NOVEL O-ALPHA-GLUCOSYL FLAVONOIDS ON CYCLE B, PROCESS FOR OBTAINING AND USES

专利摘要:
The present invention relates to a process for producing O-α-glucosylated flavonoid derivatives comprising at least one step of incubating at least one glucan-saccharase with a flavonoid and at least one sucrose, the flavonoid being of formula ( I): It also relates to new O-α-glucosyl flavonoid derivatives and their use.
公开号:FR3018822A1
申请号:FR1456417
申请日:2014-07-03
公开日:2015-09-25
发明作者:Sandrine Morel；Isabelle Andre；Yoann Brison；Emmanuelle Cambon；Yannick Malbert；Denis Pompon；Magali Remaud-Simeon；Philippe Urban
申请人:INST NAT SCIENCES APPLIQ；Centre National de la Recherche Scientifique CNRS；Institut National des Sciences Appliquees de Toulouse；Institut National de la Recherche Agronomique INRA；
IPC主号:

专利说明:

[0001] FIELD OF THE INVENTION The present invention relates to the field of the glucosylation of flavonoids and more particularly to the α-glucosylation of certain flavonoids, in order to obtain 0-α-glucosyl flavonoid derivatives, in particular on their aromatic ring B at the level of non-vicinal hydroxyl functions. The present invention also relates to the 0-α-glucosyl compounds obtained from a glucosylation process of the invention and the use of these compounds for different purposes, in particular cosmetic or therapeutic purposes.
[0002] PRIOR ART Flavonoids are compounds having a C6-C3-C6 carbon structure whose backbone is a 1-benzopyran type ring system, in which the aromatic ring is defined as ring A and the pyranic ring is defined as than cycle C, which also includes a phenyl substituent, on the pyranic ring, as cycle B. They constitute a group of 8000 compounds widely spread in the plant kingdom where they are responsible for the color of part of the flowers and fruits. They can thus intervene in the protection against solar radiation, in the resistance against pathogenic micro-organisms of the plant and against the herbivorous animals, as well as in the relations of interaction with the other organisms of the environment, such as symbiotic fungi, bacteria, or even insects (Quideau S et al., Angew Chem Int.End., 2011 50: 586-621). It is also attributed to them numerous biological properties including antioxidant, anti-hepatotoxic, antiallergic, anti-inflammatory, antiulcer and anti-tumor properties (Harborne J et al., Phytochemistry 2000 55: 481-504, Quideau S et al., Angew Chem Int.And.U. 2011 50: 586-621). The flavonoids can be hydroxylated in many positions, and these hydroxyl groups are frequently methylated, acetylated, prenylated or sulfated. In plants, they are most often present in the form of soluble C- or O-glycosylated glycosides. To date, there are several routes for obtaining glucosylated flavonoids.
[0003] Many flavonoids naturally exist in the form of glycosides. In vivo, glucosylation relies on the use of Leloir type glucosyltransferases, capable of transferring the glucosyl residue of a sugar nucleotide (UDP-glucose) to the flavonoid skeleton. These enzymes, which contribute to the synthesis of secondary metabolites in plants, are known to have a broad spectrum of acceptor substrates. However, their levels of production by plant cells are very low and the 3-glucosylation reaction is the most common compared to α-glucosylation. Cellular glucosylation can have various effects and affect the addressing and / or toxicity of the products obtained. Thus, although this is not an absolute rule, it should be noted that generally the glycosylation of flavonoids makes it possible to increase the stability and the solubility, and consequently the availability, of these molecules. Several UDP glycosyl transferases have been isolated and cloned into different microorganisms. The natural or recombinant forms of these enzymes can thus be used in vitro for the production of glucosylated flavonoids.
[0004] For example, the UDP glycosyl transferase (UGT) of Bacillus cereus was expressed in Escherichia neck (E. neck). This glucosyl enzyme apigenin, genistein, kaempferol, luteolin, naringenin and quercetin. Position 3 is the preferentially glucosylated position, but in the absence of hydroxyl function at this position, glucosylation takes place at position 7. The products obtained by the recombinant enzyme are identical to those produced by the wild-type enzyme (Ko JH et al., FEMS Microbiol Lett, 2006, 258: 263-268). Similarly, UDP-glucosyltransferase YjiC from Bacillus licheniformis DSM 13 was used to glucosylate apigenin. Two 13-mono-glucosyl forms, at the 4 'or 7 positions, were obtained. A 13-diglucosylated form at positions 4 'and 7 has also been structurally characterized (Gurung, R.B. et al., Mol Cells 2013, 36 (4): 355-361). Oleandomycin glycosyl transferase (01eD GT) from Streptomyces antibioticus was expressed in E. neck BL 21. The purified enzyme catalyzes the glucosylation of several flavonoids apigenin, chrysin, daidzein, genistein, kaempferol, luteolin, naringenin and quercetin, to from UDP-glucose. The best conversion (90%) was obtained with naringenin at 1.11 in 4 hours. No indication of the position of glucosylation is specified in the publication. (Choi S H et al., Biotechnol Lett, 2012, 34: 499-505).
