Method for producing macrocyclic cyclodextrins

Abstract

The present disclosure relates to a synthetic method for producing δ-cyclodextrin that is scalable and does not require chromatography for product isolation.

Description

[Technical Field]

[0001] The present invention relates to the production of macrocyclic organic compounds, specifically cyclodextrins. [Background technology]

[0002] Cyclodextrins (CDs) are a family of cyclic oligosaccharides consisting of a macrocyclic ring of glucopyranose subunits linked by α-1,4 glycosidic bonds.

[0003] Cyclodextrins are used to improve the water solubility and bioavailability of drugs. Because of their diverse uses, several types of pharmaceuticals may contain cyclodextrin. These include tablets, aqueous parenteral solutions, nasal sprays, and eye drops. Examples of cyclodextrins used in pharmaceuticals on the European market include β-CD in cetirizine tablets and cisapride suppositories, and γ-CD in minoxidil solutions. Examples of β-cyclodextrin derivatives used include SBE-β-CD in the intravenous antifungal voriconazole, HP-β-CD in intravenous and oral solutions of the antifungal itraconazole, and RM-β-CD in nasal sprays for hormone replacement therapy with 17β-estradiol. In Germany and Japan, infusion products containing alprostadil (prostaglandin E1, PGE1) with α-CD (EMA, 2017) are commercially available.

[0004] Cyclodextrins typically contain six to eight glucose subunits and are called α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin (also abbreviated as CD6, CD7, and CD8). Cyclodextrins are commercially produced by the enzymatic degradation of starch or amylose.

[0005] New methods for producing cyclodextrins larger than the conventional α-, β-, and γ-CDs in high yield and purity without the need for column chromatography are highly desirable. Such methods could potentially make macrocyclic CDs commercially available, preferably at low cost, and thus enable their use as excipients in new pharmaceutical formulations. Macrocyclic CDs also have potential applications in cosmetics and nutrition. Summary of the Invention

[0006] The present disclosure provides a method for producing δ-cyclodextrin and its derivatives in good yield and high selectivity, thereby enabling their isolation without the need for chromatography.

[0007] One aspect of the present disclosure provides a method for producing a δ-cyclodextrin or a δ-cyclodextrin derivative, the method comprising: a. glucose-based compounds, b. Enzymes such as cyclodextrin glucanotransferase, c.Formula (B 12 Cl n H 12-n ) 2- ions of the formula (wherein n is 1 to 12), and d. Mix the solvents forming a mixture; and allowing the mixture to incubate; Thereby, δ-cyclodextrin or a δ-cyclodextrin derivative is obtained.

[0008] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula I: [ka]

[0009] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula I, or a salt thereof: Each R is CH2OR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of: Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl.

[0010] In one aspect of the disclosure, the glucose-based compound has the structure of Formula II: [ka]

[0011] In one embodiment of the present disclosure, the glucose-based compound has the structure of Formula II: Each R is CH2OR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a, H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl; and m is an integer of 2 or greater.

[0012] One aspect of the present disclosure is a compound of formula (B 12 Cl n H 12-n ) 2- In one embodiment, n is 1 to 12. In one embodiment, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one embodiment, n is 9, 10, 11, or 12. In one embodiment, a compound of formula [B 12 Cl n H 12-n ] 2- The ions of (B 12 Cl 12 ) 2- , (B 12 Cl 11 H) 2- , (B 12 Cl 10 H2) 2- , (B 12 Cl9H3) 2- , (B 12 Cl8H4) 2- , (B 12 Cl7H5) 2- , (B 12 Cl6H6) 2- , (B 12 Cl5H7) 2- , (B 12 Cl4H8) 2- , (B 12 Cl3H9) 2- , (B 12 Cl2H10 ) 2- and (B 12 ClH 11 ) 2- In one embodiment, the compound of formula [B 12 Cl n H 12-n ] 2- The ions of (B 12 Cl 12 ) 2- , (B 12 Cl 11 H) 2- , (B 12 Cl 10 H2) 2- and (B 12 Cl9H3) 2- In one embodiment, the compound of formula (B 12 Cl n H 12-n ) 2- The ions of (B 12 Cl 12 ) 2- is.

[0013] In one embodiment of the disclosure, the enzyme, such as cyclodextrin glucanotransferase, is a cyclodextrin glucanotransferase as defined by Enzyme Commission (EC) number 2.4.1.19.

[0014] In one particular aspect, the method comprises: a. i. glucose-based compounds, ii. cyclodextrin glucanotransferase, iii.Formula (B 12 Cl n H 12-n ) 2- ions of the formula (wherein n is 1 to 12), and iv. Mixing the solvents forming a mixture; b. heating and incubating the mixture; c. partially evaporating the solvent from the mixture to form a solid phase and a liquid phase; d. Separating the solid and liquid phases of the mixture; e. adding an anti-solvent to the liquid phase to precipitate δ-cyclodextrin or a δ-cyclodextrin derivative; f. filtering the precipitated δ-cyclodextrin or δ-cyclodextrin derivative.

[0015] The template ions used in the disclosed methods can be recovered and reused to perform the disclosed methods again. Thus, in one aspect, the method comprises: 12 Cl n H 12-n ) 2- and recovering the ions of a. Obtaining a supernatant from the δ-cyclodextrin or δ-cyclodextrin derivative precipitation step; b. evaporating the anti-solvent under reduced pressure; c. adding hydrochloric acid, for example 37% hydrochloric acid, to obtain a pH of 1 to 4, for example 1.5 to 2.5; d. Adding a base, such as an amine base, for example an alkylamine base, for example a trialkylamine compound, for example triethylamine, to form a compound of formula (B 12 Cl n H 12-n ) 2- as a salt of said base; e. filtering the precipitate; As a result, the formula (B 12 Cl n H 12-n ) 2- ions as the salt of the base.

[0016] One aspect of the present disclosure provides a δ-cyclodextrin or δ-cyclodextrin derivative obtained from the method disclosed herein.

[0017] One aspect of the present disclosure provides a composition, the composition comprising: a. a glucose-based compound disclosed herein; and b. an enzyme, such as a cyclodextrin glucanotransferase, disclosed herein; c. A compound of formula (B 12 Cl n H 12-n ) 2- and ions of

[0018] One aspect of the present disclosure provides a kit of parts, the kit of parts comprising: a. a glucose-based compound disclosed herein; and b. an enzyme, such as a cyclodextrin glucanotransferase, disclosed herein; c. A compound of formula (B 12 Cl n H 12-n ) 2- and a salt containing the ion. [Brief explanation of the drawings]

[0019] [Figure 1] HPLC chromatogram showing that treatment of CD6 with CGTase in the absence of a template converts CD6 to a mixture of CD6, CD7, and CD8. [Figure 2] When CD6 is treated with CGTase without a template, the distribution of CD and linear α-1,4-glucans (G1 to G4) produced over time is shown. [Figure 3] HPLC chromatogram showing the conversion of CD6 primarily to CD9 when CD6 is treated with CGTase in the presence of Na2B12Cl12. [Figure 4-1] When CD6 was treated with CGTase in the presence of 4 mM Na2B12Cl12 at 25°C, the distribution of CDs and linear α-1,4-glucans formed over time was shown. The distribution of various CDs formed. [Figure 4-2] Distribution of CD and linear α-1,4-glucan produced over time when CD6 was treated with CGTase in the presence of 4 mM Na2B12Cl12 at 25°C. Concentration of all glucan products formed. [Figure 5-1] When CD6 was treated with CGTase in the presence of 4 mM Na2B12Cl12 at 5°C, the distribution of CDs and linear α-1,4-glucans formed over time was shown. The distribution of various CDs formed. [Figure 5-2] Distribution of CD and linear α-1,4-glucan produced over time when CD6 was treated with CGTase in the presence of 4 mM Na2B12Cl12 at 5°C. Concentration of all glucan products formed. [Figure 6-1] When CD6 was treated with CGTase in the presence of 4 mM Na2B12Cl12 at 40°C, the distribution of CDs and linear α-1,4-glucans formed over time was shown. The distribution of various CDs formed. [Figure 6-2] Distribution of CD and linear α-1,4-glucan produced over time when CD6 was treated with CGTase in the presence of 4 mM Na2B12Cl12 at 40°C. Concentration of all glucan products formed. [Figure 7-1] When CD6 was treated with CGTase in the presence of 2 mM Na2B12Cl12 at 25°C, the distribution of CDs and linear α-1,4-glucans formed over time was shown. The distribution of various CDs formed. [Figure 7-2] Distribution of CD and linear α-1,4-glucan produced over time when CD6 was treated with CGTase in the presence of 2 mM Na2B12Cl12 at 25°C. Concentration of all glucan products formed. [Figure 8-1] This is the distribution of CDs produced over time when CD6 is treated with CGTase in the presence of 30 mM Na2B12Cl12 at 25°C. This is the distribution of various CDs formed. [Figure 8-2] Distribution of CDs produced over time when CD6 was treated with CGTase in the presence of 30 mM Na2B12Cl12 at 25°C. Concentration of all glucan products formed. [Figure 9] 1 is an HPLC-ESLD chromatogram showing the purity of isolated CD9. [Figure 10] 1H NMR spectrum of isolated CD9. [Figure 11-1] 1H NMR spectra of Na2B12Cl12 in D2O before recovery from enzymatic CD9 production. Note that no peaks are expected; in fact, only solvent peaks are observed in both cases. [Figure 11-2] 1H NMR spectra of Na2B12Cl12 in D2O after recovery from enzymatic CD9 production. Note that no peaks are expected, and indeed only solvent peaks are observed in both cases. [Figure 12-1] 11B NMR spectrum of Na2B12Cl12 in D2O before recovery from enzymatic CD9 production. [Figure 12-2] 11B NMR spectrum of Na2B12Cl12 in D2O after recovery from enzymatic CD9 production. [Figure 13-1] Mass spectrum (MALDI-TOF, positive mode) of Na2B12Cl12 from enzymatic CD9 synthesis before recovery. The major peak corresponds to the sodium adduct. "60k" means "60000", etc. [Figure 13-2] Mass spectrum (MALDI-TOF, positive mode) of Na2B12Cl12 after recovery from enzymatic CD9 production. The major peak corresponds to the sodium adduct. "60k" means "60000", etc. [Figure 14] MALDI-TOF-MS of the reaction mixture after treatment of mono-6-deoxy-CD6 in the presence of Na2B12Cl12 in phosphate buffered water at room temperature for 20 days. [Figure 15] 1 is an HPLC-ELS chromatogram of the reaction mixture after treatment of mono-6-deoxy-CD6 in the presence of Na2B12Cl12 in phosphate buffered water at room temperature for 20 days. [Figure 16] 1 is a chromatogram (ELSD) showing the distribution of linear and cyclic α-1,4-glucans in the reaction of starch treated with CGTase in the presence of Na2B12Cl12. [Figure 17] Mass spectrum (MALDI-TOF) of partially chlorinated chlorododecaborate Na2B12HxCl12-x. [Figure 18] Chromatogram (HPLC-ELSD) of the reaction mixture after 24 hours of CGTase treatment of CD6 in the presence of the partially chlorinated closo-dodecaborate template Na2B12HxCl12-x. DETAILED DESCRIPTION OF THE INVENTION

[0020] definition Cyclodextrin glucanotransferase refers to an enzyme capable of catalyzing the formation of cyclodextrins from starch, glucose, and similar substrates. Synonyms and specific variants of cyclodextrin glucanotransferase include 1,4-α-D-glucopyranosyltransferase, acrilex C cyclodextrin glycosyltransferase, α-1,4-glucan 4-glycosyltransferase, cyclization, α-cgt, α-CGTase, α-cyclodextrin glucanotransferase, α-cyclodextrin glucosyltransferase, α-cyclodextrin glycosyltransferase, Bacillus macerans amylase, β-CGTase, β-cyclodextrin glucanotransferase, β-cyclodextrin glucosyltransferase, β-cyclodextrin glycosyltransferase, β-cyclodextrinase, BMA, C-CGTase, CD glucanotransferase, CGT, CGT13, CGTase, cgtS, CGTse Examples of suitable cyclodextrin glucanotransferases include ET1, CGT_TK, cyclodextrin β-glucanotransferase, cyclodextrin glucosyltransferase, cyclodextrin glycosyltransferase, cyclodextrin glycosyltransferase, cyclodextrin glycosyltransferase, cyclodextrinase, cyclomaltodextrin glucanyltransferase, cyclomaltodextrin glucosyltransferase, cyclomaltodextrin glycosyltransferase, γ-CGTase, γ-cyclodextrin glycosyltransferase, Contizyme, M-CGTase, neutral cyclodextrin glycosyltransferase, PFCGT, and Torzyme. Cyclodextrin glucanotransferase is any enzyme defined by the Enzyme Commission (EC) number 2.4.1.19 or 2.4.1.25.

