4-HO-NMT in Magic Mushrooms could be if they decarboxylate Tryptophan into Tryptamine first,
then Methylate that Tryptamine into DMT and then 4-hydroxylate that DMT into Psilocin.

In this case you would find both dmt and nmt traces, which you don't atm.

Shulgin specificly mentions the Magic Mushrooms 4-Hydroxylase enzyme being not-too-discriminating on what it 4-hydroxylates.
He also sais that sprinkling DET into the mycelium, makes the mushrooms growing from it produce 4-HO-DET.
I suppose he didn't make that up. I wish he had gone in more detail about this.

I don't argue about the specificity of th 4-hydroxlase. It is well accepted that it can process a variety of substrates. I argue about the specificity of methyltransferase. I said above that this is possibly why you find nor baeocystin and baeocystin but not NMT, DMT, and 4-HO-NMT.

Are we hypothising about how mushrooms make psilocin because none of us is a biochemist? Or because simply no one really knows?

It's because none of us really knows. I am more likely to believe (from what we know so far) that psilocin comes after psilocybin (via dephosphorylation) rather than the opposite. This might be wrong, but that's what our current knowledge on the intermediates suggests.

 

[SKA]

So do you think baeocystin and norbaeocystin are by products or stepping stones in the mushroom's enzymatic process to change a certain starting material into psilocyn?

I was just looking on Wikipedia at the Chemical constituents of Brown Rice & Barley; 2 plants often used to grow potent Psilocybe Mushrooms.
Both have a very high Niacin content(Vitamin B3) content. Looking at the moleculair structure of Niacin, it shows to bare a vague resemblence to
a Tryptamine-structure.

Could there be an enzymatic process in the Mushroom that turns Niacin into Psilocin stepwise and happens to form norbaeocystine & baeocystine in the process?
Niacin, in comparison to the Indole-ring structure, is missing the Benzene ring that Tryptamines have.
Could it be that the mushroom draws Benzene rings from other compounds in it's diet that it enzymatically breaks down and merges these Benzene rings onto Niacin molecules to form an Indole structure?

 

[Infundibulum]

So do you think baeocystin and norbaeocystin are by products or stepping stones in the mushroom's enzymatic process to change a certain starting material into psilocyn?

They're intermediates, or stepping stones as you say.

I was just looking on Wikipedia at the Chemical constituents of Brown Rice & Barley; 2 plants often used to grow potent Psilocybe Mushrooms.
Both have a very high Niacin content(Vitamin B3) content. Looking at the moleculair structure of Niacin, it shows to bare a vague resemblence to
a Tryptamine-structure.

Could there be an enzymatic process in the Mushroom that turns Niacin into Psilocin stepwise and happens to form norbaeocystine & baeocystine in the process?
Niacin, in comparison to the Indole-ring structure, is missing the Benzene ring that Tryptamines have.
Could it be that the mushroom draws Benzene rings from other compounds in it's diet that it enzymatically breaks down and merges these Benzene rings onto Niacin molecules to form an Indole structure?

Nah, it seems unlikely that niacin can be used as a precursor indole, let alone to psilocin/psilocybin. You may want to check the Skikimate pathway tryptophan is made in cells.

 

[SKA]

The original papers can be found on Shroomery over here, they documented up to a 300% increase in alkaloid production, iirc. There was a member of the nexus who successfully added tryptamine to his substrate about a year or a year-and-a-half ago and reported, in addition to an increase in potency, that the fruits were greatly mutated and dwarfed. His results seemed to confirm the old experiment and sounded rather impressive to me.

Wait Whaaaat? alkaloid production increases of up to 300%?!!

Can't believe I missed that before.
That nexus member that added Tryptamine to his mushroom substrate you mention;
Did he confirm that the Psilocybin/Psilocin content of the resulting mushrooms
was markedly increased?


Back to the topic.
Here's some information on 4-HO-DET;

Wikipedia 4-HO-DET article
TIHKAL 4-HO-DET entry

The second link goes in great detail describing it's effects in different dosages & routes of
administration. I believe these various reports also come from different people.
Sounds very interresting.