[0005] Rosa hybrida UDP-glycosyltransferase RhGT1 was tested on a collection of 24 flavonoids. It shows results comparable to those obtained with oleandomycin glycosyl transferase in terms of acceptor recognition (Wang L et al., Carbohydr Res 2013, 368: 73-77).
[0006] To date, six microbial UDP-glycosyl transferases are known to have flavonoid glucosylation activity (Wang L et al., Carbohydr Res 2013, 368: 73-77). The glycosylation of flavonoids in vitro can be carried out by the use of glycoside hydrolase enzymes, cyclodextrin glucanotransferase transglycosylases or glycoside phosphorylases. More particularly, the enzymatic glycosylation of flavonoids in vitro can be carried out via the use of glucan saccharases. Such a synthetic route leads to the production of α-glucosylated flavonoids, and relies on the use of glucan-saccharases belonging to families 13 or 70 of glycoside hydrolases (GH 13 and GH 70) (CAZy classification - Henrissat B Davies, GJ, Curr Op, Struct Biol., 1997, 7: 637644). Glucansucrases are transglucosylases which catalyze from sucrose the synthesis of homopolymers, consisting of α-D-glucosyl units, called glucans. These glucans are generally of very high molar mass (108 Da), and have various structures due to the presence of different types of saccharide bonds (α-1,2, α-1,3, α-1,4, and or a-1,6) as well as their location in the polymer. Isomers of sucrose and glucose are also produced from sucrose but in very small amounts compared to the polymer. More particularly, these enzymes are capable of glucosylating so-called "acceptor" hydroxylated molecules, introduced into the reaction medium in addition to sucrose, such as flavonoids. The degree of glucosylation of the acceptor depends on its structure as well as that of the enzyme. Thus, an effective acceptor, or good acceptor, will be able to divert polymer synthesis almost completely for the benefit of its own glucosylation. On the contrary, an ineffective acceptor, or bad acceptor, can only very slightly divert the synthesis of polymers and therefore will be very little, if any, glucosylated.
[0007] This is why these enzymes have been studied for many years in order to offer innovative enzymatic tools, effective for the synthesis of original molecules, and meeting industrial needs including the synthesis of new gluco-conjugates of interest. Indeed, for obvious reasons, the industry is in constant search for new compounds, which can in particular be produced in sufficient quantities, and in particular at the lowest possible cost. As early as 1995, the glucosylation of catechin with a glucosyltransferase of Streptococcus sobrinus 6715 (serotype g) was carried out in a 100 mM phosphate buffer (pH 6) in the presence of 1 g / l of catechin and 2% of sucrose (Nakahara et al., Appl., Environ Microbiol., 1995, 61: 2768-2770). The monoglucosylated product obtained with a yield of 13.7% is 4'-O-α-D-glucopyranosyl - (+) - catechin. A similar enzyme, Streptococcus mutans GS-5 glucosyltransferase-D, was also tested a few years later on the same substrate (Meulenbeld G et al., Appl., Microbiol., 1999, 65: 4141-4147). Two monoglucosylation products were thus isolated: 4'-O-α-D-glucopyranosyl- (+) - catechin and 7-O-α-D-glucopyranosyl- (+) - catechin, as well as a di-glucosylated product. 4 ', 7-O-α-D-glucopyranosyl - (+) - catechin. A study was conducted in 2000 to determine the specificity of Streptococcus mutans GS-5 glucosyltransferase-D. Several acceptors have been tested (catechol, 3-methoxycatechol, 3-methylcatechol, 4-methylcatechol, phenol, 3-hydroxyphenol, benzyl alcohol, 2-hydroxybenzyl alcohol, 2-methoxybenzyl alcohol, 1-phenyl-1,2-ethanediol, 4 methylphenol, 3-methylphenol, 3,5-dihydroxybenzyl alcohol, 2-methoxy-4-methylphenol, 2-methoxybenzyl alcohol, 3-methoxybenzyl alcohol and catechin) (Meulenbeld G Hartmans S., Biotechnol Bioeng 2000, 70: 363 -369). Only acceptors possessing two adjacent and therefore vicinal hydroxyl groups on the aromatic ring B were glucosylated. A few years later, the enzymatic glucosylation of a flavone (luteolin) and two flavanols (quercetin and myricetin) was performed using two glucansucrases: Leuconostoc mesenteroides dextransucrase NRRL B-512F and alternansucrase of Leuconostoc mesenteroides NRRL B-23192 (Bertrand A et al., Carbohydr Res 2006, 341: 855-863). The reactions were carried out in a mixture of aqueous-organic solvents to improve the solubility of the substrates. A conversion of 44% was achieved after 24 hours of dextransucrase catalyzed reaction in 70% aqueous acetic acid / sodium acetate and 30% bis (2-methoxyethyl) ether. Two products have been characterized by NMR: 3'-O-α-D-glucopyranosylluteolin and 4'-O-α-D-glucopyranosylluteolin. In the presence of one alternansucrase, three additional products, namely 4'-O-α-D-triglucopyranosylluteolin and two forms of 4'-O-α-D-diglucopyranosylluteolin, were obtained with a luteolin conversion of 8%.
