Novel cannabinoid oligosaccharides

Abstract

Described herein are novel oligosaccharides of cannabidiol and other cannabinoids and methods of production and uses thereof. The production method includes contacting cannabidiol with UDP-carbohydrate dependent glycosyltransferases (UGT73A class) to produce a cannabinoid-β-D-glycoside, then contacting the product with a cyclomaltodextrin glucanotransferase and a cyclodextrin to elongate the sugar moiety with (1-4)-α-D-glucopyranoside, thereby producing a mixed 0-a-structure in a recombinant host that has an amphiphilic structure as a surfactant solubilizer and different enzymatic degradability of α- and β-D-glucopyranose residues by amylases and β-glycosidases, thereby allowing targeted drug delivery.

Description

TECHNICAL FIELD

[0001] The present invention describes novel oligosaccharides of cannabidiol and other cannabinoids and special methods of their production, isolation and application, which are easy due to their specific properties. BACKGROUND

[0002] Cannabinoids are secondary metabolites of Cannabis (Cannabis sativa) and are isoprenylated phenols. The main cannabinoid compounds are Δ 9 -tetrahydrocannabinol (THC), cannabidiol (CBD) Figure 1 ), cannabichromene (CBC) and cannabigerol (CBG). Cannabinol (CBN) is known from some cultivars of Cannabis and originates from THC. The relative proportions of these main cannabinoid compounds vary in different Cannabis varieties. In addition to neutral cannabinoids, cannabinoid acids with carboxyl groups are present and dominant in fresh plant material. Acidic cannabinoids are precursors of cannabinoids and can also be converted non-enzymatically into the latter. Up to about 60 different cannabinoid compounds are known from Cannabis varieties. Non-acidic cannabinoids are lipophilic compounds, usually with a phenolic structure, and are mainly C 21 compounds. Part of the structure comes from fatty acid and isoprenoid biosynthesis as well as polyketide pathways.

[0003] Cannabinoids are consumed worldwide in various forms. It is estimated that 2.6-5.0% of the world population uses cannabis as a herbal (marijuana) or resin (hashish) (UNODC, 2012). In some countries, cannabinoid-containing medicines are available for medical use, either with natural cannabis extracts (Sativex®), or with synthetic cannabinoids (nabiximols, dronabinol). The Food and Drug Administration (FDA) has approved THC extracted from cannabis for the control of chemotherapy-related nausea and vomiting, and recently also for stimulating the appetite of AIDS patients. In Germany, the flowers of cannabis ("cannabis flowers") are used as a medicine, and dronabinol can be prescribed as a preparation with a specific indication for treatment. Other biological activities allow therapeutic applications, such as the treatment of glaucoma, migraine, spasticity, anxiety and as an analgesic.

[0004] There is substantial evidence that agents that activate cannabinoid receptors in the body, such as cannabinoids and endocannabinoids, can modulate appetite, as well as alleviate nausea, vomiting, pain (Martin B. R. and Wiley, J. L, Mechanism of action of cannabinoids: how it may lead to treatment of cachexia, emesis, and pain, Journal of Supportive Oncology 2: 1 - 10, 2004), multiple sclerosis (Pertwee, R. G., Cannabinoids, and multiple sclerosis, Pharmacol. Ther. 95, 165 - 174, 2002) and epilepsy (Wallace, M. J., Blair, R. E., Falenski, K. W W., Martin, B. R., and DeLorenzo, R. J. Journal Pharmacology and Experimental Therapeutics, 307: 129- 137, 2003).Furthermore, CB2 receptor agonists have been shown to be effective in animal models for the treatment of pain (Clayton N., Marshall F. H., Bountra C, O'Shaughnessy C. T., 2002. CB1 and CB2 cannabinoid receptors are implicated in inflammatory pain. 96, 253 - 260; Malan T. P., Ibrahim M. M., Vanderah T. W., Makriyannis A., Porreca F., 2002. Inhibition of pain responses by activation of CB (2) cannabinoid receptors. Chemistry and Physics of Lipids 121, 191 - 200; Malan T. P., Jr., Ibrahim M. M., Deng H., Liu Q., Mata H. P., Vanderah T., Porreca F., Makriyannis A., 2001. CB2 cannabinoid receptor-mediated peripheral antinociception. 93, 239- 245.; Quartilho A., Mata H. P., Ibrahim M. M., Van derah T. W., Porreca F., Makriyannis A., Malan T. P., Jr., 2003. Inhibition of inflammatory hyperalgesia by activation of peripheral CB2 cannabinoid receptors. Anesthesiology 99, 955 - 960) and multiple sclerosis (Pertwee, R. G. Cannabinoids and multiple sclerosis, Pharmacol. Ther. 95, 165 174, 2002).

[0005] Recently, the scope of approval of cannabis and cannabinoid products for medical use has expanded. There is a need to develop more efficient methods of producing and isolating cannabinoid compounds. Traditional methods of cannabinoid production generally focus on extraction and purification of cannabinoids from harvested raw cannabis. However, traditional methods of cannabinoid extraction and purification face technical and practical issues, primarily the need for non-aqueous solvents (organic solvents (U.S. Patent No. 6,403,126 (Webster et al.); U.S. Patent Application No. 20160326130 (Lekhram et al.); butane) or supercritical CO2 (CA2424356 (Muller et al.)) for extraction and purification of lipophilic cannabinoids. There is also a similar need for water-soluble forms of cannabinoids in their applications, primarily due to the instability (short shelf life) and emulsion turbidity of such water-based products.

[0006] Glycosides of cannabinoids have been investigated. Tanaka et al. (Journal of Natural Products (1993) 56(12): 2068-2072) describe cannabielosh derived by bioconversion with in vitro plant tissue. Tanaka et al. (Plant Cell Reports (1996) 15: 819-823) describe beta-D-glucopyranosides of cannabidiol and cannabidiolic acid obtained by bioconversion with Pinellia ternata plant tissue.

[0007] US20190085347A1 discloses “High level in vivo biosynthesis and isolation of water-soluble cannabinoids in a plant system”. Water-solubility of cannabinoids is achieved via glycosylation of cannabinoids in the last biosynthetic step by heterologous expression of suitable plant UDP-dependent glycosyltransferases or glucuronosyltransferases, resulting in beta-D-glucopyranosides or beta-D-glucuronosides, respectively.

[0008] WO2020239784A1 describes “Genetically modified host cells for producing glycosylated cannabinoids” which rely on UDP-dependent glycosyltransferases and thus produce beta-D-glycosides broadly, except for one example of alpha-L-rhamnosides (a rare representative of L-carbohydrates).

[0009] WO2021146687A1 claims “A cannabinoid glycoside composition and method of production thereof”; this is by feeding animals (in particular insects / orthopterans, crickets). Their account includes beta-D-glucopyranosides of cannabinoid compounds obtained with up to 6 glycoside residues.

[0010] WO2021173130A1 discloses "Novel cannabinoid glycosides and uses thereof". The cannabinoid glucopyranoside structures given are all ß-anomers with up to four glucopyranoside residues.

[0011] WO2022099078A1 describes "Production of glycosylated cannabinoids by UDP-dependent glycosyltransferases from Arabidopsis thaliana and Helianthus annuus".