[0021] The terms delta-cyclodextrin, delta-cyclomaltodextrin and CD9 are used synonymously herein.

[0022] A δ-cyclodextrin derivative refers to a macrocyclic compound consisting essentially of nine glucopyranose moieties, or a derivative of glucopyranose. This means that the ring is composed of neither more nor less than nine pyranose moieties, but the moiety need not strictly be glucopyranose, but may be a derivative thereof, for example, by replacing one or more OH groups with other moieties. Those skilled in the art will readily recognize δ-cyclodextrin derivatives based on these considerations.

[0023] The following terms have the following meanings unless otherwise specified: Terms not defined have their art-recognized meanings.

[0024] As used herein, the term "alkyl," by itself or as part of another substituent, refers to a branched or straight-chain monovalent hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of a parent alkane, alkene, alkyne, etc. Where a specific level of saturation is intended, the nomenclature "alkanyl," "alkenyl," or "alkynyl" is used. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g., propan-1-yl or propan-2-yl), butyl (e.g., butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, or 2-methyl-propan-2-yl), and the like. In some embodiments, an alkyl group contains 1 to 20 carbon atoms. In other embodiments, an alkyl group contains 1 to 10 carbon atoms. In still other embodiments, an alkyl group contains 1 to 6 carbon atoms, e.g., 1 to 4 carbon atoms.

[0025] "Alkanyl" by itself or as part of another substituent refers to a saturated branched, straight-chain, or cyclic alkyl radical derived by removing one hydrogen atom from a single carbon atom of an alkane. Typical alkanyl groups include, but are not limited to, methanyl, ethanyl, propanyl, e.g., propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc., butanyl, e.g., butan-1-yl, butan-2-yl (sec-butyl), 2-methylpropan-1-yl (isobutyl), 2-methylpropan-2-yl (t-butyl), cyclobutan-1-yl, etc., and the like.

[0026] "Alkylene" typically refers to a branched or unbranched saturated hydrocarbon chain having from 1 to 40 carbon atoms, more typically from 1 to 10 carbon atoms, and even more typically from 1 to 6 carbon atoms. This term is exemplified by groups such as methylene (-CH-), ethylene (-CHCH-), propylene isomers (e.g., -CHCHCH- and -CH(CH)CH-).

[0027] "Alkenyl," by itself or as part of another substituent, refers to an unsaturated branched, straight-chain, or cyclic alkyl radical having at least one carbon-carbon double bond derived by the removal of a hydrogen atom from a single carbon atom of an alkene. The group can be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl (vinyl), propenyl such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl, cycloprop-2-en-1-yl, butenyl such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc., and the like.

[0028] "Alkynyl" by itself or as part of another substituent refers to an unsaturated branched, straight-chain, or cyclic alkyl radical having at least one carbon-carbon triple bond derived by removing one hydrogen atom from a single carbon atom of an alkyne. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc., butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc., and the like.

[0029] "Acyl" by itself or as part of another substituent refers to the radical -C(O)R a In the formula, R ais hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, and substituted versions thereof, as defined herein. Representative examples include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, piperonyl, succinyl, malonyl, and the like.

[0030] "Alkoxy" by itself or as part of another substituent means the radical -OR b In the formula, R b represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy, and the like.

[0031] "Alkoxycarbonyl" by itself or as part of another substituent means the radical -C(O)OR c In the formula, R c represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, cyclohexyloxycarbonyl, and the like.

[0032] "Aryl" by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of an aromatic ring system. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and the like. In certain embodiments, an aryl group contains 6 to 20 carbon atoms. In certain embodiments, an aryl group contains 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl. In one embodiment, "aryl" refers to "heteroaryl."

[0033] "Arylalkyl" by itself or as part of another substituent means an alkyl group having a carbon atom (typically a terminal or sp 3 "aryl" refers to an alkyl radical in which one of the hydrogen atoms bonded to a carbon atom is replaced by an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethene-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like. In certain embodiments, an arylalkyl group is a C7-C 30 Arylalkyl, for example, the alkyl, alkenyl, or alkynyl portion of the arylalkyl group is C1-C 10 and the aryl moiety is (C6-C 20 In certain embodiments, the arylalkyl group is a C7-C 20 Arylalkyl, for example, the alkyl, alkenyl, or alkynyl portion of the arylalkyl group is (C1-C8) and the aryl portion is (C6-C 12)

[0034] "Arylaryl," by itself or as part of another substituent, refers to a monovalent hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of a ring system in which two or more identical or non-identical aromatic ring systems are directly linked by a single bond, where the number of such direct ring connections is one less than the number of aromatic ring systems involved. Typical arylaryl groups include, but are not limited to, biphenyl, triphenyl, phenylnaphthyl, binaphthyl, biphenylnaphthyl, and the like. When the number of carbon atoms in an arylaryl group is specified, that number refers to the carbon atoms comprising each aromatic ring. For example, C5-C 14 Arylaryl is an arylaryl group in which each aromatic ring contains 5 to 14 carbons, such as biphenyl, triphenyl, binaphthyl, phenylnaphthyl, etc. In certain embodiments, each aromatic ring system of the arylaryl group independently contains C5 to C6 14 In certain embodiments, each aromatic ring system of the arylaryl group is independently C5-C 10 In certain embodiments, each aromatic ring system is identical, for example, biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.

[0035] "Cycloalkyl" by itself or as part of another substituent refers to a saturated or unsaturated cyclic alkyl radical. Where a specific level of saturation is intended, the nomenclature "cycloalkanyl" or "cycloalkenyl" is used. Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane, and the like. In certain embodiments, cycloalkyl groups are C3-C6 10 In certain embodiments, the cycloalkyl group is a C3-C7 cycloalkyl.

[0036] "Cycloheteroalkyl" or "heterocyclyl," by itself or as part of another substituent, refers to a saturated or unsaturated cyclic alkyl radical in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatoms. Typical heteroatoms replacing the carbon atom(s) include, but are not limited to, N, P, O, S, Si, and the like. Where a specific level of saturation is intended, the nomenclature "cycloheteroalkanyl" or "cycloheteroalkenyl" is used. Typical cycloheteroalkyl groups include, but are not limited to, groups derived from epoxides, azirines, thiiranes, imidazolidines, morpholines, piperazines, piperidines, pyrazolidines, pyrrolidines, quinuclidines, and the like.

[0037] "Heteroalkyl, heteroalkanyl, heteroalkenyl, and heteroalkynyl," by themselves or as part of another substituent, refer to alkyl, alkanyl, alkenyl, and alkynyl groups, respectively, in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced with the same or different heteroatom groups. Exemplary heteroatom groups that can be included in these groups are -O-, -S-, -SS-, -OS-, -NR d R e -, =NN=, -N=N-, -N=N-NR f R g -PR h -, -P(O)2-, -P(O)R i -, -OP(O)2-, -SO-, -S(O)-, -S(O)2-, -SnR j R k -, etc., where R d , R e , R f , R g , R h , R i , R j and R kis independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, or substituted heteroarylalkyl.

[0038] "Heteroaryl" by itself or as part of another substituent refers to a monovalent heteroaromatic radical derived by removing one hydrogen atom from a single atom of a heteroaromatic ring system. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, arsindole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, benzodioxole, and the like. In certain embodiments, the heteroaryl group is a 5- to 20-membered heteroaryl. In certain embodiments, the heteroaryl group is a 5- to 10-membered heteroaryl. In certain embodiments, the heteroaryl group is a group derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole, and pyrazine.

[0039] "Heteroarylalkyl" by itself or as part of another substituent means an alkyl group having a carbon atom (typically a terminal or sp 3"Heteroaryl" refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom is replaced by a heteroaryl group. When specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylalkenyl, and / or heteroarylalkynyl is used. In certain embodiments, a heteroarylalkyl group is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl, or alkynyl portion of the heteroarylalkyl is 1-10 membered, and the heteroaryl moiety is a 5-20 membered heteroaryl. In certain embodiments, a heteroarylalkyl group is a 6-20 membered heteroarylalkyl, e.g., the alkanyl, alkenyl, or alkynyl portion of the heteroarylalkyl is 1-8 membered, and the heteroaryl moiety is a 5-12 membered heteroaryl.

[0040] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced with the same or different substituent(s). Exemplary substituents are alkylenedioxy (such as methylenedioxy), -M, -R l , -O - , =O, -OR l , -SR l , -S - , =S, -NR l R m , =NR l , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2O - , -S(O)2OH, -S(O)2R l , -OS(O)2O - , -OS(O)2R l , -P(O)(O - )2, -P(O)(OR l )(O - ), -OP(O)(OR l )(OR m ), -C(O)R l , -C(S)R l , -C(O)OR l , -C(O)NR l R m , -C(O)O - , -C(S)OR l , -NRn C(O)NR l R m , -NR n C(S)NR l R m , -NR n C(NR o )NR l R m and -C(NR n )NR l R m where M is a halogen and R l , R m , R n and R o are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, or optionally, R l and R m together with the nitrogen atom to which they are attached form a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, the substituents are -M, -R l , =O, -OR l , -SR l , -S - , =S, -NR l R m , =NR l , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2R l , -OS(O)2O - , -OS(O)2R l , -P(O)(O - )2, -P(O)(OR l )(O - ), -OP(O)(OR l )(OR m ), -C(O)R l , -C(S)R l , -C(O)OR - , -C(O)NR l R m , -C(O)O - , -NR n C(O)NR l Rm In certain embodiments, the substituents include -M, -R l , =O, -OR l , -SR l , -NR l R m , -CF3, -CN, -NO2, -S(O)2R l , -P(O)(OR l )(O - ), -OP(O)(OR l )(OR m ), -C(O)R l , -C(O)OR l , -C(O)NR l R m , -C(O)O - In certain embodiments, the substituents include -M, -R l , =O, -OR l , -SR l , -NR l R m , -CF3, -CN, -NO2, -S(O)2R l , -OP(O)(OR l )(OR m ), -C(O)R l , -C(O)OR l , -C(O)O - wherein R l , R m and R n is as defined above. For example, the substituent may be a methylenedioxy substituent, or have 1, 2, or 3 substituents selected from a halogen atom, a C1-C4 alkyl group, and a C1-C4 alkoxy group.