[0008] Both enzymes were also used to glucosylate quercetin and myricetin with respective conversion rates of 4% and 49%. However, no glucosylation was observed when these two enzymes were used with diosmetin, diosmin and 7-3-D-glucopyranosyldiosmetin. Glucosylation of quercetin in the presence of sucrose and glucansucrase of the strain Leuconostoc mesenteroides NRRL B-1299 has also been described in Korean Application KR20060063703. Epigallocatechin gallate was also glucosylated in the presence of sucrose and glucansucrase of Leuconostoc mesenteroides B-1299CB (Moon et al., Journal of Molecular Catalysis B: Enzymatic, 2006, 40: 1-7). A mixture of three products was obtained: - a product of monoglucosylation: 4 "-O-α-D-glucopyranosylepigallocatechin gallate (15.7%) and - two products of di-glucosylation: 7.4" -0-aD -glucopyranosylepigallocatechin (22.7%) and 4 ', 4' gallate (23.8%). Glucosylation of quercetin was performed in 2007 in the presence of sucrose and glucansucrase of Leuconostoc mesenteroides B-1299CB (Moon Y H et al., Enzyme Microb Technol 2007, 40: 1124-1129). A mixture of two monoglucosyl products is obtained: 4'-O-α-D-glucopyranosylquercetin and 3'-O-α-D-glucopyranosylquercetin.
[0009] The amylosucrase of Deinococcus geothermalis was expressed in E. neck and studied for the glucosylation of (+) - catechin and 3'-O-α-D-maltosylcatechine (Cho HK et al., Enzyme Microb Technol 2011, 49 (2): 246-253). In US Patent Application 20110183930A1, Auriol et al. have described the preparation of phenolic derivatives obtained by enzymatic condensation between phenolic compounds selected from pyrocatechols or their derivatives, and the glucosyl residue from sucrose. The production of these phenol compounds derivatives is carried out with a glucosyltransferase (EC 2.4.1.5). O-α-D-glucosides of synthesized phenolic compounds have a higher solubility in water than their parent polyphenol. These compounds are especially described for their use as antioxidant, antiviral, antibacterial, immunostimulant, antiallergic, antihypertensive, anti-ischemic, antiarrhythmic, antithrombic, hypocholesterolemic, antilipoperoxidant, hepatoprotective, anti-inflammatory, anticarcinogenic, antimutagenic, antineoplastic and vasodilators. Glucosylation of astragalin in the presence of sucrose and glucansucrase of Leuconostoc mesenteroides B-512FMCM has also been performed (Kim G E et al., Enzyme Microb Technol., 2012, 50: 50-56). Nine products were isolated, namely: - two products of monoglucosylation: kaempferol-3-0-3-D-glucopyranosyl- (1-> 6) -O-α-D-glucopyranoside and kaempferol-3-0-3- D-glucopyranosyl- (1 → 3) -O-α-D-glucopyranoside; and seven astragalin polyglucosylation products (glucosylation linkages of ampelopsin was further carried out in the presence of sucrose and glucansucrase of Leuconostoc mesenteroides B-1299CB4.) Five glucosylation products were isolated and the product of monoglucosylation characterized: it is 4'-O-α-D-glucopyranosylampelopsin (Woo HJ et al., Enzyme Microb Technol, 2012 51: 311-318) However, to the knowledge of the inventors, and despite the very large number of experiments carried out for many years in the field, the glucosylation of monohydroxy or hydroxyl flavonoids in a non-vicinal way, on cycle B, has never been carried out.
[0010] There is consequently a need, in the state of the art, for the availability of α-glucosyl flavonoids, and in particular 0-α-glucosylated flavonoids, on non-vicinal hydroxyl groups, in particular on cycle B.