[0012] WO2022126028A1 reports a "Continuous enzyme perfusion reactor system for production of cannabinoid glycosides".

[0013] In summary, all reports focus on the production and / or handling of ß-D-glycosides, mainly ß-D-glucopyranosides of cannabinoids, which mainly have one to four carbohydrate units and various intercarbohydrate linkages (1-3, 1-4, 1-6).

[0014] A general and important requirement for cannabinoid glycosides is that they are degradable by enzymatic hydrolysis (like the widely used ß-glucosidases) after consumption of the cannabinoid containing product.

[0015] Object of the invention

[0016] The task of the present invention is to find an efficient method for the production of highly glycosylated cannabidiol derivatives, which will have excellent water solubility and also the typical structure of surfactants / solubilizers, designed to allow solubilization or emulsification of smaller glycosylated or free cannabinoids, and also a structure that allows enzymatic cleavage. This will facilitate the development of better isolation methods and promote new and improved forms of application of cannabinoids. Cannabidiol as the main cannabinoid other than THC is just one example.

[0017] The present invention must solve the problem of producing such highly glycosylated cannabinoid derivatives, which should also be degradable by enzymes when ingested by humans. However, in addition to the prior art, the demand for water-soluble cannabinoid derivatives can also be met by α-D-glucopyranosides of cannabinoids. In addition, α-D-glucopyranosides can be hydrolyzed by amylases, which are typical enzymes of the human digestive tract. For very good water solubility and the typical structure of surfactants / solubilizers, a production method for highly glycosylated cannabinoid derivatives must be invented.

[0018] Cyclomaltodextrin glucanotransferases are a well-known class of enzymes. They not only produce a-, b-, g- (6, 7, 8-membered rings) or higher cyclodextrins from starch, but also perform transglycosylation reactions with carbohydrate acceptors, and hydrolysis with water as acceptor (https: / / www.cazypedia.org / index.php / Glycoside_Hydrolase_Family_13). However, it is reported that this is mainly used for transfer to other carbohydrates / oligosaccharides / polysaccharides, with only a small amount of activity for direct transglycosylation of non-carbohydrate acceptor substrates. Unlike UDP-a-glucose dependent glycosyltransferases, the stereochemistry of the carbohydrate is not transformed by a displacement mechanism to build a glucopyranoside, but is preserved by a double displacement of the intermediate bound to the enzyme. Thus, a-D-glucopyranosides are formed by cyclomaltodextrin glucanotransferases with (1-4)-a-D-glucopyranose residues as co-substrates (like from cyclodextrins). Some reactions with these enzymes, like partial transglycosylation, partial transglycosylation again (dissimilation) or partial hydrolysis (e.g. Ara et al. Glycobiology, 2015, Vol. 25, No. 5, 514-523, doi: 10.1093 / glycob / cwu182) can result in a lower number of (1-4)-a-D-glucopyranose residues than expected from simple cyclodextrin residue transfer. This lower number of a-D-glucopyranose residues can also be obtained by transglycosylation reactions of glucansucrases (https: / / www.cazypedia.org / index.php / Glycoside_Hydrolase_Family_70), which use disaccharides, mainly sucrose, as co-substrates instead of cyclodextrins.

[0019] Steviol glycosides and rebaudioside A show transglycosylation of the natural glycosyl donor and it was found that it improves the taste quality of the derivatized compounds (Muñoz-Labrador et al. Foods 2020, 9, 1753; doi:10.3390 / foods9121753). Considering this substrate acceptability and UDP-dependent glycosyltransferases (once the enzymes with the appropriate specificity for the aglycon are identified), one possible strategy is to perform an initial and direct backbone glycosylation of the beta-D-glucopyranoside by a UDP-dependent glycosyltransferase and then to transfer six to eight units (by a cyclic maltodextrin glucanotransferase) or a single (by a dextransucrase) (1-4) alpha-D-glucopyranoside residue stepwise, thus presenting a highly glycosylated cannabinoid derivative. The dextransucrase from Liquorilactobacillus nagelii and Liquorilactobacillus hordei known for the present invention is a dextransucrase (Bechtner et al. 2022, Gels 2022, 8, 171. https: / / doi.org / 10.3390 / gels8030171). This strategy will allow the formation of cannabinoid oligosaccharides with the expected mixed beta / alpha structure. SUMMARY

[0020] The present invention relates to methods for the production and isolation of cannabinoid oligosaccharides, exemplified by cannabidiol oligosaccharides, based on the use of specific plant UDP-glucose dependent glycosyltransferases of the UGT73a class in a first step and cyclic maltodextrin glucanotransferases and / or dextransucrases in a second step to form cannabinoid oligosaccharides with an amphiphilic structure typical for water-soluble compounds and emulsifiers / solubilizers. Preferred embodiments are based on methods for the heterologous expression in microorganisms like E. coli or cannabis; the dependency on heterologous expression in cannabis allows the use of glucosyl residues as a protective group against the biosynthesis of tetrahydrocannabinol and provides the option of water extraction as the first step of production. Further subjects of the invention are the compositions obtained by these methods, methods for the isolation of cannabidiol oligosaccharides from these compositions and the use of these cannabinoid oligosaccharides as active ingredients. Concentrated solutions as active ingredients for pharmaceutical or veterinary drugs are also advantageous embodiments of the invention. Glycosidases, especially beta-glucosidases and amylases can be applied with the various forms of cannabinoid oligosaccharides to trigger the release of the aglycon. The difference in enzymatic degradability of alpha-D-glucopyranoside residues versus beta-D-glucopyranoside residues by amylases and beta-glucosidases allows for targeted drug delivery.

[0021] More specifically, the present invention relates to the following:

[0022] (1) A method of producing a cannabinoid-oligosaccharide, such as cannabidiol (CBD)- oligosaccharide, comprising the step of contacting a cannabinoid, such as CBD, with an isolated UDP-carbohydrate dependent glycosyltransferase, preferably of the UGT73A class, preferably with the glycosyltransferases identified as [Seq1 (DNA), Seq2 (AA) = NbGT-fc6] and [Seq3 (DNA), Seq4 (AA) = CaGT2], or with a UDP-carbohydrate dependent glycosyltransferase showing or having at least 70%, at least 80%, at least 90%, at least 95%, at least 98% nucleic acid sequence identity to [Seq1 = CaGT2], or at least 70%, at least 80%, at least 90%, at least 95%, at least 98% amino acid sequence identity to [Seq2 = CaGT2], in a reaction mixture under conditions including any co-substrates and co-factors necessary for the glucosyltransferase activity, effective to produce a cannabinoid-β-D-glycoside, and subsequently or in parallel, contacting this or a cannabinoid-β-D-glycoside from another source, with at least one of a cyclomaltodextrin glucanotransferase and its substrate cyclodextrin or substituted cyclodextrin (e.g. hydroxypropyl-, methyl-), linear maltodextrin, starch or glycogen, and a further step of purifying the cannabinoid oligosaccharide from the reaction mixture to obtain an isolated cannabinoid oligosaccharide.