[0041] δ-Cyclodextrin and its derivatives The action of cyclodextrin glycosyltransferase enzymes on substrates containing glucopyranose polysaccharides produces almost exclusively small-cyclic cyclodextrins, i.e., α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, as kinetically trapped products. Very little macrocyclic cyclodextrins are formed. Isolation of the macrocyclic cyclodextrin products, if any, requires extensive chromatography and results in very low yields. The methods disclosed herein are capable of producing δ-cyclodextrin as the major cyclodextrin product in the reaction mixture, with little or no formation of other cyclodextrin products. This allows for the isolation of δ-cyclodextrin in high yield.

[0042] Separation of different sized cyclodextrins is difficult, if not impossible, due to similar solubilities and hydrophilicities, making separation by column or precipitation difficult. Disclosed herein is a method for producing δ-cyclodextrin and its derivatives. The method provides a crude reaction mixture containing δ-cyclodextrin or its derivatives as the predominant cyclodextrin product, e.g., δ-cyclodextrin or its derivatives is the only cyclodextrin product. Thus, the method disclosed herein allows for purification of the reaction mixture using conventional precipitation / recrystallization.

[0043] Thus, one embodiment of the present disclosure provides a method for producing a δ-cyclodextrin or a δ-cyclodextrin derivative, said method comprising: a. glucose-based compounds, b. enzymes such as cyclodextrin glucanotransferase, and c.Formula (B 12 Cl n H 12-n ) 2- ions (wherein n is 1 to 12), d. Mix the solvents forming a mixture; and allowing the mixture to incubate; Thereby, δ-cyclodextrin or a δ-cyclodextrin derivative is obtained.

[0044] The methods disclosed herein have been shown to be useful for providing both δ-cyclodextrin and its derivatives. Thus, in one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula I: [ka]

[0045] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula I, or a salt thereof: Each R is CH2OR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of: Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl.

[0046] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula Ia: [ka]

[0047] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula Ib: [ka]

[0048] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of formula Ib: Each R' is CH2OR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of Each R is OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of: Each R ais independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl.

[0049] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula Ic: [ka]

[0050] In one embodiment, R is CHOR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of: Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl.

[0051] The glucopyranose moieties comprising a δ-cyclodextrin or derivative thereof can have different substitution patterns, i.e., one or more of the glucopyranose moieties of a δ-cyclodextrin or derivative thereof can be different. Thus, in one embodiment of the present disclosure, a δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula I or Formula Ib, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 of the R and / or R' moieties is selected from the group consisting of OR, ... a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a and Si(R a ) 3.

[0052] In one embodiment of the disclosure, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula I or Formula Ib, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 of the R and / or R′ moieties is OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a and Si(R a )3, the remainder being selected from the group consisting of CHOR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a, Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a is selected from the group consisting of:

[0053] In one embodiment of the disclosure, the δ-cyclodextrin derivative has the structure of Formula Ia, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the R moieties is CHOR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a is selected from the group consisting of:

[0054] In one embodiment of the disclosure, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula Ic, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the R′ moieties is CHOR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, Ra , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a is selected from the group consisting of:

[0055] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula Ib, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the R moieties is OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a and Si(R a ) 3.

[0056] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula Ia, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the R moieties is CHOH.

[0057] In one embodiment of the disclosure, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of formula Ib, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the R moieties is OH.

[0058] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula Ib, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the R' moieties is CH2OH.

[0059] In one embodiment of the disclosure, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of formula Ic, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the R' moieties is CH2OH.

[0060] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula Ib, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the R′ moieties is CHOR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a is selected from the group consisting of:

[0061] In one embodiment, the δ-cyclodextrin or δ-cyclodextrin derivative has the structure of Formula Ic, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the R′ moieties is CHOR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNRa is selected from the group consisting of:

[0062] In one embodiment of the present disclosure, the purity of the δ-cyclodextrin or derivative thereof obtained thereby is at least 50%, such as 55%, for example 60%, for example 65%, for example 70%, for example 75%, for example 80%, for example 85%, for example 90%, for example 91%, for example 92%, for example 93%, for example 94%, for example 95%, for example 96%, for example 97%, for example 98%, for example 99%. In one embodiment of the present disclosure, the isolated δ-cyclodextrin or derivative thereof is (B 12 Cl n H 12-n ) 2- Contains ions of.

[0063] In one embodiment, the disclosed method provides an isolated yield of δ-cyclodextrin or a derivative thereof of at least 1%, such as 2%, for example 3%, for example 4%, for example 5%, for example 6%, for example 7%, for example 8%, for example 9%, for example 10%, for example 15%, for example 20%, for example 25%, for example 30%, for example 35%, for example 40%, for example 45%, for example 50%.

[0064] Glucose-based compounds As shown in the examples herein, the methods disclosed herein can produce δ-cyclodextrin or its derivatives starting from other cyclodextrins and / or linear glucopyranose species. Thus, in one embodiment, the glucose-based compound is linear or cyclic. The glucose-based compound preferably refers to a compound containing one or more glucopyranose moieties. The glucose-based compound preferably refers to a compound containing glucopyranose moieties linked by 1,4-linkages.

[0065] In one embodiment of the present disclosure, the glucose-based compound has the structure of Formula II. [ka]

[0066] In one embodiment of the present disclosure, the glucose-based compound has the structure of Formula II: Each R is CH2OR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl; and m is an integer of 2 or greater.

[0067] Cyclodextrin glucanotransferase (CGTase) can act on substrates containing two or more glucopyranose subunits, such as substrates containing two or more glucopyranose subunits linked by 1,4-glycosyl bonds. Thus, in one embodiment, m is an integer greater than or equal to 2.

[0068] In one embodiment, the glucose-based compound has the structure of Formula IIa. [ka]

[0069] In one embodiment, the glucose-based compound has the structure of Formula IIb: [ka]

[0070] In one embodiment, the glucose-based compound has the structure of Formula IIb: Each R' is CH2OR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of Each R is OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a and Si(R a ) 3 independently selected from the group consisting of: Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl; and m is an integer of 2 or greater.

[0071] In one embodiment, the glucose-based compound has the structure of Formula IIc. [ka]

[0072] As shown in the examples herein, the disclosed method can convert other cyclodextrins to δ-cyclodextrin or its derivatives. Such other cyclodextrins can be α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or dextrins with ring sizes of 10 or greater. Thus, in one embodiment, the glucose-based compound has the structure of Formula III: [ka]

[0073] In one embodiment, the glucose-based compound has the structure of Formula III: Each R is CH2OR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl; and k is an integer of 6, 7, 8, 10, or greater than 10.

[0074] Structures such as those shown in Formula III are intended to represent macrocycles of 1,4-linked pyranose moieties.

[0075] In one embodiment of the present disclosure, k is an integer of 6, 7, or 8. In one embodiment, k is an integer of 10 or greater.

[0076] In one embodiment, the glucose-based compound has the structure of Formula IIIa. [ka]

[0077] In one embodiment, the glucose-based compound has the structure of Formula IIIb. [ka]

[0078] In one embodiment, the glucose-based compound has the structure of Formula IIIb: Each R' is CH2OR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of Each R is OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a)2, SR a and Si(R a )3 independently selected from the group consisting of: Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl; and k is an integer of 6, 7, 8, 10, or greater than 10.

[0079] In one embodiment, the glucose-based compound has the structure of Formula IIIc. [ka]

[0080] In one embodiment of the present disclosure, each R is CHOR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of: Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl.

[0081] The mixtures described herein may include more than one glucose-based compound. Thus, in embodiments of the present disclosure, the mixture further includes a second glucose-based compound.

[0082] In one embodiment, the mixture further comprises a second glucose-based compound having the structure of Formula IV. [ka]

[0083] In one embodiment, the mixture further comprises a second glucose-based compound having the structure of Formula IV: Each R is CH2OR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl; and j is an integer of 1 or greater.

[0084] In one embodiment of the present disclosure, the second glucose-based compound has the structure of Formula IVa: [ka]

[0085] In one embodiment of the present disclosure, the second glucose-based compound has the structure of Formula IVb: [ka]

[0086] In one embodiment of the present disclosure, the second glucose-based compound has the structure of formula IVb: Each R' is CH2OR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a are independently selected from the group consisting of Each R is OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a and Si(R a ) 3 independently selected from the group consisting of: Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl; and j is an integer of 1 or greater.

[0087] In one embodiment, the second glucose-based compound has the structure of Formula IVc: [ka]

[0088] In one embodiment of the present disclosure, each R' is CHOR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a In one embodiment of the present disclosure, each R is independently selected from the group consisting of OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a and Si(R a In one embodiment of the present disclosure, each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl. In one embodiment of the present disclosure, j is an integer greater than or equal to 1.

[0089] In one embodiment, the glucose-based compound is selected from the group consisting of glucose, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, second cyclodextrin, second macrocyclic cyclodextrin, amylose, synthetic amylose, glycogen, maltodextrin, dextrin, cycloamylose, amylopectin, starch, modified starch such as soluble starch, limit dextrins, and other α-glucans such as pullulan, or any mixture of all of the above. In the context of the substrate, macrocyclic cyclodextrin refers to a cyclodextrin consisting of 10 or more glucopyranose moieties.

[0090] In one embodiment, the glucose-based compound is glucose, a glucose derivative, a modified glucose, a glucose-based polysaccharide, a glucose-based polysaccharide derivative, a modified glucose-based polysaccharide, a second cyclodextrin, or a second cyclodextrin derivative.

[0091] In one embodiment, the second cyclodextrin is α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin. In one embodiment, the second macrocyclic cyclodextrin is a cyclodextrin having a size of 10 or more glucose moieties, or a derivative of a cyclodextrin having a size of 10 or more glucose moieties.

[0092] In one embodiment, the second cyclodextrin derivative is an α-cyclodextrin derivative, a β-cyclodextrin derivative, or a γ-cyclodextrin derivative. In one embodiment, the second cyclodextrin derivative is a modified cyclodextrin, such as a modified α-cyclodextrin, a modified β-cyclodextrin, or a modified γ-cyclodextrin.

[0093] As shown in the Examples, the methods disclosed in the present invention can produce δ-cyclodextrin derivatives by using modified glucose-based compounds. In one embodiment, the modification involves replacing one or more of the OH moieties present on the glucose-based compound with an OR a, H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a and / or the modification is to replace one or more of the CH2OH moieties present on the glucose-based compound with a moiety independently selected from the group consisting of CH2OR a , CH3, CH2F, CH2Cl, CH2Br, CH2I, CH2N3, CH2CN, CH2R a , CH2N(R a )2, CH2SR a , CH2Si(R a )3, CH2CHO, CH2COOH, CH2COOR a , CH2CON(R a )2, CH2CHNR a , OR a , H, F, Cl, Br, I, N3, CN, R a , N(R a )2, SR a , Si(R a )3, CHO, COOH, COOR a , CON(R a )2 and CHNR a and substituting with a moiety independently selected from the group consisting of: Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl.

