The present disclosure provides compounds and methods that are useful for the preparation of compounds useful as orexin-2 receptor antagonists.

The invention relates to a non-natural cannabinoid synthase comprising at least one amino acid variation as compared to a wild type cannabinoid synthase Δ9-tetrahydrocannabinolic acid synthase (THCAS), comprising three alpha helices (αA, αB and αC) where a disulfide bond is not formed between alpha helix αA and alpha helix αC, wherein the non-natural cannabinoid synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into a cannabinoid. The invention further relates to a non-natural Δ.sup.9-tetrahydrocannabinolic acid synthase (THCAS), a non-natural cannabidiolic acid synthase (CBDAS), and a non-natural cannabichromenic acid synthase (CBCAS) comprising at least one amino acid variation as compared to a wild type THCAS, CBDAS, or CBCAS, respectively, comprising three alpha helices (αA, αB and αC) and wherein a disulfide bond is not formed between alpha helix αA and alpha helix αC. The invention also relates to a nucleic acid, expression construct, and engineered cell for making the non-natural THCAS, CBDAS, and/or CBCAS. Also provided are compositions comprising the non-natural THCAS, CBDAS, and/or CBCAS; isolated non-natural THCAS, CBDAS, and/or CBCAS enzymes; methods of making the isolated enzymes; cell extracts comprising cannabinoids; and methods of making cannabinoids.

The invention relates to a non-natural cannabinoid synthase comprising at least one amino acid variation as compared to a wild type cannabinoid synthase Δ9-tetrahydrocannabinolic acid synthase (THCAS), comprising three alpha helices (αA, αB and αC) where a disulfide bond is not formed between alpha helix αA and alpha helix αC, wherein the non-natural cannabinoid synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into a cannabinoid. The invention further relates to a non-natural Δ.sup.9-tetrahydrocannabinolic acid synthase (THCAS), a non-natural cannabidiolic acid synthase (CBDAS), and a non-natural cannabichromenic acid synthase (CBCAS) comprising at least one amino acid variation as compared to a wild type THCAS, CBDAS, or CBCAS, respectively, comprising three alpha helices (αA, αB and αC) and wherein a disulfide bond is not formed between alpha helix αA and alpha helix αC. The invention also relates to a nucleic acid, expression construct, and engineered cell for making the non-natural THCAS, CBDAS, and/or CBCAS. Also provided are compositions comprising the non-natural THCAS, CBDAS, and/or CBCAS; isolated non-natural THCAS, CBDAS, and/or CBCAS enzymes; methods of making the isolated enzymes; cell extracts comprising cannabinoids; and methods of making cannabinoids.

Provided are: a protein complex capable of selectively and asymmetrically oxidizing an enantiomer of a secondary alcohol without adding a coenzyme and having an asymmetric oxidation activity in a water-soluble solvent system in the presence of oxygen; a method for producing the same; and a method for coating the protein complex with a high molecular weight compound. The method for producing the protein complex includes: (1) enclosing a crude water-soluble protein in a gel, air-oxidizing the gel, and eluting the protein complex into an aqueous solution; and (2) applying gravity to concentrate and precipitate the protein complex, redissolving the precipitate in an aqueous glycine sodium hydroxide solution of about 0.5 mM and allowing the same to homogeneously coexist with a high molecular weight compound, and re-precipitating the solution and dehydrating and drying the same to yield a protein complex coated with a high molecular weight compound.

Provided are: a protein complex capable of selectively and asymmetrically oxidizing an enantiomer of a secondary alcohol without adding a coenzyme and having an asymmetric oxidation activity in a water-soluble solvent system in the presence of oxygen; a method for producing the same; and a method for coating the protein complex with a high molecular weight compound. The method for producing the protein complex includes: (1) enclosing a crude water-soluble protein in a gel, air-oxidizing the gel, and eluting the protein complex into an aqueous solution; and (2) applying gravity to concentrate and precipitate the protein complex, redissolving the precipitate in an aqueous glycine sodium hydroxide solution of about 0.5 mM and allowing the same to homogeneously coexist with a high molecular weight compound, and re-precipitating the solution and dehydrating and drying the same to yield a protein complex coated with a high molecular weight compound.

Provided herein are processes, such as commercially viable processes, of producing alkyl resorcinols, such as olivetol and olivetolic acid, and analogs of each thereof. Certain of these processes utilize a recombinant, heterologous host microorganism. Certain of the heterologous microorganisms include a Cannabis sativa olivetol synthase (which is a tetraketide synthase, csOLS). Certain of the heterologous microorganisms include a Cannabis sativa olivetolic acid cyclase (csOAC). Certain of the heterologous microorganisms include a Cannabis sativa acyl activating enzyme (csAAE), such as, without limitation, csAAE1. In certain of these processes, glucose is fermented. In certain of these processes, the fermentation further comprises a carboxylic acid, RCO.sub.2H where R is defined as herein, or a salt thereof. Certain of these processes provide olivetol and olivetolic acid in a combined amount of at least 3 g/liter.

Provided herein are processes, such as commercially viable processes, of producing alkyl resorcinols, such as olivetol and olivetolic acid, and analogs of each thereof. Certain of these processes utilize a recombinant, heterologous host microorganism. Certain of the heterologous microorganisms include a Cannabis sativa olivetol synthase (which is a tetraketide synthase, csOLS). Certain of the heterologous microorganisms include a Cannabis sativa olivetolic acid cyclase (csOAC). Certain of the heterologous microorganisms include a Cannabis sativa acyl activating enzyme (csAAE), such as, without limitation, csAAE1. In certain of these processes, glucose is fermented. In certain of these processes, the fermentation further comprises a carboxylic acid, RCO.sub.2H where R is defined as herein, or a salt thereof. Certain of these processes provide olivetol and olivetolic acid in a combined amount of at least 3 g/liter.

A recombinant micro-organism such as Saccharomyces cerevisiae which produces and excretes into culture medium a stilbenoid metabolite product when grown under stilbenoid production conditions, which expresses in above native levels a ABC transporter which transports said stilbenoid out of said micro-organism cells to the culture medium. The genome of the Saccharomyces cerevisiae produces an auxotrophic phenotype which is compensated by a plasmid which also expresses one or more of said enzymes constituting said metabolic pathway producing said stilbenoid, an expression product of the plasmid is genetically modified to include a ubiquitination tag sequence. Expression of an enzyme participating in catabolism of phenylalanine by the Ehrlich pathway is optionally reduced compared to its native expression level.

A recombinant micro-organism such as Saccharomyces cerevisiae which produces and excretes into culture medium a stilbenoid metabolite product when grown under stilbenoid production conditions, which expresses in above native levels a ABC transporter which transports said stilbenoid out of said micro-organism cells to the culture medium. The genome of the Saccharomyces cerevisiae produces an auxotrophic phenotype which is compensated by a plasmid which also expresses one or more of said enzymes constituting said metabolic pathway producing said stilbenoid, an expression product of the plasmid is genetically modified to include a ubiquitination tag sequence. Expression of an enzyme participating in catabolism of phenylalanine by the Ehrlich pathway is optionally reduced compared to its native expression level.

The present invention provides a method for preparing salidroside. The present invention uses β-glucoside and CoFe.sub.2O.sub.4 particles to form a cross-linked aggregate capable of effectively catalyzing the reaction of β-D-glucose and tyrosol, thereby increasing the yield of the salidroside. The steps of the preparation method of the present invention are simple and short, and the method is easy to operate and readily applicable to industrial production.