[0023] (2) The method according to (1), wherein the isolated UDP-carbohydrate dependent glycosyltransferase uses a cannabinoid, such as CBD, and as co-substrate UDP-glucose or fructose, glucose, galactose, mannose, rhamnose, ribose, arabinose, xylose, activated forms thereof, or activated oligosaccharides, or activated aldoses or uronic acids, such as glucuronic acid, glucoronic acid or galacturonic acid, or N-substituted derivatives of said carbohydrates, such as 2-N-acetylglucosamine, and wherein the cyclomaltodextrin glucanotransferase uses the intermediate product (or added lychnophore from another source) cannabinoid-β-D-glycoside as substrate to form higher glycosylated oligosaccharides (the latter having an alpha-anomeric pyranoglucose residue), and the product is a corresponding cannabidiol oligosaccharide with a backbone β-D-glycoside structure and branching alpha-pyranoglucose oligomers.

[0024] (3) The method according to (1) or (2), wherein the isolated UDP-carbohydrate dependent glycosyltransferase and the cyclomaltodextrin glucanotransferase are obtained by heterologous expression in an organism of one or both of the species different from the original species of the glycosyltransferase or the cyclomaltodextrin glucanotransferase; preferably, the conversion of a cannabinoid into a cannabinoid-oligosaccharide is dependent on the cellular supply of a co-substrate identified as UDP-glucose or other activated carbohydrate and presumably on the cellular supply or import of at least one of the substrates cyclodextrin or substituted cyclodextrins (e.g. hydroxypropyl-, methyl-), linear maltodextrins, starch or glycogen and occurs in the same heterologous organism which can be optimized for this purpose by genetic and / or genetic engineering means.

[0025] (4) The method according to any one of (1) to (3), wherein instead of or in addition to the cyclomaltodextrin glucanotransferase and its substrates (cyclodextrin or substituted cyclodextrins (e.g. hydroxypropyl-, methyl-), linear maltodextrins, starch or glycogen) a glucansucrase and its substrate sucrose (or other compatible disaccharide) is used to add (1-2)-, (1-3)-, (1-4)-, or (1-6)-linked a-D-glucopyranoside residues to produce a branched cannabinoid oligosaccharide.

[0026] (5) The method according to any one of (1) to (4), wherein a cannabinoid oligosaccharide is obtainable, e.g. but not limited to, wherein a CBD-2ßG-8aG and / or higher reaction product or branched cannabinoid oligosaccharide is obtained from cannabidiol with GT40 / UDP-glucose and with cyclomaltodextrin glucanotransferase / gamma-cyclodextrin or with glucansucrase and sucrose (or other disaccharide).

[0027] (6) A cannabinoid oligosaccharide obtained by the method according to any one of (1) to (5) having a glycosylation pattern comprising zero or one to four beta-D-glucopyranosyl backbone residues and one or more additional ((1-4)-alpha-D-glucopyranosyl) 5、6、7或>7 (1-4)-alpha-D-glucopyranoside) n oligosaccharide residues or branched cannabinoid oligosaccharide, which can also be derived by other glycosyltransferases or transglycosylation reactions than cyclomaltodextrin glucanotransferase, such as glucansucrase, and in this case not only (1-4)- but also (1-2)-, (1-3)- or (1-6)-linked, (1-4)-alpha-D-glucopyranoside residues can be attached directly to the cannabinoid, its beta-D-glucopyranose residue, or, if more than one, also to each other.

[0028] (7) The CBD oligosaccharides obtained according to (6) comprise zero or one to four beta-D-glucopyranosyl stem residues and one or more additional ((1-4)-alpha-D-glucopyranosyl) 5、6、7或>7 (1-4)-alpha-D-glucopyranoside) n The oligosaccharide residue is either a CBD oligosaccharide or a CBD oligosaccharide which can be (1-2)-(1-3)-(1-4)- or (1-6)-alpha-linked.

[0029] (8) The compounds CBD-2pG-6aG, CBD-(pG-6aG)2, CBD-2pG-12aG, CBD-2pG-7aG, CBD-(pG-7aG)2, CBD-2pG-14aG, CBD-2pG-8aG, CBD-(pG-8aG)2, CBD-2pG-16aG, CBD-2pG-(6 / 7 / 8aG) n and CBD oligosaccharides which can be (1-2)-, (1-3)-, (1-4)- or (1-6)-alpha-linked, or esters thereof, or acetal, or hemiacetal derivatives thereof or esters of the latter.

[0030] (9) The method according to any one of (1) to (5), wherein E. coli or a yeast strain, such as S. cerevisiae or P. pastoris, is used as production microorganism.

[0031] (10) The method according to any one of (1) to (5), wherein UDP-carbohydrate dependent glycosyltransferases and a cyclic maltodextrin glucanotransferase and / or a glucansucrase are heterologously expressed in cannabis and cannabinoids such as cannabidiol from the secondary metabolism of the plant are used as substrates for conversion into the respective cannabinoid oligosaccharides and are obtainable in this way.

[0032] (11) The method according to any one of (1) to (5) and (9), wherein UDP-carbohydrate dependent glycosyltransferases are heterologously expressed in cannabis and wherein the glucose residues on the 2’ and 6’ hydroxyl groups of cannabinoids such as cannabichromene serve as protecting groups against or reduce metabolism and cyclization into tetrahydrocannabinol.

[0033] (12) A composition obtainable by the method according to any one of (1) to (11), comprising

[0034] - a cannabinoid oligosaccharide, such as a cannabidiol oligosaccharide,

[0035] - a component of the reaction medium according to (1) to (3), or

[0036] - a component of the organism according to (3), (9), (10), (11), and / or

[0037] - ingredients of the culture medium according to (3), (9) and degradation products thereof;

[0038] The composition can optionally contain unconverted cannabinoid substrates, such as cannabidiol and by-products derived from the cannabinoid oligosaccharides, for example acetates with variable substitution sites on the glucopyranosyl residues.

[0039] (13) Further method, by which the cannabinoid oligosaccharides obtained by the method according to any one of (1) to (11) are enriched and purified from the composition according to (12) in a second step and in which extraction with an organic solvent, preferably ethyl acetate, and adsorption to a lipophilic solid phase, preferably on a polystyrene matrix, are used for enrichment and purification and elution from the matrix using an organic solvent, preferably methanol.

[0040] (14) Method according to any one of (1) to (8) and (10) to (13), wherein the first step for the subsequent enrichment and purification of the cannabinoid oligosaccharides obtained from the organism Cannabis is an extraction with an aqueous phase, or a hot aqueous phase (water or steam), or a polar solvent, or a mixture of polar solvents.

[0041] (15) Kit for the production, purification and analysis of cannabinoid oligosaccharides, such as cannabidiol oligosaccharides, according to (1) to (14), comprising the genes and / or enzymes and and / or DNA sequences, or codon-optimized DNA sequences, or amino acid sequences, or isolated DNA, or DNA in a vector or expression vector, or enzymes or enzyme compositions containing the same, or the genes and / or enzymes of the cyclomaltodextrin glucanotransferase and / or the glucansucrase in the form of transgenic microorganisms or transgenic plants, as well as specific means and instructions for carrying out the production and purification processes and for the analysis and quality control of the products.