[0094] In certain embodiments, the modified cyclodextrin derivative is a deoxycyclodextrin, such as a monodeoxycyclodextrin, such as a 6-deoxycyclodextrin, such as a 6-deoxy α-cyclodextrin, such as mono-6-deoxy α-cyclodextrin.

[0095] Templates The disclosed method is so-called template synthesis, in which reactants do not necessarily form covalent bonds with other reactants but induce product formation through non-covalent interactions with other reaction partners. The inventors have discovered that certain boron-halogen clusters can serve as templates for the synthesis of δ-cyclodextrin. Templates can influence the formation of specific products, for example, by stabilizing the desired product, i.e., by making its formation thermodynamically favorable. Templates can also influence the reaction rate, increasing the rate of formation of certain products and thereby making their formation more favorable. In certain embodiments, the presently disclosed method is believed to be controlled by the thermodynamic template effect.

[0096] One embodiment of the present disclosure is a compound of formula (B 12 Cl n H 12-n ) 2- In one embodiment, n is 1 to 12. In one embodiment, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one embodiment, n is 9, 10, 11, or 12. In one embodiment, a compound of formula [B 12 Cl n H 12-n ] 2- The ions of (B 12 Cl 12 ) 2- , (B 12 Cl 11 H) 2- , (B 12 Cl 10 H2) 2- , (B 12 Cl9H3) 2- , (B 12 Cl8H4) 2- , (B 12 Cl7H5) 2- , (B 12 Cl6H6) 2- , (B 12 Cl5H7) 2- , (B 12 Cl4H8) 2- , (B 12 Cl3H9)2- , (B 12 Cl2H 10 ) 2- and (B 12 ClH 11 ) 2- In one embodiment, the compound of formula [B 12 Cl n H 12-n ] 2- The ions of (B 12 Cl 12 ) 2- , (B 12 Cl 11 H) 2- , (B 12 Cl 10 H2) 2- and (B 12 Cl9H3) 2- In one embodiment, the compound of formula (B 12 Cl n H 12-n ) 2- The ions of (B 12 Cl 12 ) 2- is.

[0097] In one embodiment of the present disclosure, a compound of formula (B 12 Cl n H 12-n ) 2- The ions of formula M2(B 12 Cl n H 12-n ) or M(B 12 Cl n H 12-n ) wherein M is a monovalent or divalent cation. In one embodiment, M is Li + , Na + , K. + , Cs + Alkali metal ions such as Mg 2+ Ya Ca 2+ Earth alkali metal ions such as NH4 + , (R p )4N + and (R p )4P + is selected from the group consisting of Each R pis independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl.

[0098] In one embodiment, the compound of formula M2(B 12 Cl n H 12-n ) salt is Na2(B 12 Cl 12 )

[0099] enzyme In one embodiment of the present disclosure, the enzyme, such as a cyclodextrin glucanotransferase, is a cyclodextrin glucanotransferase as defined by Enzyme Commission (EC) No. 2.4.1.19. Other enzymes may be capable of catalyzing the hydrolysis and / or condensation of 1,4-glycosidic bonds. Thus, in one embodiment, the enzyme, such as a cyclodextrin glucanotransferase, is an enzyme capable of catalyzing the reaction catalyzed by an enzyme defined by Enzyme Commission (EC) No. 2.4.1.19. In one embodiment, the enzyme, such as a cyclodextrin glucanotransferase, is selected from the group of enzymes defined by Enzyme Commission (EC) No. 2.4.1.25. In one embodiment, the cyclodextrin glucanotransferase is selected from the group of enzymes defined by Enzyme Commission (EC) No. 3.2.1.54. In one embodiment, the cyclodextrin glucanotransferase is a CGTase from Amano Enzyme Europe Limited, catalog number 970390. In one embodiment, the enzyme is a cyclodextrin glucanotransferase disclosed herein.

[0100] In one embodiment, the cyclodextrin glucanotransferase is Alkalihalobacillus alcalophilus, Alkalihalobacillus clausii, Alkalihalobacillus lehensis, Alkalihalobacillus pseudalcaliphilus, Anaerobranca gottschalkii, Bacillus autolyticus, Bacillus cereus, Bacillus licheniformis, Bacillus ohbensis, Bacillus sp, Bacillus subtilis, Brevibacillus brevis, Brevibacterium sp, Cytobacillus firmus, E. coli, Evensella clarkii, Geobacillus stearothermophilus, Haloferax mediterranei, Klebsiella oxytoca, Klebsiella pneumoniae, Lederbergia lentus, Lysinibacillus sphaericus, Microbacterium terrae, Micrococcus luteus, Micrococcus sp., Niallia circulans, Nostoc sp., Paenibacillus barengoltzii, Paenibacillus compinasensis, Paenibacillus graminis, Paenibacillus illinoisensis, Paenibacillus macerans, Paenibacillus pabuli, Paenibacillus sp., Parageobacillus thermoglucosidasius, Priestia flexa, Priestia megaterium, Pyrococcus furiosus, Saccharomyces cerevisiae, Salimicrobium halophilum, Salipaludibacillus agaradhaerens, Streptococcus pyogenes, Sus scrofa, Thermoactinomyces vulgaris, Thermoanaerobacter sp., Thermoanaerobacterium cyclodextrin glucanotransferase of thermosulfurigenes, Thermococcus kodakarensis, Thermococcus sp., Trichoderma viride, Carboxydocella sp., Weizmannia coagulans, Xanthomonas axonopodis or Xanthomonas campestris. In certain embodiments, the cyclodextrin glucanotransferase is Paenibacillus macerans cyclodextrin glucanotransferase.

[0101] In one embodiment, this enzyme is 4-α-glucose transferase. In one embodiment, 4-α-glucose is transferase. reinhardtii、Clostridium butyricum、Corynebacterium glutamicum、Culex quinquefasciatus、Daucus carota、Dictyoglumus thermophilum、E. coli, Gallus gallus, Haemophilus influenzae, Homo sapiens, Hordeum vulgare, Ipomoea batatas, Manihot esculenta, Mycobacterium tuberculosis, Oryctolagus cuniculus, Oryza sativa, Paenibacillus macerans, Pisum sativum, Pseudomonas stutzeri, Pyrobaculum aerophilum, Pyrococcus furiosus, Pyrococcus sp., Rattus norvegicus, Saccharolobus sulfataricus, Saccharomyces cerevisiae, Solanum lycopersicum, Solanum tuberosum, Spinacia oleracea, Squalus acanthias, Streptococcus equinus, Streptococcus mitis, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus sp., Sus scrofa, Synechocystis sp., Thermococcus kodakarensis, Thermococcus literalis, Thermotoga maritima, Thermotoga neapolitana, Thermus aquaticus, Thermus brockianus, Thermus filiformis, Thermus Scotoductus, Thermus thermophilus, Triticum aestivum, Vicia faba, Vigna radiate, or Zea mays 4-α-glucanotransferase. In one embodiment, the disclosed method is an in vitro method.

[0102] In one embodiment, the enzyme is cyclomaltodextrinase. In one embodiment, the cyclomaltodextrinase is the cyclomaltodextrinase of Alicyclobacillus acidocaldarius, Anoxybacillus flavithermus, Archaeoglobus fulgidus, Bacillus licheniformis, Bacillus sp., Bacillus subtilis, Bacillus thermoalkalophilus, Bacteroides ovatus, Escherichia coli, Evansella clarkia, Flavobacterium sp., Geobacillus stearothermophilus, Klebsiella oxytoca, Laceyella sacchari, Lactobacillus sp., Lactococcus lactis, Lysinibacillus sphaericus, Massilia timonae, Niallia circulans, Nostoc punctiforme, Paenibacillus macerans, Paenibacillus sp., Palaeococcus pacificus, Parabacteroides distasonis, Pyrococcus furiosus, Thermoactinomyces vulgaris, Thermoanaerobacter ethanolicus, Thermoanaerobacter thermohydrosulfuricus, Thermococcus kodakarensis, Thermococcus sp., Thermofilum pendens, Thermoplasma volcanium, Thermotoga maritima, Thermotoga neapolitana, Thermus, Weizmannia coagulans or Xanthomonas campestris.

[0103] Solvent The method is carried out in a solvent. In one embodiment, the solvent is a protic solvent. In one embodiment, the solvent is a polar solvent. In one embodiment, the solvent is a protic polar solvent. In one embodiment, the solvent comprises two or more solvents. A mixture of solvents may be used in the methods disclosed herein. Thus, in one embodiment, the solvent comprises water and an organic solvent. The organic solvent may be polar and / or protic. In certain embodiments, the solvent comprises or is dimethylformamide, dimethyl sulfoxide, acetone, acetonitrile, isopropanol, ethanol, or methanol.

[0104] In one embodiment, the solvent comprises or is an aqueous solvent, hi one embodiment, the aqueous solvent comprises or is water, such as deionized water, e.g., Milli-Q water.

[0105] In one embodiment, the solvent comprises or is an aqueous buffer solution. In one embodiment, the aqueous buffer solution is a phosphate buffer solution or a sulfate buffer solution. In one embodiment, the concentration of the aqueous buffer solution is 1 to 500 mM, for example, 1 to 250 mM, for example, 5 to 150 mM, for example, 10 to 100 mM, for example, 20 to 100 mM, for example, about 50 mM.

[0106] In one embodiment, the solvent has a pH of 3.5 to 11.0, such as 4.0 to 9.0, for example, 4.5 to 9.0, for example, 5.5 to 8.5, for example, 5.5 to 8.5, for example, 6.0 to 8.5, for example, 6.5 to 8.5, for example, 7.0 to 8.0, for example, about 7.5. In one embodiment, the solvent has a pH within the active pH range of an enzyme, such as cyclodextrin glucanotransferase. In one embodiment, the solvent has a pH of 4.0 to 4.5, for example, 4.5 to 5.0, for example, 5.0 to 5.5, for example, 5.5 to 6.0, for example, 6.0 to 6.5, for example, 6.5 to 7.0, for example, 7.0 to 7.5, for example, 7.5 to 8.0, for example, 8.0 to 8.5, for example, 8.5 to 9.0. In one embodiment, the mixture has a pH of 3.5 to 11.0, such as 4.0 to 9.0, for example, 4.5 to 9.0, for example, 5.5 to 8.5, for example, 5.5 to 8.5, for example, 6.0 to 8.5, for example, 6.5 to 8.5, for example, 7.0 to 8.0, for example, about 7.5, or is pH-adjusted to 3.5 to 11.0. In one embodiment, the mixture has a pH within the pH range of action of an enzyme, such as cyclodextrin glucanotransferase, or is pH-adjusted to that range. In one embodiment, the mixture has a pH of 4.0 to 4.5, for example, 4.5 to 5.0, for example, 5.0 to 5.5, for example, 5.5 to 6.0, for example, 6.0 to 6.5, for example, 6.5 to 7.0, for example, 7.0 to 7.5, for example, 7.5 to 8.0, for example, 8.0 to 8.5, for example, 8.5 to 9.0, or is pH-adjusted to 4.0 to 4.5.

[0107] incubation Incubation is carried out under conditions until sufficient product is produced.