Now in that TIHKAL excerpt I found this:

Some fascinating studies have been done in Germany where the metabolically active mycelium of some Psilocybe species have been administered diethyltryptamine as a potential diet component. Normally, this mushroom species dutifully converts N,N-dimethyltryptamine (DMT) to psilocin, by introducing a 4-hydroxyl group into the molecule by something that is probably called an indole 4-hydroxylase by the biochemists. You put DMT in, and you get 4-hydroxy-DMT out, and this is psilocin. Maybe if you put Mickey Mouse in, you would get 4-hydroxy-Mickey Mouse out. It is as if the mushroom psyche didn't really care what it was working with, it was simply compelled to do its sacred duty to 4-hydroxylate any tryptamine it came across. It was observed that if you put N,N-diethyltryptamine (DET, not a material found in nature) into the growing process, the dutiful and ignorant enzymes would hydroxylate it to 4-hydroxy-N,N-diethyltryptamine (4-HO-DET) a potent drug also not known in nature. This is the title drug of this commentary. What a beautiful burr to thrust into the natural versus synthetic controversy. If a plant (a mushroom mycelium in this case) is given a man-made chemical, and this plant converts it, using its natural capabilities, into a product that had never before been known in nature, is that product natural? What is natural? This is the stuff of many long and pointless essays.

Obviously we need to find published test results of these german studies!
Anyone who has access to publications of german research in the area of
DET, DMT, 4-HO-DET & Psilocybin Mushrooms?






PS: Would it be possible for a noob kitchen chemist to synth DET from something like DMT or Tryptamine?

 

[benzyme]

Sister has also read about mushroom growers adding either tryptamine or DMT to their growing medium to make shrooms more potent, but has no first hand experience with it.

benzyme can confirm this, having experimented with a 10 mM conc. of tryptamine hcl.
the result was a wild-type strain of cubensis which was comparable to panaeolus cyanescens.

 

[SKA]

I have found the German study that Shulgin mentioned! And what a treasure it is!

http://www.scribd.com/doc/8329963/Jochen-Gartz-Biotransformation-of-Tryptamine-Derivatives-in-Mycelial-Cultures-of-Psilocybe

Great read! This is about adding Tryptamine & DMT to mushroom substrates to improve yields
and it works great with Both, suggesting that DMT indeed IS an intermediary step to psilocybin
after all.

It could just be that after the first type enzyme makes DMT, the next enzyme immediately
hydroxylises that DMT into 4-OH-DMT? That the 2 enzymatic processes flow over into eachother so seemlessly that, at no time, will detectable amounts of DMT be present?

Well here's the other study, about adding DET to mushroom substrate:

http://countyourculture.com/2012/02/17/biosynthesis-4-substituted-tryptamine-derivatives/

And according to Jochen's experiments it indeed works!

Interresting guy, this Jochen Gartz!

 

[SKA]

These studies by Jochen Gartz got me really inspired.

SWIM wanted to grow mushrooms anyways, but when he starts
he will experiment with adding different Tryptamines to
the developping Mycelia of various separate grow-bags.

Tryptamines to feed Mycelia & what compounds could be made:
-Tryptamine => [increased 4-HO-DMT & 4-PO-DMT content] Tried & True
-Tryptophan => [increased 4-HO-DMT & 4-PO-DMT content] " "
-5-Hydroxy-Tryptophan (5-HTP)=> [4,5-Hydroxy-DMT & 4,5-PO-DMT???] Needs Research.
-5-methoxy-N-acetyltryptamine (Melatonin) => [4-HO-5-MeO-N-acetyl-DMT???] Needs Research.
-5-MeO-DMT => [4-HO-5-MeO-DMT?] Hypothised by Jochen Gartz & Alexander Shulgin. Needs Research.


An answer of Shulgin to a question regarding 4-HO-5-MeO-DMT;

Ask Dr. Shulgin Online: 4-Hydroxy-5-methoxy-N,N-dimethyltryptamine, Psilocybe mushrooms, Psilocin

Not all people can easily get their hands on Tryptamines like DET, DiPT & such,
but Melatonin & 5-HTP can be bought over the counter of most drugstores and
Tryptamine, Tryptophan, 5-MeO-DMT should be obtainable for the relatively
experienced kitchen chemist.

As soon as SWIM's ready to start growing mushrooms he'll buy a bottle of
Melatonin pills, crush some pills and feed to the substrate before innoculating.
Next up will be extracting some 5-MeO-DMT and adding that to the substrate.

Anyone willing to give this a try? (especially if you have easy access to DET,
DiPT or 5-MeO-DMT and Mushroom spores)

 

[Dante]

Wow, this is so interesting! I might give it a try in the future...

Please, post the tek and the results!!

 

[InMotion]

Not to dive too deep into speculation.

However the hydrolysis of melatonin yielding mexamine(5-meo-t) is another interesting and simple idea.