[0042] (16) Cannabinoid oligosaccharides, such as optionally substituted cannabidiol oligosaccharides, such as acetylated or peracetylated cannabidiol oligosaccharides, according to any one of (1) to (14), for use as biologically active compounds, as drugs or veterinary drugs, which can have improved water solubility compared to the corresponding free cannabinoids, can have solubilizing or emulsifying effects on other glycosides of cannabidiol or other cannabinoids with fewer carbohydrate residues (e.g. CBD-1 βG, CBD-2 βG) or free cannabinoids or other components of the composition with low solubility in a composition, such as aroma compounds or other biologically active compounds in a beverage, have a different taste or mouthfeel, different physical absorption, altered metabolism, altered activity or altered pharmacokinetics.

[0043] (17) Composition, comprising

[0044] - cannabinoid oligosaccharides, such as cannabidiol oligosaccharides; and

[0045] - at least one glycosidase, preferably at least one ß-glycoside enzyme, or a glycosidase composition, preferably a ß-glycoside enzyme; and / or

[0046] - a microorganism producing a glycosidase, preferably a ß-glycoside enzyme; and / or

[0047] - amylase and / or

[0048] - encapsulated form of the single, some or all of the components of the composition,

[0049] for use as an active pharmaceutical ingredient, a drug or a veterinary drug.

[0050] (18) A method for obtaining a concentrate in an aqueous solution using the cannabinoid oligosaccharides, such as cannabidiol oligosaccharides and / or substituted derivatives according to claim 16 and / or the compositions with these substances according to (17), whereby an intermediate product for the purposes of (16) and (17) is obtained, which can advantageously be used for the production of the corresponding end product, since such concentrates are more durable or better stored or transported due to their smaller volume and weight; such concentrates show a concentration of at least twice the cannabinoid oligosaccharides or their derivatives compared to the concentration in the corresponding end product (e.g. a beverage), more preferably at least a five-fold concentration in the concentrate.

[0051] (19) Use of cannabinoid oligosaccharides with mixed a- and ß-pyranoglucosyl structures for differential release of the aglyca depending on the presence of organ-specific (e.g. oral cavity, stomach, small intestine and large intestine) hydrolytic enzymes / enzymes with glycosidase activity (e.g. various human amylases, e.g. microbial ß-glucosidases) for the purpose of targeted drug delivery. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 Chemical structure of cannabidiol (top), note that positions 2' and 6' are equivalent by rotation around the resorcinol-cyclohexene linkage. Chemical structure of CBD-2ßG (bottom) ("ß-backbone glycosylation").

[0053] Figure 2Cannabidiol-2'6'-di-β-D-glucopyranoside, a key cannabinoid oligosaccharide, was obtained by reaction of cannabidiol-2'6'-di-β-D-glucopyranoside with γ-cyclodextrin under catalysis of a cyclomaltodextrin glucanotransferase. It consists of one 2',6'-β-main glycosylation and one modular α-glycosylation consisting of a chain of 6, 7 or 8 or >8 α-glucopyranosyl residues, in this example 8 (n=7, n+1 is the terminal glucopyranosyl). A lower number of (1-4)-α-D-glucopyranoside residues is often observed, due to the previously reported disproportionating and hydrolyzing activity of the cyclomaltodextrin glucanotransferase (“interleaved cannabinoid oligosaccharides”). R1 and R2 can also each be such a modular α-glucopyranosyl chain. The modular α-glucopyranosyl chains will have a starch-like helical structure. Cannabinoid oligosaccharides obtained with a glucan sucrose enzyme can have other linkages than (1-4)-α, like the dextransucrase of Example 6, which produces (1-6)-α-linkages.

[0054] Figure 3: GT73A-gene (NbGT-fc6; Seq1 (DNA) (=SEQ ID NO: 1), see Figure 3A Seq2 (AA) (=SEQ ID NO: 2), see Figure 3B which was used in Example 1 for the heterologous expression and biotransformation of cannabidiol to CBD-2βG, and a second gene GT73A-gene (CaGT2 Seq3 (DNA) (=SEQ ID NO: 3), see Figure 3C Seq4 (AA) (=SEQ ID NO: 4), see Figure 3D from Catharanthus roseus), which also allows the production of CBD-2βG.

[0055] Figure 4 : Synthesis of CBD oligosaccharides from CBD-2βG (Example 1), aliquots were analyzed by HPLC. Enzymatic conversion of CBD-2βG with CGTase and (figure from top to bottom) a-, β-, γ-cyclodextrin and starch as co-substrates, and a control with CBD-2βG only. Products were observed in all co-substrates. The peak eluting before the CBD-2βG peak (4.2 min) is CBD-2βG-1αG (3.9 min). The peaks eluting before this (at about 3.6 min) are CBD-2βG-2αG and CBD-2βG-3αG (minor peak at about 3.4 min).

[0056] Figure 5 : Analysis of aliquots by HPLC of the synthesis of the CBD-2βG interdigitated CBD oligosaccharides (Example 2). Bottom trace: Control: CBD-2βG and γ-cyclodextrin substrates, no CGTase enzyme reaction. Top trace: Peaks formed eluting before CBD-2βG (at ~4.1 min) are CBD-2βG-1αG (at ~3.9 min), CBD-2βG-2αG (at ~3.6 min) and CBD-2βG-3αG (at ~3.4 min minor peak) representing part of the products present CBD2βG-8αG.

[0057] Figure 6: LC-MS analysis of the interdigitated CBD oligosaccharides (Example 3). Top graph ( Figure 6a ): Top box: Total ion current chromatogram. Lower boxes: Chromatograms of individual compounds in negative mode at their respective m / z, CBD-2βG m / z 683 [M+HCOOH-H] - from formic acid from the solvent (second box), CBD-2βG-1αG m / z 799 [M-H] - (third box), CBD-2βG-2αG m / z 961 [M-H]- (fourth box), CBD-2βG-3αG m / z 1123 [M-H] - (fifth box). Bottom graph ( Figure 6b ): MS / MS2 spectra of CBD-2βG m / z 683 [M+HCOOH-H] - (first box), CBD-2βG-1αG m / z 799 [M-H] - (second box), CBD-2βG-2αG m / z 961 [M-H]- (third box), CBD-2βG-3αG m / z 1123 [M-H] - (fourth box). MS / MS2 spectra of all compounds show the expected fragmentation, losing one or more glucopyranose residues (time (-162 D).

[0058] Figure 7Hydrolytic cleavage of the interleaved CBD oligosaccharide composition CBD-2βG-1αG, CBD-2βG-2αG, and CBD-2βG-3αG using saliva (top panel, Example 4) or β-glycosomal enzymes (middle panel, Example 5), and a control without hydrolytic enzymes (substrate shown, bottom panel). The CBD-2βG peak eluted at 4.2 min, followed by CBD-2βG-1αG (3.9 min), CBD-2βG-2αG (3.6 min), and CBD-2βG-3αG (previously eluted in small amounts). The hydrolysates were CBD-2βG and CBD-1βG (5.5 min).

[0059] Figure 8 HPLC analysis of the formation of interleaved CBD oligosaccharides from CBD-2βG by dextran sucrase of *Lactobacillus licorice*™ 1.1822 (top box) and *Lactobacillus nereis*™ 1.1827 (bottom box) (Example 6). The curves from bottom to top represent the control (0), 1, 2, and 4 hours of incubation for the enzyme reaction. The substrate CBD-2βG eluted at approximately 4.2 minutes, and the products CBD-2βG-1αG, CBD-2βG-2αG, and CBD-2βG-3αG (all with (1-6)-α linkages) eluted prior to this with decreasing elution time. Detailed Implementation

[0060] Surprisingly, the discovery of substances such as cannabidiol (CBD) Figure 1 The cannabinoids at the top contain only small amounts of β-D-glucopyranose residues, such as CBD-1βG or CBD-2βG ( Figure 1 The bottom layer is an excellent substrate for cyclomaltodextrin glucan transferase.