[0108] In one embodiment of the present disclosure, incubation is carried out at 0 to 120°C, for example, 0 to 100°C, for example, 0 to 80°C, for example, 0 to 70°C, for example, 0 to 60°C, for example, 5 to 45°C, 10 to 40°C, for example, 15 to 40°C, or for example, 20 to 35°C. In one embodiment, incubation is carried out at 0 to 5°C, for example, 5 to 10°C, for example, 10 to 15°C, for example, 15 to 20°C, for example, 20 to 25°C, for example, 25 to 30°C, for example, 30 to 35°C, for example, 35 to 40°C, or for example, 40 to 45°C.

[0109] In one embodiment, the incubation is carried out for 10 to 200 hours, for example, 10 to 150 hours, for example, 10 to 100 hours, for example, 10 to 80 hours, for example, 20 to 70 hours, for example, 30 to 60 hours, for example, 35 to 55 hours, for example, about 40 to 50 hours. In one embodiment, the incubation is carried out for 10 to 15 hours, for example, 15 to 20 hours, for example, 20 to 25 hours, for example, 25 to 30 hours, for example, 30 to 35 hours, for example, 35 to 40 hours, for example, 40 to 45 hours, for example, 45 to 50 hours, for example, 50 to 55 hours, for example, 55 to 60 hours, for example, 60 to 65 hours, for example, 65 to 70 hours, for example, 70 to 75 hours, for example, 75 to 80 hours, for example, 80 to 85 hours, for example, 85 to 90 hours, for example, 90 to 95 hours, or for example, 95 to 100 hours. In certain embodiments, the incubation is for at least 24 hours, such as at least 36 hours, such as at least 42 hours, such as at least 48 hours. In certain embodiments, the incubation is for about 42 hours or about 48 hours.

[0110] The inventors believe that the optimal incubation time for the disclosed method will vary depending on the incubation temperature, the concentrations of the enzyme, the glucose-based compound, and the template. Those skilled in the art will know how to follow the progress of the reaction and determine when to stop the incubation using well-known methods shown in the examples.

[0111] Reagent stoichiometry As outlined herein, the formula (B 12 Cl n H 12-n ) 2- The ions act as templates and therefore do not necessarily need to be present in stoichiometric amounts relative to the δ-cyclodextrin or derivative thereof, or the product δ-cyclodextrin or derivative thereof.

[0112] In one embodiment of the present disclosure, a glucose-based compound and a compound of formula (B 12 Cl n H12-n ) 2- The ratio of the ions to the ions is 99:1 to 20:80 (w / w), for example, 90:10 to 65:35.

[0113] In one embodiment of the present disclosure, a glucose-based compound and a compound of formula (B 12 Cl n H 12-n ) 2- The ratio of the ions to the ions is 99:1 to 98:2 (w / w), for example, 98:2 to 95:5, for example, 95:5 to 90:10, for example, 90:10 to 85:15, for example, 85:15 to 80:20, for example, 80:20 to 75:25, for example, 75:25 to 70:30, for example, 70:30 to 65:35, for example, 65:35 to 60:40, for example, 60:40 to 55:45, for example, 55:45 to 50:50, for example, 50:50 to 45:55, for example, 45:55 to 40:60, for example, 40:60 to 35:65, for example, 35:65 to 30:70, for example, 30:70 to 25:75, for example, 25:75 to 20:80.

[0114] In one embodiment, a compound of formula (B 12 Cl n H 12-n ) 2- The amount of the ion of formula (B) is 2 to 50 mol %, for example, 2 to 45 mol %, for example, 3 to 40 mol %, for example, 4 to 35 mol %, for example, 5 to 30 mol %, for example, 6 to 25 mol % per monosaccharide moiety in the glucose-based compound. 12 Cl n H 12-n ) 2- The amount of the ions is 2 to 3 mol%, for example, 3 to 4 mol%, for example, 4 to 5 mol%, for example, 5 to 6 mol%, for example, 6 to 7 mol%, for example, 7 to 8 mol%, for example, 8 to 9 mol%, for example, 9 to 10 mol%, for example, 10 to 11 mol%, for example, 11 to 12 mol%, for example, 12 to 13 mol%, for example, 13 to 14 mol%, for example, 14 to 15 mol%, for example, 15 to 17 mol%, for example, 17 to 20 mol%, for example, 20 to 25 mol%, for example, 25 to 30 mol%, for example, 30 to 35 mol%, for example, 35 to 40 mol%, for example, 40 to 45 mol%, for example, 45 to 50 mol%.

[0115] In one embodiment, the mixture contains cyclodextrin glucanotransferase in an amount corresponding to 0.1 to 100% (v / v) cyclodextrin glucanotransferase stock solution, e.g., 0.1 to 60%, e.g., 0.1 to 50%, e.g., 0.5 to 40%, e.g., 0.5 to 30%, e.g., 1.0 to 20%, e.g., 1 to 15%, e.g., 1 to 10% stock solution. The reaction can be carried out in a pure enzyme stock solution. Thus, in one embodiment, the solvent is a cyclodextrin glucanotransferase stock solution. In one embodiment of the present disclosure, the mixture comprises 0.1-0.2% (v / v) cyclodextrin glucanotransferase stock solution, for example 0.2-0.3%, for example 0.3-0.4%, for example 0.4-0.5%, for example 0.5-0.6%, for example 0.6-0.7%, for example 0.7-0.8%, for example 0.8-0.9%, for example 0.9-1.0%, for example 1.0-1.5%, for example 1.5-2.0%, for example 2.0-2.5%, for example 2.5-3.0%, For example, the cyclodextrin glucanotransferase stock solution contains an amount of cyclodextrin glucanotransferase equivalent to 3.0-4.0%, for example, 4.0-5.0%, for example, 5.0-6.0%, for example, 6.0-7.0%, for example, 7.0-8.0%, for example, 8.0-9.0%, for example, 9.0-10%, for example, 10-15%, for example, 15-20%, for example, 20-25%, for example, 25-30%, for example, 30-40%, or for example, 40-50% of the cyclodextrin glucanotransferase stock solution. When cyclodextrin glucanotransferase is mentioned herein, it is understood that any of the enzymes disclosed herein can be used in place of the cyclodextrin glucanotransferase. In one embodiment, the cyclodextrin glucanotransferase stock solution corresponds to or is cyclodextrin glucanotransferase catalog number 970390 from Amano Enzyme Europe Limited.

[0116] In one embodiment of the present disclosure, the concentration of the glucose-based compound in the solvent is 0.1 to 200 g / L, for example, 0.1 to 150 g / L, for example, 0.1 to 100 g / L, for example, 0.2 to 90 g / L, for example, 0.5 to 80 g / L, for example, 0.5 to 70 g / L, for example, 1 to 60 g / L, for example, 1 to 50 g / L, for example, 1 to 50 g / L, for example, 1 to 40 g / L, for example, 1 to 30 g / L, for example, 1 to 25 g / L. In one embodiment, the concentration of the glucose-based compound in the solvent is 0.1 to 0.2 g / L, for example, 0.2 to 0.5 g / L, for example, 0.5 to 1 g / L, for example, 1 to 2 g / L, for example, 2 to 5 g / L, for example, 5 to 10 g / L, for example, 10 to 15 g / L, for example, 15 to 20 g / L, for example, 20 to 25 g / L, for example, 25 to 30 g / L, for example, 30 to 40 g / L, for example, 40 to 50 g / L, for example, 50 to 60 g / L, for example, 60 to 70 g / L, for example, 70 to 80 g / L, for example, 80 to 90 g / L, for example, 90 to 100 g / L.

[0117] In one embodiment, a compound of formula (B) in a solvent 12 Cl n H 12-n ) 2- In one embodiment, the concentration of the ion of formula (B) in the solvent is 0.1 to 300 mM, for example, 0.1 to 250 mM, for example, 1 to 200 mM, for example, 1 to 150 mM, for example, 1 to 100 mM, for example, 0.2 to 90 mM, for example, 0.3 to 80 mM, for example, 0.4 to 70 mM, for example, 0.5 to 60 mM, for example, 0.6 to 50 mM, for example, 0.7 to 40 mM, for example, 0.8 to 30 mM, for example, 0.9 to 25 mM, for example, 1.0 to 20 mM, for example, 1.0 to 15 mM, for example, 1.0 to 10 mM. 12 Cl n H 12-n ) 2-The concentration of the ions is 0.1 to 0.2 mM, for example, 0.2 to 0.3 mM, for example, 0.3 to 0.4 mM, for example, 0.4 to 0.5 mM, for example, 0.5 to 0.6 mM, for example, 0.6 to 0.7 mM, for example, 0.7 to 0.8 mM, for example, 0.8 to 0.9 mM, for example, 0.9 to 1.0 mM, for example, 1.0 to 1.5 mM, for example, 1.5 to 2.0 mM, for example, 2 to 3 mM, for example, 3 to 4 mM, for example, 4 to 5 mM, for example, 5 to 6 mM, for example, 6 to 7 mM, for example, 7 to 8 mM, for example, 8 to 9 mM, for example, 9 to 10 mM, for example, 10 to 15 mM, for example, 15 to 20 mM, for example, 20 to 25 mM, for example, 25 to 30 mM, for example, 30 to 40 mM, for example, 40 to 50 mM, for example, 50 to 60 mM, for example, 60 to 70 mM, for example, 70 to 80 mM, for example, 80 to 90 mM, for example, 90 to 100 mM.

[0118] Further method conditions The methods of the present disclosure can be carried out using the exemplary combinations of conditions outlined below.

[0119] One embodiment provides a method of the present disclosure, comprising: a. Incubation is carried out for 30 to 60 hours, and b. Incubation is carried out at 0-45°C.

[0120] One embodiment provides a method of the present disclosure, comprising: a. Incubation is carried out for 40 to 56 hours, and b. Incubation is carried out at 20-30°C.

[0121] One embodiment provides a method of the present disclosure, comprising: a. the glucose-based compound is a starch, a cyclodextrin such as α-cyclodextrin, or a modified cyclodextrin such as mono-6-deoxy α-cyclodextrin, and b.Formula (B 12 Cl n H 12-n ) 2- The ions of (B 12 Cl 12 ) 2- , (B12 Cl 11 H) 2- , (B 12 Cl 10 H2) 2- , or (B 12 Cl9H3) 2- is.

[0122] One embodiment provides a method of the present disclosure, comprising: a. The concentration of the glucose-based compound in the solvent is 1 to 50 g / L; b. Formula (B 12 Cl n H 12-n ) 2- The concentration of the ion is 0.5 to 25 mM, and c. The mixture contains cyclodextrin glucanotransferase in an amount equivalent to 0.1-50% (v / v) cyclodextrin glucanotransferase stock solution described herein.

[0123] One embodiment provides a method of the present disclosure, comprising: a. The concentration of the glucose-based compound in the solvent is 5 to 25 g / L; b. Formula (B 12 Cl n H 12-n ) 2- The concentration of the ion is 0.5 to 10 mM, and c. The mixture contains cyclodextrin glucanotransferase in an amount equivalent to 1-10% (v / v) cyclodextrin glucanotransferase stock solution described herein.

