User:Ccdietz/sandbox/Psilocybin Synthesis

Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) is an organic compound biosynthesized by organisms of the Psilocype genus, as well as certain species of several other  fungal genera. Psilocybin is the prodrug of the psychoactive compound psilocin (4-hydroxy-N,N-dimethyltryptamine) which is structurally similar to the neurotransmitter serotonin (5-hydroxytryptamine). When present in the brain, psilocin interferes with neuron signal transmission in pathways using serotonin through agonistic binding to neural receptors, especially 5-HT2C receptors, which results in various physical and psychological effects. Due to the psychotropic properties of psilocin, fungal species that produce psilocybin are commonly known as “magic mushrooms” and are frequently consumed as recreational drugs.

The biological pathway of psilocybin synthesis in producing organisms was first characterized in 1968 and further research has identified the specific enzymes involved as well as the precise mechanism of reactions. Various in vitro biotechnological and total organic synthetic pathways have also been reported.

History
A group led by chemist Albert Hofmann first determined the structures of psilocybin and psilocin from samples extracted from Psilocybe mexicana in 1958. In 1959, Hofmann published the first known total synthesis of the molecules from indoles using traditional organic synthetic methods. Since then, a number of improvements to the original process have been reported, including some recent transition metal-catalyzed syntheses.

The biological pathway of psilocybin synthesis in vivo was first suggested by Stig Agurell and J. Lars Nilsson in 1968. The development of genomic sequencing technology since has allowed researchers to isolate a sequence of DNA found in various psilocybin-producing species which encode enzymes found to catalyze the formation of the compound. This discovery elucidated the most probable biosynthetic mechanism and disproved the previous 1968 proposal. This has led to further research into enzymatic pathways using psilocybin-producing enzymes to efficiently synthesize psilocybin and psilocin in vitro through various methods.

Synthetic Mechanisms
Multiple pathways for the synthesis of psilocybin have been studied and reported since its chemical structure was identified in 1958. Initial interest was placed largely on its biosynthetic mechanisms but this quickly gave way to multiple studies on organic synthetic routes and into enzymatic in vitro methods of production.

Biosynthetic
The since disproven biosynthetic pathway suggested by Agurell and Nilsson identified tryptophan [2-amino-3-(1H-indole-3-yl)propanoic acid] as the biological precursor molecule of psilocybin in producing organisms. The mechanism proposed consisted of decarboxylation of tryptophan to tryptamine [2-(1H-indole-3-yl)ethanamine], followed by two rounds of methylation to form N,N-dimethyltryptamine then a hydroxylation to produce psilocin which would then be phosphorylated into psilocybin, as shown below.



The mechanism proposed by Agurell and Nilsson was corrected by Janis Fricke, Felix Blei, and Dirk Hoffmeister in 2017. They were able to identify a series of four amino acid sequences encoded on samples of psilocybin-producing P. cubensis and  P. cyanescens DNA which corresponded to enzymes that successfully synthesized psilocybin at biological conditions when supplied with biologically available precursor molecules. The group identified Psilocybe tryptophan decarboxylase (PsiD), Psilocybe P450 monooxygenase (PsiH), Psilocybe kinase (PsiK), and Psilocybe methyltransferase (PsiM) as the enzymes responsible for psilocybin biosynthesis. This served to elucidate a new, highly probable, biosynthetic mechanism. First, PsiD decarboxylates tryptophan to tryptamine. The phosphorylation step occurs next, with PsiH catalyzing the formation of 4-hydroxytryptamine which is then converted into 4-phosphoryloxytryptamine by PsiK. Finally, two rounds of methylation catalyzed by PsiM produce psilocybin, as shown below.



Enzymatic
In the same study, Fricke et al. Identified and successfully tested an in vitro enzymatic one-pot synthesis of psilocybin using the same Psilocybe enzymes but with 4-hydroxytryptophan used as the starting molecule. The first step to occur, shown below, is decarboxylation to 4-hydroxytryptamine catalyzed by the PsiD enzyme. The remainder of the synthesis follows the same biosynthetic route previously identified.



Organic
The first total organic synthesis of psilocybin was reported by Hofmann et al. in 1959. Benzyl-protected 4-hydroxyindole was reacted with oxalyl chloride to form 5-benzyloxy-3-indoleglyoxylyl chloride. Dimethylamine was added, reacting with the acid chloride to produce 5-benzyloxy-3-indole-N,N-dimethylglyoxylamide, which was then reduced by lithium aluminum hydride to form 4-benzyloxy-N,N-dimethyltryptamine. Following catalytic deprotection, the resulting psilocin was phosphorylated into psilocybin using O,O-dibenzylphosphoryl chloride with transition metal catalysis, as shown below.



Hoffman’s original procedure was modified by David Nichols and Stewart Frescas who proposed a different method of psilocin phosphorylation capable of generating 66% yield of psilocybin. They reacted psilocin with n-butyllithium and tetra-O-benzylpyrophosphate to generate 4-(O-monobenzylphosphoryloxy)-N,N-dimethyltryptamine, which was then reducted using a Palladium catalyst and hydrogen gas to produce psilocybin at elevated yields.



Further improvement was made when Osamu Shirota, Wataru Hakamata, and Yikihiro Goda utilized an acetyl protecting group in place of the benzyl group used previously. This allowed for deprotection and reduction to occur simultaneously, revealing a shorter synthetic path to Psilocybin. In their study, 5-acetyloxy-3-indole-N,N-glyoxylamide was reacted with lithium aluminum hydride in tetrahydrofuran to produce psilocin in 85% yield.



In 2016, a novel method for psilocybin total synthesis employing a borrowing hydrogen process was reported by Silvia Bartolucci, Michele Mari, Giovanni Di Gregorio, and Giovanni Piersanti. Using an iridium catalyst, a pentamethylcyclopentadienyliridium chloride dimer along with CS2CO3, they were able to attach a pre-tailored dimethylamine side chain to the correct position on an Benzyl-protected 5-hydroxyindole to produce psilocin. This synthetic mechanism was found to be highly efficient and yielded low waste, formally generating only water as a byproduct.