[0061] CBD-2βG was chosen for demonstration because it has β-D-glucopyranosyl residues on both hydroxyl functional groups, making it the simplest and most representative structure of this type. This allows for the specific transfer of six, seven, or eight (or more) (1-4)-α-D-glucopyranosyl residues from linear substrates with a polymerized (1-4)-α-D-glucopyranosyl structure (such as starch or glycogen) from cyclodextrins, substituted cyclodextrins (such as hydroxypropyl-, methyl-), or mixtures of these residues. Figure 2 ).

[0062] Therefore, highly glycosylated cannabinoids will be obtained, possessing the inherent and expected amphiphilic structure characteristic of surfactants / solvents, as well as a mixed end-isomer structure (β / α). Consequently, not only excellent water solubility can be expected, but also solubilization of compounds with lower water solubility, such as cannabinoids, or those with lower glycosylation patterns, and the glycoside release mechanism will be altered.

[0063] As with the β-D-glucopyranoside residues under the action of β-D-glucosidases, the α-D-glucopyranoside residues can be cleaved by the activity of amylases found in the human digestive tract (saliva, small intestine), allowing a stepwise and presumably more targeted degradation and release of the glycoside aglycone for the purpose of targeted drug delivery.

[0064] A positive influence on the taste profile of the product seems to be achievable as well. Such derivatives with long hydrophilic residues (as the well-known polysorbates) have optimal properties for various water-based products in the pharmaceutical and other fields.

[0065] Definitions

[0066] For the description of the present application, the terms are understood as follows:

[0067] Amylase

[0068] Specific glycosidase for the hydrolysis of (1-4)-a-D-oligo- or polysaccharides such as starch or compounds with (1-4)-a-linked oligosaccharide residues. The Greek prefix refers to the enzyme subclass, not the anomeric form of the glycoside. a-Amylase cleaves randomly and produces dextrins ((1-4) a-linked oligosaccharides), maltotriose (a trisaccharide unit of three 1-4-linked a-D-glucose molecules), maltose (a disaccharide unit of two 1-4-linked a-D-glucose molecules), and glucose. ß-Amylase end-cleaves maltose, and g-Amylase end-cleaves the glucose monosaccharide unit. a-Amylases are found in animals (salivary glands, pancreas), plants, microorganisms. ß-Amylases are found in plants (seeds, fruits) and microorganisms. g-Amylases are found in animals (small intestine) and microorganisms.

[0069] Anomer

[0070] Diastereoisomer of a carbohydrate, here in particular D-glucose, refers to the acetylhydroxyl function on carbon atom 1 in the glycoside, corresponding to the a- or ß-form of the glycoside.

[0071] Amphiphile

[0072] Molecules with both hydrophilic and lipophilic parts are called amphiphilic molecules.

[0073] Cannabidiol

[0074] 2-[(1R,6R)-3-methyl-6-prop-1-en-2-yl-1-cyclohex-2-enyl]-5-pentylbenzo-1,3-diol

[0075] Cannabinoid 2-[(1R,6R)-3-methyl-6-prop-1-en-2-yl-1-cyclohex-2-enyl]-5-pentylbenzo-1,3-diol

[0076] As used herein, it refers to a secondary metabolite specific to Han- f a (Cannabis) and is a pentylphenol isoprenylated.

[0077] Cannabinoid oligosaccharide

[0078] As used herein, the term refers to cannabinoids with zero to four beta-D- glucopyranosyl residues and one or more additional ((1-4)-alpha-D- glucopyranosyl) 5、6、7或>7 (1-4)-alpha-D-glucopyranosyl oligosaccharide residues. Figure 2 Key exemplary structures are given in the

[0079] Interleaved cannabinoid oligosaccharide

[0080] Cannabinoid oligosaccharides can have a lower number of (1-4)-alpha-D- glucopyranose residues compared to simple cyclodextrin residue transfer by partial transglycosylation, partial retransglycosylation (dissimilation) or partial hydrolysis. They can also be obtained by using a dextransucrase and a disaccharide such as sucrose as co-substrate, in which case they can not only be (1-4)-alpha-linked, but also (1-2)-, (1-3)- or (1-6)-alpha-linked.

[0081] Cyclomaltodextrin glucanotransferase

[0082] Transglycosylation by ring opening from alpha-, beta-, gamma- or higher cyclodextrin (here the Greek prefix refers to the cyclodextrin type) or by helix opening from starch to ((1-4)-alpha-D-glucopyranosyl) 5、6、7或>7 (1-4)-alpha-D-glucopyranoside oligosaccharides. Definition follows https: / / www.cazypedia.org / index.php / Glycoside_Hydrolase_Family_13.

[0083] Glycoside

[0084] Chemical structure formed from a carbohydrate and an alcohol, amine or thiol (corresponding to an O-, N- or S-glycoside). Here only O-glycosides are considered.

[0085] Glucansucrase

[0086] GH70 dextransucrase (GS) family catalyzes the synthesis of alpha-dextran from sucrose. Definition follows https: / / www.cazypedia.org / index.php / Glycoside_Hydrolase_Family_7.

[0087] https: / / www.cazypedia.org / index.php / Glycoside_Hydrolase_Family_7.

[0088] Glucansucrases

[0089] Dextran sucrose can form the alternating saccharide (alteran) a-(1-3), (1-6), dextran a-(1-6), reuteran a-(1-4) or mutan a-(1-3).

[0090] Glycoside

[0091] Specific glycosides with glucose as the binding carbohydrate. As with glycosides, a- and ß-D-glycosides are also distinguished, depending on the position of the oxygen atom forming the glycosidic bond in the standard projection relative to the carbohydrate (a-glycoside: oxygen is up relative to the ring, in ß-glycosides it is down) (other definitions relate to the configuration of C5).

[0092] Glycosidase

[0093] Enzyme with hydrolytic activity on glycosides, which is the cleavage of a glycoside with uptake of one water molecule. ß-glycosidases can cleave ß-glycosides such as ß-glycosides. Glycoside enzymes are special glycosidases or their specific activity.

[0094] Lipophilic

[0095] Respectively, have an affinity for fats or oils. The term describes molecules with low polarity.

[0096] Transglycosylation

[0097] Transfer of a carbohydrate residue from one glycoside to another.

[0098] Example

[0099] Specific chemicals used

[0100] ß-amylase was obtained from Roth (8717.1), Germany. The cyclomaltodextrin glucanotransferase (CGTase) was obtained from Amano, Japan. The dextran sucrose was obtained directly from the culture of the Lactobacillus. Rapidase (a mixture of four ß-glycosidases) was obtained from DSM Food specialties B.V., The Netherlands, under the trade name Rapidase AR 2000.