[0124] One embodiment provides a method of the present disclosure, comprising: a. Incubation is carried out for 30 to 60 hours, b. Incubation is carried out at 0-45°C. c. The concentration of the glucose-based compound in the solvent is 1 to 50 g / L; d. A compound of formula (B 12 Cl n H 12-n ) 2-The concentration of the ion is 0.5 to 25 mM, and e. The mixture contains cyclodextrin glucanotransferase in an amount equivalent to 0.1-50% (v / v) cyclodextrin glucanotransferase stock solution described herein.

[0125] One embodiment provides a method of the present disclosure, comprising: a. Incubation is carried out for 40 to 56 hours, b. Incubation is carried out at 20-30°C. c. The concentration of the glucose-based compound in the solvent is 5 to 25 g / L; d. A compound of formula (B 12 Cl n H 12-n ) 2- The concentration of the ion is 0.5 to 10 mM, and e. The mixture contains cyclodextrin glucanotransferase in an amount equivalent to 1-10% (v / v) cyclodextrin glucanotransferase stock solution.

[0126] One embodiment provides a method of the present disclosure, comprising: a. Incubation is carried out for 30 to 60 hours, b. Incubation is carried out at 0-45°C. c. The concentration of the glucose-based compound in the solvent is 1 to 50 g / L; d. A compound of formula (B 12 Cl n H 12-n ) 2- The concentration of the ions is 0.5 to 25 mM, e. the mixture comprises cyclodextrin glucanotransferase in an amount equivalent to 0.1 to 50% (v / v) cyclodextrin glucanotransferase stock solution described herein; f. the glucose-based compound is selected from glucose, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, modified α-cyclodextrin, modified β-cyclodextrin, modified γ-cyclodextrin, starch such as soluble starch, and glucan; and g.Formula [B 12 Cl n H 12-n ] 2- The ions of (B 12 Cl 12 ) 2- , (B 12 Cl 11 H) 2- , (B 12 Cl 10 H2) 2- , (B 12 Cl9H3) 2- , (B 12 Cl8H4) 2- , (B 12 Cl7H5) 2- , (B 12 Cl6H6) 2- , (B 12 Cl5H7) 2- , (B 12 Cl4H8) 2- , (B 12 Cl3H9) 2- , (B 12 Cl2H 10 ) 2- and (B 12 ClH 11 ) 2- is selected from the group consisting of:

[0127] One embodiment provides a method of the present disclosure, comprising: a. Incubation is carried out for 40 to 56 hours, b. Incubation is carried out at 20-30°C. c. The concentration of the glucose-based compound in the solvent is 5 to 25 g / L; d. A compound of formula (B 12 Cl n H 12-n ) 2- The concentration of the ions is 0.5 to 10 mM. e. The mixture contains cyclodextrin glucanotransferase in an amount equivalent to 1-10% (v / v) cyclodextrin glucanotransferase stock solution. f. the glucose-based compound is selected from glucose, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, modified α-cyclodextrin, modified β-cyclodextrin, modified γ-cyclodextrin, starch such as soluble starch, and glucan; and g.Formula [B 12 Cl n H 12-n ] 2- The ions of (B 12 Cl 12 ) 2- , (B 12 Cl 11 H) 2- , (B 12 Cl 10 H2) 2- and (B 12 Cl9H3) 2- is selected from the group consisting of:

[0128] Further method steps The δ-cyclodextrin or derivative thereof obtained by the methods disclosed herein may be further modified, e.g., chemically modified. Thus, in one embodiment, the methods of the present disclosure further comprise chemically modifying the δ-cyclodextrin or δ-cyclodextrin derivative obtained from the methods of the present disclosure.

[0129] The reaction occurring during the incubation step of the disclosed method can be terminated by denaturing the enzyme. Such denaturation can be carried out using methods known to those skilled in the art. One way to denature the enzyme is by heating. Thus, in one embodiment, the method further comprises heating the mixture to at least 70°C, such as at least 75°C, such as at least 80°C, such as at least 85°C, such as at least 90°C, such as at least 95°C, such as at least 100°C, such as at least 110°C, such as at least 120°C, such as at least 130°C. In one embodiment, the heating of the mixture is for 1 to 60 minutes, such as 5 to 50 minutes, such as 5 to 40 minutes, such as 5 to 30 minutes, such as 5 to 20 minutes, such as about 15 minutes. In one embodiment, the heating of the mixture is for at least 5 minutes, such as at least 10 minutes, such as at least 15 minutes. In one embodiment, the heating step is carried out after the incubation step.

[0130] δ-cyclodextrin or δ-cyclodextrin derivatives can be isolated as outlined herein below.

[0131] In one embodiment, the method further comprises partially evaporating the solvent from the mixture. In one embodiment, evaporating the mixture comprises removing 10-20%, e.g., 20-30%, e.g., 30-40%, e.g., 40-50%, e.g., 50-60%, e.g., 60-70%, e.g., 70-80%, e.g., 80-90% of the solvent volume. In one embodiment, partially evaporating the solvent from the mixture is performed under reduced pressure and / or by heating the mixture. In one embodiment, partially evaporating the solvent from the mixture is performed under atmospheric pressure and / or without heating the mixture. In one embodiment, partially evaporating the solvent from the mixture is effective to precipitate cyclodextrin glucanotransferase.

[0132] Because the disclosed method can form δ-cyclodextrin and its derivatives without forming other cyclodextrins, the δ-cyclodextrin and its derivatives can be isolated using precipitation and / or reprecipitation. Thus, in one embodiment, the mixture comprises a solid phase containing cyclodextrin glucanotransferase and a liquid phase containing δ-cyclodextrin or a δ-cyclodextrin derivative, and the method further comprises separating the solid and liquid phases of the mixture. In one embodiment, the step of separating the solid phase containing cyclodextrin glucanotransferase from the liquid phase containing δ-cyclodextrin or a δ-cyclodextrin derivative is performed using centrifugation, filtration, and / or decantation. In one embodiment, the method further comprises precipitating the δ-cyclodextrin or a δ-cyclodextrin derivative from the liquid phase.

[0133] In one embodiment, precipitating the δ-cyclodextrin or δ-cyclodextrin derivative comprises adding an anti-solvent to the liquid phase, thereby precipitating the δ-cyclodextrin or δ-cyclodextrin derivative. In one embodiment, the anti-solvent is a protic solvent or an aprotic polar solvent. In one embodiment, the anti-solvent is selected from the group consisting of acetone, ethanol, isopropanol, and acetonitrile. In one embodiment, the method further comprises filtering the precipitated δ-cyclodextrin or δ-cyclodextrin derivative.

[0134] In one embodiment, the method comprises: a. dissolving δ-cyclodextrin or a δ-cyclodextrin derivative in water and / or making a slurry in water; b. adding an anti-solvent, such as a protic solvent or an aprotic polar solvent, such as acetone, ethanol, isopropanol, or acetonitrile, thereby precipitating the δ-cyclodextrin or δ-cyclodextrin derivative; c. and filtering the precipitated δ-cyclodextrin or δ-cyclodextrin derivative.

[0135] Illustrative Embodiments In one embodiment, the method comprises: a. v. glucose-based compounds, vi. cyclodextrin glucanotransferase, vii.Formula (B 12 Cl n H 12-n ) 2- ions of the formula (wherein n is 1 to 12), and viii. Mixing the solvents forming a mixture; b. heating and incubating the mixture; c. partially evaporating the solvent from the mixture to form a solid phase and a liquid phase; d. Separating the solid and liquid phases of the mixture; e. adding an anti-solvent to the liquid phase to precipitate δ-cyclodextrin or a δ-cyclodextrin derivative; f. filtering the precipitated δ-cyclodextrin or δ-cyclodextrin derivative.

[0136] The template ions used in the disclosed methods can be recovered and reused to perform the disclosed methods again. In one embodiment, the method comprises: 12 Cl n H 12-n ) 2- and recovering the ions of a. Obtaining a supernatant from the δ-cyclodextrin or δ-cyclodextrin derivative precipitation step; b. evaporating the anti-solvent under reduced pressure; c. adding hydrochloric acid, for example 37% hydrochloric acid, to obtain a pH of 1 to 4, for example 1.5 to 2.5; d. Adding a base, such as an amine base, for example an alkylamine base, for example a trialkylamine compound, for example triethylamine, to form a compound of formula (B 12 Cl n H 12-n ) 2- precipitating the ions of as a salt of a base; e. filtering the precipitate; As a result, the formula (B 12 Cl n H 12-n ) 2- ions as the salt of the base.

[0137] One embodiment of the present disclosure is a compound of formula (B 12 Cl n H 12-n ) 2- from a mixture of components, the isolation method comprising: a.Formula (B 12 Cl n H 12-n ) 2- obtaining an aqueous liquid phase containing ions of b. adding hydrochloric acid, for example 37% hydrochloric acid, to obtain a pH of 1 to 4, for example 1.5 to 2.5; c. Adding a base, such as an amine base, for example an alkylamine base, for example a trialkylamine compound, for example triethylamine, to form a compound of formula (B 12 Cl n H 12-n ) 2- as a salt of said base; d. filtering the precipitate; As a result, the formula (B 12 Cl n H 12-n ) 2- ions as the salt of the base.

[0138] One embodiment of the present disclosure provides a method for producing a δ-cyclodextrin or a δ-cyclodextrin derivative, the method comprising: a. i. glucose-based compounds, ii. cyclodextrin glucanotransferase, iii.Formula (B 12 Cl n H 12-n ) 2- ions of the formula (wherein n is 1 to 12), and iv. Mixing the solvents forming a mixture; b. heating and incubating the mixture to at least 70°C for at least 5 minutes; c. partially evaporating the solvent from the mixture, thereby precipitating the cyclodextrin glucanotransferase, thereby forming a solid phase and a liquid phase; d. Separating the solid and liquid phases of the mixture; e. adding an anti-solvent, such as a protic solvent or an aprotic polar solvent, to the liquid phase, thereby precipitating δ-cyclodextrin or a δ-cyclodextrin derivative; f. filtering the precipitated δ-cyclodextrin or δ-cyclodextrin derivative; Thereby, δ-cyclodextrin or a δ-cyclodextrin derivative is obtained.

[0139] One embodiment of the present disclosure provides a δ-cyclodextrin or δ-cyclodextrin derivative obtained from the method disclosed herein.

[0140] One embodiment of the present disclosure provides a composition, the composition comprising: a. a glucose-based compound disclosed herein; and b. an enzyme, such as a cyclodextrin glucanotransferase, disclosed herein; c. A compound of formula (B 12 Cl n H 12-n ) 2- and ions of Such compositions are useful in the disclosed methods.

[0141] One embodiment of the present disclosure provides a kit of parts, comprising: a. a glucose-based compound disclosed herein; and b. an enzyme, such as a cyclodextrin glucanotransferase, disclosed herein; c. A compound of formula (B 12 Cl n H 12-n ) 2- and a salt containing the ion. [Example]

[0142] Example 1. Analytical-scale procedure for CD6 using CGTase without template Materials and Methods All solutions were prepared in buffered water (50 mM sodium phosphate, pH 7.5). 50 μL of α-CD solution (20 mg / mL) was added to a 0.5 mL vial at 25 °C, followed by the addition of buffer (43.5 μL) to bring the total volume to 93.5 μL. The reaction was initiated by adding 6.5 μL of CGTase stock solution* to obtain 100 μL of CD6 solution (10 mg / mL). (*The commercial supplier did not disclose the concentration of CGTase in the stock solution.) CGTase was purchased from Amano Enzyme Europe Limited (product number 970390). The reaction was monitored at various time points. An analytical aliquot (3 μL) was removed, and the enzymatic reaction was stopped by immediately adding 90 μL of a 1% (v / v) solution of trifluoroacetic acid in acetonitrile / water (3:1). Samples were analyzed using high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELS). Separation was achieved using gradient elution on a HILIC type column.