[0101] Abbreviations

[0102] The abbreviations used in the present invention are:

[0103] CBD Cannabidiol

[0104] CBD-1 βG Cannabidiol-2'-ß-D-glucopyranoside

[0105] CBD-2 βG Cannabidiol-2',6'-ß-D-glucopyranoside

[0106] CBD-2 βG-6 αG Cannabidiol-2'-ß-D-glucopyranosyl-((1-4)-a-D-glucopyranosyl)) 5-(1-4)-a-D-glucopyranoside, 6'-ß-D-glucopyranoside

[0107] CBD-(βG-6αG)2 Cannabidiol-2'-ß-D-glucopyranosyl-((1-4)-a-D-glucopyranosyl)) 5-(1-4)-a-D-glucopyranoside, 6'-ß-D-glucopyranosyl-((1-4)-a-D-glucopyranosyl)) 5-(1-4)-a-D-glucopyranoside

[0108] CBD-2 βG-12 αG Cannabidiol-2'-ß-D-glucopyranosyl-((1-4)-a-D-glucopyranosyl)) 11-(1-4)-a-D-glucopyranoside, 6'-ß-D-glucopyranoside

[0109] CBD-2 βG-7 αG Cannabidiol-2'-ß-D-glucopyranosyl-((1-4)-a-D-glucopyranosyl)) 6-(1-4)-a-D-glucopyranoside, 6'-ß-D-glucopyranoside

[0110] CBD-(βG-7αG)2 Cannabidiol-2'-ß-D-glucopyranosyl-((1-4)-a-D-glucopyranosyl)) 6-(1-4)-a-D-glucopyranoside, 6'-ß-D-glucopyranosyl-((1-4)-a-D-glucopyranosyl)) 6-(1-4)-a-D-glucopyranoside

[0111] CBD-2 βG-14 αG Cannabidiol-2'-ß-D-glucopyranosyl-((1-4)-a-D-glucopyranosyl)) 13-(1-4)-a-D-glucopyranoside, 6'-ß-D-glucopyranoside

[0112] CBD-2 βG-8 αG Cannabidiol-2'-ß-D-glucopyranosyl-((1-4)-a-D-glucopyranosyl)) 7-(1-4)-a-D-glucopyranoside, 6'-ß-D-glucopyranoside Figure 2 )

[0113] CBD-2βG-16αG Cannabidiol-2'-beta-D-glucopyranosyl-((1-4)-alpha-D- glucopyranosyl))15-(1-4)-alpha-D-glucopyranoside, 6'-beta-D-glucopyranoside

[0114] CBD-2βG-16αG Cannabidiol-2'-beta-D-glucopyranosyl-((1-4)-alpha-D- glucopyranosyl))15-(1-4)-alpha-D-glucopyranoside, 6'-beta-D-glucopyranoside

[0115] CBD-2βG-(6 / 7 / 8αG) n Cannabidiol-2'-beta-D-glucopyranosyl-((1-4)-alpha-D-glucopyranosyl)) 5,6,7 -(1-4)-alpha-D-glucopyranoside, 6'-beta-D-glucopyranoside (a mixture of compounds with variable chain length obtained by using a starch, glycogen, dextrin or cyclodextrin mixture as substrate), which includes compounds with multiple (n) such substituents in different positions.

[0116] CGTase Cyclomaltodextrin glucanotransferase

[0117] GT Glycosyltransferase

[0118] mg milligram

[0119] min minute

[0120] mL milliliter

[0121] mM millimolar

[0122] rpm revolutions per minute, centrifugation speed

[0123] w / v weight / volume; concentration value

[0124] Example 1

[0125] CBD-2βG with cyclomaltodextrin glucanotransferase forms CBD-oligosaccharides

[0126] The glycosyltransferase UGT73A gene from tobacco (SEQ 1 (Figure 3)) in an expression vector was used for glycosylation of cannabidiol to CBD-2βG by bioconversion in E. coli BL21 (DE3) pLysS (Novagen, Schwalbach, Germany). Pre-cultures were grown overnight at 37°C in Luria Bertani (LB) medium containing 100 μg / mL ampicillin and 23 μg / mL chloramphenicol.

[0127] The next day, 50 mL M9-minimal medium containing 1% glucose and the antibiotics were inoculated with 1 mL of the pre-culture and incubated at 37°C and 160 rpm until an OD600 (optical density at 600 nm) of 1 was reached. Subsequently, incubation was performed at 18°C and induction was performed by supplying 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside). After 6 hours, 5 mg of the substrate cannabidiol (CBD isolate 99.034%, DB labs, Las Vegas, NV, USA) was added. At fixed time intervals, 200 μL samples were taken from the culture. Cells were removed from the samples by centrifugation and diluted 1 :2 for HPLC analysis to control the progress of the bioconversion.

[0128] CBD-2βG was purified by polystyrene matrix adsorption and methanol elution.

[0129] For the cyclomaltodextrin glucanotransferase reaction, each 0.0175 mg CBD-2βG from the stem solution was incubated with 2.25 U CGTase (Amano) in 25 mM sodium acetate buffer (pH 5.5) and 0.056 mg a-, β-, γ-cyclodextrin or starch from the stem solution (final volume 0.14 mL) and incubated overnight at room temperature. Aliquots of each reaction were analyzed by HPLC Figure 4 ) at different time points.

[0130] Example 2

[0131] Synthesis of CBD-2βG-derived oligosaccharides

[0132] CBD-2βG, the intermediate product of Example 1, was used for the planned synthesis of CBD-2βG-8aG. For this, 5 mg CBD-2βG was incubated with 450 U CGTase (Amano) in 25 mM sodium acetate buffer (pH 5.5) and 15 mg γ-cyclodextrin (final volume 5 mL) and incubated overnight at room temperature. Aliquots of each reaction were analyzed by HPLC Figure 5).

[0133] Example 3

[0134] LC-MS-MS2 analysis of CBD-2βG derived oligosaccharides

[0135] The analysis was performed by liquid chromatography coupled to mass spectrometry using a Bruker esquire 3000 plus (Bruker Daltonics, Bremen, Germany) mass spectrometer equipped with an Agilent 1100 HPLC system, an Agilent 1100 / 1200 microplate autosampler 1 and an Agilent 1100 / 1200 diode array detector SL 1. For mass spectrometry, a dual ion source (DUIS) was used and operated in negative mode of ionization.

[0136] Separation was performed on a reversed phase column LUNA C18 100A 150 x 2 mm (Phenomenex, Aschaffenburg, Germany). Milli-Q purified water (A) and methanol (B) were used as mobile phases, each containing 0.1 % formic acid. The flow rate was 0.2 mL min −1 , with a gradient of: 0 min 40% B, 15 min 100% B, 20 min 40% B, 30 min 40% B. The sample injection volume was 5 μL. The mass spectrometry flow was adjusted to 0.2 mL by a flow splitter. The mass spectra were recorded from 50 m / z to 1000 m / z, negative ionization, with a scan speed of 4000 V and an interface voltage of -500 V, using nitrogen as collision gas.

[0137] Figure 3 shows the LC-MS analysis of the biotransformation products of cannabidiol to CBD oligosaccharides of Example 2. The results show that the anastrophic cannabinoid oligosaccharides CBD-2βG-1αG, CBD-2βG-2αG and CBD-2βG-3αG have been obtained as a mixture (Figure 6). The hydrolytic cleavage experiments (Example 4) further support the structures regarding the anomeric character of the glucopyranose residues.

[0138] Example 4

[0139] Hydrolytic cleavage of the anastrophic oligosaccharides CBD-2βG-1αG, CBD-2βG-2αG and CBD-2βG-3αG with saliva

[0140] A 0.2 mL solution of 0.5 mg / mL of the interlaced oligosaccharides CBD-2βG-1αG, CBD-2βG-2αG, CBD-2βG-3αG and 0.5 mg / mL CBD-2βG (remaining substrate) from Example 2 was mixed with 0.1 mL of saliva and incubated at 30°C for 24 hours. The sample was analyzed by HPLC (Figure 6). Figure 7 Top graph, control at bottom graph.

[0141] The results show that saliva hydrolyzes the α-glucopyranosyl residues of this cannabinoid oligosaccharide composition faster than the β-glucopyranosyl backbone glycosylation (remaining as CBD-2βG) in this assay.

[0142] Example 5

[0143] ß-glycosidase hydrolytic cleavage of the interlaced oligosaccharides CBD-2βG-1αG, CBD-2βG-2αG and CBD-2βG-3αG

[0144] To a 0.2 mL solution of 0.5 mg / mL of the interlaced oligosaccharides CBD-2βG-1αG, CBD-2βG-2αG, CBD-2βG-3αG and 0.5 mg / mL CBD-2βG (remaining substrate) from Example 2 was added 5 mg Rapidase (a mixture of β-glycosidases) dissolved in 0.1 mL water and incubated at 30°C for 24 hours. The control used 0.1 mL water instead of the enzyme solution. The sample was analyzed by HPLC (Figure 7). Figure 7 Middle graph, control at bottom graph.

[0145] The results show that degradation of CBD-2βG at the backbone glycosylation and removal of the glucopyranosyl residue is possible, as foreseen in the use of glycosidic aglycone-releasing cannabinoid diterpene oligosaccharide compounds and compositions. The free aglycone is insoluble in water and cannot be determined with this assay, only the degradation intermediate CBD-1βG, which is still soluble in water, can be observed.

[0146] Example 6

[0147] CBD-2βG with dextransucrase to form CBD-oligosaccharides

[0148] Strains Lactobacillus bifidus TM 1.1822 and Lactobacillus namurensis TM 1.1827, which form the backbone of dextransucrase (dextran sucrose), were inoculated each with 50 mL of MRS medium and incubated overnight at 35°C and 200 rpm. Each of the two cultures of 50 mL was centrifuged at 4.700 rpm for 15 min. Each cell pellet was resuspended in 1 mL of 100 mM citrate buffer (pH 6) supplemented with 0.25 mg CBD-2pG and 10 mg sucrose, incubated at 30°C and shaken at 300 rpm. At 1, 2 and 4 hours, 0.2 mL samples were taken for analysis and centrifuged at 15.000 rpm for 15 min.

[0149] HPLC analysis of both reaction mixtures showed that dextransucrase from Lactobacillus bifidus TM 1.1822 and Lactobacillus namurensis TM 1.1827 formed alternating CBD oligosaccharides from CBD-2pG. The products were CBD-2pG-1aG, CBD-2pG-2aG and CBD-2pG-3aG ((1-6)-a-linked) Figure 8 ); with lower efficiency than the a-(1-4)-linked products produced with CGTase. From the known product specificity of Lactobacillus bifidus TM 1.1822 and Lactobacillus namurensis TM 1.1827 as dextran sucrose, the linkage of the a-glucopyranosyl residues must be a-(1-6), unlike the CGTase products with a-(1-4) linkages.

Claims

1. A method of producing a cannabinoid-oligosaccharide, such as cannabidiol (CBD)- oligosaccharide, comprising the step of contacting a cannabinoid, such as CBD, with an isolated UDP-carbohydrate dependent glycosyltransferase, preferably of the UGT73A class, preferably with a glycosyltransferase encoded by the nucleic acid sequence of SEQ ID NO: 1 or having the amino acid sequence of SEQ ID NO: 2 or a glycosyltransferase encoded by the nucleic acid sequence of SEQ ID NO: 3 or having the amino acid sequence of SEQ ID NO: 4, or with a UDP-carbohydrate dependent glycosyltransferase having at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% nucleic acid sequence identity to SEQ ID NO: 1 or at least 70%, at least 80%, at least 90%, or at least 95% amino acid sequence identity to SEQ ID NO: 2, or with a UDP-carbohydrate dependent glycosyltransferase having at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% nucleic acid sequence identity to SEQ ID NO: 3 or at least 70%, at least 80%, at least 90%, at least 95%, at least 98% amino acid sequence identity to SEQ ID NO: 4, in a reaction mixture under conditions including any co-substrates and cofactors necessary for the glucosyltransferase activity, effective to produce a cannabinoid-beta-D-glycoside, and subsequently or in parallel, contacting this or a cannabinoid-beta-D-glycoside from another source, with at least one of a cyclomaltodextrin glucanotransferase and its substrate cyclodextrin or substituted cyclodextrin (e.g. hydroxypropyl-, methyl-), linear maltodextrin, starch or glycogen, and purifying the cannabinoid oligosaccharide from the reaction mixture to obtain an isolated cannabinoid oligosaccharide.

2. The method of claim 1, wherein the isolated UDP-carbohydrate dependent glycosyltransferase uses a cannabinoid, such as CBD, and as co-substrate UDP-glucose or fructose, glucose, galactose, mannose, rhamnose, ribose, arabinose, xylose, activated forms thereof, or activated oligosaccharides, or activated aldoses or uronic acids, such as glucuronic acid, glucoronic acid or galacturonic acid, or N-substituted derivatives of said carbohydrates, such as 2-N-acetylglucosamine, and wherein the cyclomaltodextrin glucanotransferase uses the intermediate product (or added lychnophore from another source) cannabinoid-beta-D-glycoside as substrate to form higher glycosylated oligosaccharides (the latter having alpha-anomeric pyranoglucose residues), and the product is a corresponding cannabidiol oligosaccharide with a backbone beta-D-glycoside structure and branching alpha-pyranoglucose oligomers.

3. The method according to claim 1 or 2, wherein the isolated UDP-carbohydrate dependent glycosyltransferase and the cyclomaltodextrin glucanotransferase are obtained by heterologous expression in an organism of one or both species different from the original species of the glycosyltransferase or the cyclomaltodextrin glucanotransferase; preferably, the conversion of a cannabinoid into a cannabinoid-oligosaccharide is dependent on the cellular supply of a co-substrate determined to be UDP-glucose or other activated carbohydrates and presumably on the cellular supply or import of at least one of the substrates cyclodextrin or substituted cyclodextrins (e.g. hydroxypropyl-, methyl-), linear maltodextrins, starch or glycogen and occurs in the same heterologous organism which can be optimized for this purpose by genetic and / or genetic engineering means.

4. The method according to any one of claims 1 to 3, wherein instead or in addition to the cyclomaltodextrin glucanotransferase and its substrates (cyclodextrin or substituted cyclodextrins (e.g. hydroxypropyl-, methyl-), linear maltodextrins, starch or glycogen) a glucansucrase and its substrate sucrose (or other compatible disaccharides) is used to add (1-2)-, (1-3)-, (1-4)-, or (1-6)-linked a-D-glucopyranoside residues to produce a scrambled cannabinoid oligosaccharide.