[0143] result The HPLC-ELS chromatogram shows that CD6 is converted into a mixture of CD6, CD7, and CD8 (Figure 1) and a small amount of linear α-1,4-glucan. The area under each peak can be converted into a concentration using a calibration curve constructed with authentic samples of each related species, allowing the concentrations of CD and linear α-1,4-glucan to be quantified. The time course of the reaction mixture is shown in Figure 2.

[0144] conclusion When CD6 (10 mg / mL) is treated with the commercially available enzyme cyclodextrin glucanotransferase (CGTase from Amano Enzyme) in phosphate buffer at pH 7.5 and 25°C, it is converted into a mixture of primarily CD6, CD7, and CD8 (Figs. 1 and 2).

[0145] Example 2, Na2B 12 Cl 12 Procedure for analytical-scale synthesis of CD6 to CD9 using as templates Materials and Methods All solutions were prepared in buffered water (50 mM sodium phosphate, pH 7.5). 50 μL of α-CD solution (20 mg / mL) and 8 μL of 50 mM NaB 12 Cl 12 The solution was added to a 0.5 mL vial at 25 °C. Buffer (35.5 μL) was then added to bring the total volume to 93.5 μL. The reaction was initiated by adding CGTase stock solution (6.5 μL), and 10 mg / mL α-CD and 4 mM NaB 12 Cl 12 A reaction volume of 100 μL was obtained with the template. The reaction was monitored at various time points. An analytical aliquot (3 μL) was removed, and the enzymatic reaction was stopped by immediately adding this aliquot to a 1% (v / v) solution of trifluoroacetic acid (90 μL) in acetonitrile / water (3:1). The progress of the reaction and the composition of the CD mixture were analyzed using high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELS). Separation was performed using gradient elution on a HILIC column. The same procedure was carried out separately at 5°C and 40°C. The above procedure was also used to analyze Na2B 12 Cl12 By adjusting the stock solution and buffer volumes appropriately, the final Na2B 12 Cl 12 The reaction was carried out at 25°C at a concentration of 2 mM or 30 mM.

[0146] result 4 mM Na2B at 25 °C 12 Cl 12 When reacted with , HPLC-ELS chromatograms show that CD6 is converted to a mixture consisting primarily of CD9 and a small amount of linear α-1,4-glucan (Figure 3). The area under each peak is converted to a concentration using a calibration curve constructed with authentic samples of the respective related species, allowing the concentrations of CD and linear α-1,4-glucan to be quantified over time. After 48 hours of reaction, CD9 was produced as the major product in the reaction mixture. It was produced with high selectivity, being present at a concentration of 4.0 mg / mL and accounting for over 90% of the total CD composition (Figure 4). At 5 °C, the reaction proceeded more slowly but still produced primarily CD9. After 48 hours, CD9 was produced at a concentration of 3.3 mg / mL with 59% selectivity. The selectivity for CD9 improved as the reaction progressed, and after 3 days of reaction, CD8 accounted for approximately 20% of the CD composition (Figure 5). At 40 °C, CD9 formation was more rapid, yielding a maximum concentration of CD9 (3.4 mg / mL) after 8 h. At this point, 65% selectivity for CD9 was observed. With longer reaction times, CD9 selectivity improved (up to 85%), but the overall yield decreased slightly (Figure 6). In reactions using 2 mM template at 25 °C, CD9 was again produced as the major product, reaching a maximum concentration of 1.5 mg / mL within 8 h. After 8 h, CD9 accounted for 31% of the CD composition, and after 2 days, CD6, CD7, and CD8 accounted for 32% of the CD composition. In reactions with 30 mM template at 25 °C, a maximum concentration of 2.5 mg / mL was obtained after 2 days, at which point CD9 accounted for 67% of the CD composition, and after 3 days, CD6, CD7, and CD8 accounted for 29% of the CD composition (Figure 8).

[0147] conclusion CD6 (10 mg / mL) and Na2B 12Cl 12 When treated with CGTase in the presence of 4 mM ATP in phosphate buffer at pH 7.5 at 25°C, CD9 was the major product (Figures 3 and 4). CD9 remained the major product even when higher or lower temperatures (4°C or 40°C) or different template concentrations (2 mM or 30 mM) were used.

[0148] Example 3: Large-scale synthesis procedure for CD9 Materials and Methods CD6 (10.00 g, 10.03 mmol) and Na2B 12 Cl 12 CD9 (3.00 g, 5 mmol) was dissolved in Milli-Q water (900 mL) adjusted to pH 7.5 with 1 M HCl. The mixture was transferred to a 1 L volumetric flask. Next, CGTase solution (25 mL) was added to initiate the reaction, and Milli-Q water was added up to the 1 L mark. The reaction mixture was transferred to a 2 L round-bottom flask and placed in a 30 °C water bath for 42 h. The reaction was then stopped by heating to boiling for 15 min. The reaction mixture was concentrated in vacuo to 0.2 L, centrifuged, and the supernatant was decanted, leaving the precipitated enzyme. CD9 was then precipitated by adding acetone (1.0 L) and isolated by filtration. The collected white solid was dissolved in water (100 mL), precipitated with acetone (500 mL), and isolated by filtration. This process was repeated twice, after which the white solid was dried in vacuo.

[0149] result CD9 was isolated as a white solid (4.67 g, 47% yield) with a purity of >99% (by HPLC-ELSD, Figure 9). 1 Further reprecipitation affords even greater purity, as demonstrated by H NMR spectroscopy (Figure 10). In a modification of the above procedure, by replacing acetone with other organic solvents such as ethanol, isopropanol, or acetonitrile, we found that CD9 could be isolated by precipitation from the aqueous solution described above in a similar manner.

[0150] conclusion High purity CD9 is Na2B 12 Cl12 CD6 in water was treated with CGTase in the presence of ATP and then isolated by precipitation from the aqueous solution using organic solvents.

[0151] Example 4, Na2B 12 Cl 12 Template Recovery Materials and Methods The combined filtrate and washes from the CD9 isolation procedure were concentrated under vacuum to remove acetone to a total volume of 200 mL. Next, HCl (37%) was added to pH 2, followed by the addition of triethylamine in small portions with stirring until no more gas was evolved. The white precipitate that formed was collected by filtration, washed several times with cold water, and then dried in vacuo. This Et3NHB 12 Cl 12 was converted to the sodium salt following the procedure reported by Geis et al.

[0152] result Na2B 12 Cl 12 The template was recovered from the reaction mixture in quantitative yield. The purity of the recovered template was 1 H and 11 B NMR spectroscopy (FIGS. 11 and 12) and MALDI mass spectrometry (FIG. 13) confirmed that the levels were at least the same as before recovery.

[0153] The recycled template was successfully used to generate more δ-cyclodextrin.

[0154] conclusion Na2B 12 Cl 12 The template can be directly reisolated from the reaction mixture and reused.

[0155] Example 5. Synthetic Procedure for Modified CD9 Materials and Methods NaB in buffered water (50 mM sodium phosphate at pH 7.5) 12 Cl 12To a solution of 50 mM CGTase (50 μL per mL) and mono-(6-deoxy)-CD6 (10 mg / mL), CGTase stock solution (50 μL per mL) was added. The reaction was monitored at various time points. An analytical aliquot (5 μL) was removed, and the enzymatic reaction was stopped by immediately adding this aliquot to a 1% (v / v) solution of trifluoroacetic acid (80 μL) in acetonitrile / water (3:1). The progress of the reaction and the composition of the CD mixture were analyzed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELS). Separation was performed using gradient elution on a HILIC column.

[0156] result After 20 days of reaction, the mass spectrum obtained showed that the compound was mainly CD9 ([M+Na] + The expected m / z of 1481.4) and 6-deoxy-CD9 (mono-, bis-, tris-, and tetrakis-(6-deoxy)-CD9 [M+Na] + The results show the formation of CD8 ([M+Na]) at the expected m / z 1465.5, 1449.5, 1433.5, and 1417.5 (Figure 14). + The expected m / z of 1319.4 for 6-deoxy-CD8 (mono-, bis-, and tris-(6-deoxy)-CD8 [M+Na] + The expected m / z values ​​for mono- and bis-(6-deoxy)-CD7 were 1303.4, 1287.4, and 1271.4, respectively, and 6-deoxy-CD7 ([M+Na] + The expected peaks (m / z 1141.4 and 1125.4) were also observed. These results were confirmed by HPLC-ELS chromatograms (Figure 15), in which the compounds responsible for the peaks at different retention times were identified by comparison with authentic samples (all unmodified sugars) or by MALDI-TOF-MS after fraction collection (all modified CDs).

[0157] conclusion Na2B 12 Cl 12The use of as a template also facilitates the synthesis of modified CD9.

[0158] Example 6. Procedure for Purifying CD9 from Starch Materials and Methods Soluble starch (commercial supplier Sigma-Aldrich, product number S9765) (2.00 g) was dissolved in water (80 mL, heated four times for 1 min in a microwave oven, then heated to boiling temperature in an oil bath with stirring for 20 min). 12 Cl 12 (0.300 g, 0.50 mmol) was added, and the pH was adjusted to 7.5 with 1 M HCl. The mixture was transferred to a 100 mL volumetric flask. The reaction was then initiated by adding CGTase stock solution (2.5 mL). The volume was adjusted to 100 mL with Milli-Q water, resulting in a starch concentration of 20 mg / mL. The reaction was monitored at various time points. An analytical aliquot (3 μL) was removed, and the enzymatic reaction was stopped by immediately adding this aliquot to a 1% (v / v) solution of trifluoroacetic acid in acetonitrile / water (3:1) (90 μL). The progress of the reaction and the composition of the CD mixture were analyzed using high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELS). Separation was performed using gradient elution on a HILIC column.

[0159] result Analysis of the reaction mixture from the first 3 days showed the production of some CDs and some short linear α-1,4-glucans (Figure 16). The major product was found to be CD9, which is similar to that found in reactions initiated with CD6 (see above).

[0160] conclusion Na2B 12 Cl 12 When starch is treated with CGTase in the presence of ATP, CD9 is mainly formed.

[0161] Example 7, Na2B 12 H x Cl 12-x Template-based CD9 generation procedure Materials and Methods Partially chlorinated chlorododecaborate Na2B in buffered water (50 mM sodium phosphate at pH 7.5) 12 H x Cl 12-x To a solution of CGTase (5 mM, assuming an average MW of 567 g / mol, see MALDI-MS in Figure 17) and CD6 (10 mg / mL), 50 μL of CGTase stock solution was added. The reaction was monitored at various time points. An analytical aliquot (3 μL) was removed, and the enzymatic reaction was stopped by immediately adding this aliquot to a 1% (v / v) solution of trifluoroacetic acid (90 μL) in acetonitrile / water (3:1). The progress of the reaction and the composition of the CD mixture were analyzed using high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELS). Separation was performed using gradient elution on a HILIC column.