5. The method according to any one of claims 1 to 4, wherein a cannabinoid oligosaccharide is obtainable, for example but not limited to, wherein CBD-2pG-8aG and / or higher reaction products or scrambled cannabinoid oligosaccharides are obtained from cannabidiol with GT40 / UDP-glucose and with cyclomaltodextrin glucanotransferase / gamma-cyclodextrin or with glucansucrase and sucrose (or other disaccharides).

6. Cannabinoid oligosaccharide obtained by the method according to any one of claims 1 to 5, whose glycosylation pattern comprises zero or one to four beta-D-glucopyranosyl backbone residues and one or more additional ((1-4)-alpha-D-glucopyranosyl) 5, 6, 7 or >7 (1-4)-alpha-D-glucopyranoside n Oligosaccharide residues or interlaced cannabinoid oligosaccharides, which can also be derived by other glycosyltransferases or transglycosylation reactions than the cyclomaltodextrin glucanotransferase, such as the dextransucrase, and in this case not only (1-4)- but also (1-2)-, (1-3)- or (1-6)-linked, (1-4)-alpha-D-glucopyranoside residues can be attached directly to the cannabinoid, its beta-D-glucopyranose residues, or, if more than one, also to each other.

7. The CBD oligosaccharide obtained according to claim 6 comprising zero or one to four beta-D-glucopyranosyl stem residues and one or more additional ((1-4)-alpha-D-glucopyranosyl) 5, 6, 7 or >7 (1-4)-alpha-D-glucopyranoside n The oligosaccharide residue is either a staggered cannabinoid oligosaccharide which can be (1-2)-(1-3)-(1-4)- or (1-6)-alpha-linked.

8. The compounds CBD-2βG-6αG, CBD-(βG-6αG)2, CBD-2βG-12αG, CBD-2βG-7αG, CBD-(βG-7αG)2, CBD-2βG-14αG, CBD-2βG-8αG, CBD-(βG-8αG)2, CBD-2βG-16αG, CBD-2βG-(6 / 7 / 8αG) n and interlaced cannabinoid oligosaccharides, the latter can be (1-2)-, (1-3)-, (1-4)- or (1-6)-a-linked, or esters thereof, or acetal, or hemiacetal derivatives thereof or esters of the latter.

9. The method according to any one of claims 1 to 5, wherein E. coli or a yeast strain, for example Saccharomces cerevisiae or Pichia pastoris, is used as a production microorganism.

10. The method according to any one of claims 1 to 5, wherein the UDP-carbohydrate dependent glycosyltransferase and the cyclomaltodextrin glucanotransferase and / or the glucansucrase are heterologously expressed in Cannabis sativa and a cannabinoid from plant secondary metabolism, such as cannabidiol, is used as a substrate for conversion into the corresponding cannabinoid oligosaccharide and these are obtainable by this means.

11. The method according to any one of claims 1 to 5 and 9, wherein the UDP-carbohydrate dependent glycosyltransferase is heterologously expressed in Cannabis sativa and wherein the glucose residues on the 2’ and 6’ hydroxyl groups of a cannabinoid, such as cannabidiolic acid, act as protecting groups, preventing or reducing metabolism and cyclization into tetrahydrocannabinol.

12. A composition comprising - a cannabinoid oligosaccharide, such as a cannabidiol oligosaccharide, - components of the reaction medium according to claims 1 to 3, or - a combination thereof, obtainable by the method according to any one of claims 1 to 11. ​ ​ - a composition of a biological organism according to claim 3, 9, 10, 11, and / or - a composition of a culture medium according to claim 3, 9, and degradation products thereof; The composition can optionally contain unconverted cannabinoid substrates, such as cannabidiol and side products derived from the cannabinoid oligosaccharide, for example acetates with variable substitution sites on the glucopyranosyl residues.

13. A method by which a cannabinoid oligosaccharide obtained by the method according to any one of claims 1 to 11 is enriched and purified in a second step from a composition obtained according to claim 12, and wherein extraction with an organic solvent, preferably ethyl acetate, and adsorption to a lipophilic solid phase, preferably on a polystyrene matrix, is used for enrichment and purification, and elution from the matrix using an organic solvent, preferably methanol.

14. The method according to any one of claims 1 to 8 and 10 to 13, wherein the first step for obtaining a cannabinoid oligosaccharide from the organism cannabis is an extraction with an aqueous phase, or a hot aqueous phase (water or steam), or a polar solvent, or a mixture of polar solvents.

15. A kit for the production, purification and analysis of cannabinoid oligosaccharides, such as cannabidiol oligosaccharides, according to any one of claims 1 to 14, comprising the gene and / or enzyme and and / or DNA sequence, or codon-optimized DNA sequence, or amino acid sequence, or isolated DNA, or DNA in a vector or expression vector, or enzyme or enzyme composition containing the same, or the gene and / or enzyme of the cyclomaltodextrin glucanotransferase and / or the glucansucrase in the form of a transgenic microorganism or transgenic plant, and specific means and instructions for carrying out the production and purification processes and for the analysis and quality control of the product.

16. A cannabinoid oligosaccharide, such as a cannabidiol oligosaccharide, optionally substituted on the glycoside residues, such as acetylated or peracetylated cannabidiol oligosaccharides, according to any one of claims 1 to 14, for use as a biologically active compound, as a pharmaceutical or veterinary drug, which can have improved water solubility compared to the corresponding free cannabinoid, can have an effect on other glycosides of cannabidiol or other cannabinoids with fewer carbohydrate residues (e.g. CBD-1 βG, CBD-2 βG) or free cannabidiol or other free cannabinoids, or other components in a composition with low solubility, such as solubilization or emulsification of aroma compounds or other biologically active compounds in a beverage, a different taste or mouthfeel, different physical absorption, altered metabolism, altered activity or altered pharmacokinetics.

17. A composition comprising - a cannabinoid oligosaccharide, such as a cannabidiol oligosaccharide; and - at least one glycosidase, preferably at least one β-glycoside enzyme, or a glycosidase composition, preferably a β-glycoside enzyme; and / or - a microorganism producing a glycosidase, preferably a β-glycoside enzyme; and / or - amylase and / or - an encapsulated form of the single, some or all of the components of the composition for use as an active pharmaceutical ingredient, a pharmaceutical or veterinary drug.

18. Use of cannabinoid oligosaccharides, such as cannabidiol oligosaccharides and / or substituted derivatives according to claim 16 and / or compositions with these substances according to claim 17 in concentrated aqueous solutions to obtain concentrates, whereby intermediates for the purposes according to claims 16 and 17 are obtained, which can advantageously be used for the production of the corresponding end products, since such concentrates are more durable or better stored or transported due to their smaller volume and weight; such concentrates show a concentration of cannabinoid oligosaccharides or derivatives thereof of at least twice, more preferably a concentration of at least five times in said concentrates compared to the concentration in the corresponding end product, such as a beverage.

19. Use of cannabinoid oligosaccharides with mixed alpha- and beta-glucopyranosyl structures for the differential release of the aglycone depending on the presence of organ-specific (e.g. oral cavity, stomach, small intestine and large intestine) hydrolytic enzymes / enzymes with glycosidase activity (e.g. various human amylases, such as microbial beta-glucosidases) for the purpose of targeted drug delivery.

Images