[0162] result Analysis of the reaction mixture after 24 hours confirmed the production of CD6, CD7, CD8, and CD9, as well as several short linear α-1,4-glucans (Figure 18). The major product was found to be CD9.

[0163] conclusion CD9, Na2B 12 Cl 12 It is also the major product in enzymatic reactions using similar templates.

[0164] reference Cyclodextrins used as excipients,European Medicines Agency,Report published in support of the “Questions and answers on cyclodextrins used as excipients in medicinal products for human use'EMA / CHMP / 495747 / 2013.

[0165] V. Geis, K. Guttsche, C. Knapp, H. Scherer, R. Uzun, Dalton Trans. 2009, 2687.

Claims

1. A method for producing δ-cyclodextrin or a δ-cyclodextrin derivative, wherein the method is: a. Glucose-based compounds, b. Cyclodextrin glucanotransferase, c. Formula (B １２ Cl ｎ H １２－ｎ ) ２－ The ions of (wherein n is 1 to 12), and d. Solvent, Mixing to form a mixture, The mixture is incubated, Includes, A method for obtaining the δ-cyclodextrin or δ-cyclodextrin derivative thereby.

2. The δ-cyclodextrin or δ-cyclodextrin derivative is of formula I: 【Chemistry 1】 or has the structure of a salt thereof, in the formula, Each R is independently selected from CH ２ OR ａ , CH ３ , CH ２ F, CH ２ Cl, CH ２ Br, CH ２ I, CH ２ N ３ , CH ２ CN, CH ２ R ａ , CH ２ N(R ａ ) ２ , CH ２ SR ａ , CH ２ Si(R ａ ) ３ , CH ２ CHO, CH ２ COOH, CH ２ COOR ａ , CH ２ CON(R ａ ) ２ , CH ２ CHNR ａ , OR ａ , H, F, Cl, Br, I, N ３ , CN, R ａ , N(R ａ ) ２ , SR ａ , Si(R ａ ) ３ , CHO, COOH, COOR ａ , CON(R ａ ) ２ and CHNR ａ from the group consisting of, Each R ａ This is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl. The method according to claim 1.

3. The glucose-based compound is given by formula II: 【Chemistry 2】 It has the structure, and in the formula, Each R is CH ２ OR ａ ,CH ３ ,CH ２ F, CH ２ Cl, CH ２ Br, CH ２ I, CH ２ N ３ ,CH ２ CN, CH ２ R ａ ,CH ２ N(R) ａ ) ２ ,CH ２ SR ａ ,CH ２ Si(R ａ ) ３ ,CH ２ CHO, CH ２ COOH, CH ２ COOR ａ ,CH ２ CON(R ａ ) ２ ,CH ２ CHNR ａ , OR ａ , H, F, Cl, Br, I, N ３ , CN, R ａ , N(R ａ ) ２ , SR ａ , Si(R ａ ) ３ , CHO, COOH, COOR ａ CON(R ａ ) ２ and CHNR ａ Independently selected from the group consisting of, Each R ａ This is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl. m is an integer greater than or equal to 2; or The glucose-based compound is given by formula III: 【Transformation 3】 It has the structure, and in the formula, Each R is CH 2 OR a , CH 3 , CH 2 F, CH 2 Cl, CH 2 Br, CH 2 I, CH 2 N 3 , CH 2 CN, CH 2 R a , CH 2 N(R a ) 2 , CH 2 SR a , CH 2 Si(R a ) 3 , CH 2 CHO, CH 2 COOH, CH 2 COOR a , CH 2 CON(R a ) 2 , CH 2 CHNR a , OR a , H, F, Cl, Br, I, N 3 , CN, R a , N(R a ) 2 , SR a , Si(R a ) 3 , CHO, COOH, COOR a , CON(R a ) 2 And independently selected from the group consisting of CHNR a, Each R a is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl. k is an integer greater than or equal to 10, such as 6, 7, 8, 10, or 10; The method according to claim 1.

4. The method according to claim 1, wherein the glucose-based compound is selected from the group consisting of glucose, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, second cyclodextrin, second macrocyclic cyclodextrin, amylose, synthetic amylose, glycogen, maltodextrin, dextrin, cycloamylose, amylopectin, starch, modified starch, soluble starch, limit dextrin, α-glucan, pullulan, or any mixture of all of the above.

5. The method according to claim 1, wherein the glucose-based compound is glucose, a glucose derivative, modified glucose, a glucose-based polysaccharide, a glucose-based polysaccharide derivative, a modified glucose-based polysaccharide, a second cyclodextrin, or a second cyclodextrin derivative.

6. The method according to claim 1, wherein the second cyclodextrin is α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or a cyclodextrin having a glucose moiety size of 10 or more.

7. Formula (B １２ Cl ｎ H １２－ｎ ) ２－ the ion of is (B １２ Cl １２ ) ２－ , (B １２ Cl １１ H) ２－ , (B １２ Cl １０ H ２ ) ２－ , (B １２ Cl ９ H ３ ) ２－ , (B １２ Cl ８ H ４ ) ２－ , (B １２ Cl ７ H ５ ) ２－ , (B １２ Cl ６ H ６ ) ２－ , (B １２ Cl ５ H ７ ) ２－ , (B １２ Cl ４ H ８ ) ２－ , (B １２ Cl ３ H ９ ) ２－ , (B １２ Cl ２ H １０ ) ２－ and (B １２ ClH １１ ) ２－ The method according to claim 1, which is selected from the group consisting of.

8. Formula (B １２ Cl ｎ H １２－ｎ ) ２－ The ion is given by formula M ２ (B １２ Cl ｎ H １２－ｎ ) or M(B １２ Cl ｎ H １２－ｎ The method according to claim 1, wherein the salt is derived from a salt of a, and M is a monovalent or divalent cation.

9. The method according to claim 8, wherein M is selected from the group consisting of alkali metal ions and earth alkali metal ions.

10. M is selected from the group consisting of Li+, Na+, K+, Cs+, Mg2+, Ca2+, NH4+, (Rp)4N+, and (Rp)4P+, Each R p is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, substituted acyl, aryl, substituted aryl, benzyl, and substituted benzyl. The method according to claim 8.

11. The cyclodextrin glucanotransferase is the cyclodextrin glucanotransferase defined in Enzyme Commission (EC) No. 2.4.1.19; The cyclodextrin glucanotransferase is an enzyme capable of catalyzing reactions catalyzed by enzymes as defined in Enzyme Commission (EC) No. 2.4.1.19; The cyclodextrin glucanotransferase is selected from the group of enzymes defined in Enzyme Commission (EC) No. 2.4.1.25; or The cyclodextrin glucanotransferase is selected from the group of enzymes defined in Enzyme Commission (EC) No. 3.2.1.54; The method according to claim 1.

12. The solvent is a protic solvent; The solvent is a polar solvent; The solvent is a protic polar solvent; The solvent comprises two or more solvents; The solvent comprises water and an organic solvent; The solvent comprises dimethylformamide, dimethyl sulfoxide, acetone, acetonitrile, isopropanol, ethanol, or methanol, or is dimethylformamide, dimethyl sulfoxide, acetone, acetonitrile, isopropanol, ethanol, or methanol; The solvent may include or be an aqueous solvent; The solvent contains water or is water; or The solvent includes or is an aqueous buffer, such as a phosphate buffer; The method according to claim 1.

13. Incubation is carried out at 0 to 120°C, e.g., 0 to 100°C, e.g., 0 to 80°C, e.g., 0 to 70°C, e.g., 0 to 60°C, e.g., 5 to 45°C, e.g., 10 to 40°C, e.g., 15 to 40°C, e.g., 20 to 35°C; and / or Incubation takes place for 10 to 200 hours, for example 10 to 150 hours, for example 10 to 100 hours, for example 10 to 80 hours, for example 20 to 70 hours, for example 30 to 60 hours, for example 35 to 55 hours, for example approximately 40 to 50 hours; The method according to claim 1.

14. The glucose-based compound and formula (B １２ Cl ｎ H １２－ｎ ) ２－ The ratio of ions is 99:1 to 20:80 (w / w), for example, 90:10 to 65:35; The amount of the ion of formula (B 12 Cl n H 12-n) 2- is 2 to 50 mol%, for example 2 to 45 mol%, for example 3 to 40 mol%, for example 4 to 35 mol%, for example 5 to 30 mol%, for example 6 to 25 mol%, per monosaccharide portion in the glucose-based compound; The mixture contains cyclodextrin glucanotransferase in amounts equivalent to, for example, 0.1 to 100% (v / v) cyclodextrin glucanotransferase stock solution, for example, 0.1 to 60%, for example, 0.1 to 50%, for example, 0.5 to 40%, for example, 0.5 to 30%, for example, 1.0 to 20%, for example, 1 to 15%, for example, 1 to 10% stock solution; The concentration of the glucose-based compound in the solvent is 0.1 to 200 g / L, for example 0.1 to 150 g / L, for example 0.1 to 100 g / L, for example 0.2 to 90 g / L, for example 0.5 to 80 g / L, for example 0.5 to 70 g / L, for example 1 to 60 g / L, for example 1 to 50 g / L, for example 1 to 50 g / L, for example 1 to 40 g / L, for example 1 to 30 g / L, for example 1 to 25 g / L; and / or The concentration of the ion of formula (B 12 Cl n H 12-n) 2- in the solvent is 0.1 to 300 mM, for example 0.1 to 250 mM, for example 1 to 200 mM, for example 1 to 150 mM, for example 1 to 100 mM, for example 0.2 to 90 mM, for example 0.3 to 80 mM, for example 0.4 to 70 mM, for example 0.5 to 60 mM, for example 0.6 to 50 mM, for example 0.7 to 40 mM, for example 0.8 to 30 mM, for example 0.9 to 25 mM, for example 1.0 to 20 mM, for example 1.0 to 15 mM, for example 1.0 to 10 mM; The method according to claim 1.

15. a. Incubation takes place for 30 to 60 hours. b. Incubation is carried out at 0-45°C. The method according to claim 1.

16. a. Incubation takes place for 30 to 60 hours. b. Incubation is carried out at 0-45°C. c. The concentration of the glucose-based compound in the solvent is 1 to 50 g / L. d. Formula (B) in the solvent １２ Cl ｎ H １２－ｎ ) ２－ The ion concentration is 0.5 to 25 mM. e. The mixture contains a cyclodextrin glucanotransferase in an amount equivalent to the 0.1 to 50% (v / v) cyclodextrin glucanotransferase stock solution described in any one of the prior claims. The method according to claim 1.

17. The method further comprises the step of precipitating the δ-cyclodextrin or the δ-cyclodextrin derivative from the liquid phase, The precipitation of the δ-cyclodextrin or the δ-cyclodextrin derivative involves adding a reverse solvent to the liquid phase, thereby causing the δ-cyclodextrin or the δ-cyclodextrin derivative to precipitate. The method according to claim 1.

18. The method according to claim 17, wherein the reverse solvent is a protic solvent or an aprotic polar solvent.

19. The method according to claim 17, wherein the reverse solvent is selected from the group consisting of acetone, ethanol, isopropanol, and acetonitrile.

20. A δ-cyclodextrin or δ-cyclodextrin derivative obtained by the method of Claim 1, and a compound of formula (B １２ Cl ｎ H １２－ｎ ) ２－ A composition containing ions of .

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