[0011] SUMMARY OF THE INVENTION Thus, the present invention provides a method of making 0-α-glucosylated flavonoid derivatives comprising at least one step of incubating a glucansucrase with a flavonoid and at least one sucrose, wherein: ) said flavonoid is of formula (I) below: R7 in which ring C represents a ring selected from the group consisting of the following rings of formulas (II), (III), (IV) or (V): (III) R3 R3 (IV) (V) wherein: one of R1, R2 or R3 represents a ring B of the following formula (VI): wherein: R12 (VI (a) only one of the groups chosen from R8, R0, R10, R11 and R12 represents a hydroxyl group, the other groups of R8, R9, R10, R11 and R12, which are identical or different, being chosen from the group comprising a hydrogen atom; linear or branched, saturated or unsaturated C1-C10 hydrocarbon-based hydrocarbon optionally interrupted by at least one heteroat ome selected from 0, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; -N1-12; -00NH2; -CHO; -SH; -C (O) O (C2-C3); a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); or (b) R8 and only one of the groups selected from R10, R11 and R12 represent a hydroxyl group, R9 and the other groups from R10, R11 and R12, which are identical or different, being chosen from the group comprising a hydrogen atom ; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; -N1-12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); or (c) R9 and only one of R11 and R12 is a hydroxyl group, R8, R10, and the other of R11 and R12, which are the same or different, are selected from the group consisting of hydrogen; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C 5 -C 9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; } -N 12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; a C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); Or (d) Rio and R12 represent a hydroxy group, the groups R8, R9, and R11, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C1-C19 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; } -N 12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C1-C3 amine; a C1-C3 imine; a nitrile group; a C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); or (e) R8, R10 and R12 represent a hydroxyl group, the groups R9 and R11, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; } -N 12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); the groups R1, R2 and R3 which do not represent a ring B of formula (VI), which are identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched C1-C6 alkyl; an -OH group; a C 1 -C 3 amine; a COOH group; -C (O) O (C2-C3); a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); R1 ', R2' and R3 ', which are identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C 1 -C hydrocarbon-based group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; -NH2; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C2-C3 amine; a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); or the groups R1 and R1 'when R1 does not represent a ring B of formula (VI), or R2 and R2' when R2 does not represent a ring B of formula (VI), or R3 and R3 'when R3 does not represent a ring B of formula (VI), together form a group = 0; R4, R5, R6 and R7, identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C 1 -C hydrocarbon-based group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; an -OH group; -COOH; } -N 12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); and (B) said glucansucrase being selected from the group consisting of: - a sequence having at least 80% identity with SEQ ID NO: 1, said sequence having an amino acid X1 representing an amino acid selected from the group consisting A, C, E, F, G, H, I, K, M, N, P, Q, S, T, V and Y; a sequence having at least 80% identity with SEQ ID NO: 2, said sequence having an amino acid X2 representing an amino acid selected from the group consisting of A, C, D, F, G, H, K, L, M, N, P, S, V and Y; a sequence having at least 80% identity with SEQ ID NO: 3, said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A, C, G, I, K, M, N and W; a sequence having at least 80% identity with the sequence SEQ ID NO: 4, said sequence having an amino acid X4 representing an amino acid selected from the group consisting of C, I, N, P, V and W; a sequence having at least 80% identity with the sequence SEQ ID NO: 5, said sequence having an amino acid X5 representing an amino acid selected from the group consisting of A, C, D, G, I, K, L; , M, R, V and W; a sequence having at least 80% identity with SEQ ID NO: 6, said sequence having an amino acid X6 representing an amino acid selected from the group consisting of C, G, Q, S and T; a sequence having at least 80% identity with SEQ ID NO: 7, said sequence having an amino acid X7 representing an amino acid selected from the group consisting of A and G; a sequence having at least 80% identity with SEQ ID NO: 8; a sequence having at least 80% identity with SEQ ID NO: 9; said amino acid sequence X8 representing an amino acid selected from the group consisting of C, I and L - a sequence having at least 80% identity with SEQ ID NO: 10; a sequence having at least 80% identity with SEQ ID NO: 11; and a sequence having at least 80% identity with SEQ ID NO: 12, said sequence having amino acids X9, X10, X11, X12 and X13, with: (i) X9 representing, independently of X10, Xii, X 2 and X 13, an amino acid selected from the group consisting of G, S, V, C, F, N, I, L and W; X10 representing, independently of X9, X11, X12 and X13, an amino acid selected from the group consisting of L, I, H, Y and F; except where X9 is W and X10 is F; XIII representing A; Xi2 representing F; and X13 representing L; chosen in (ii) X9 representing W; an amino acid selected from X10 representing F; an amino acid X11 representing, independently of X9, X10, X12 and X13, the group consisting of E and A; an amino acid X12 representing, independently of X9, X10, X11 and X13, the group consisting of L and F; and X13 representing L; except where X11 is A and X12 is F; or (iii) X9 representing W; X10 representing F; X11 representing A; X12 representing, independently of X9, X10, X11 and X13, selected from the list consisting of A, R, D, N, C, E, Q, G, H, I, L, K, M, P, S, T , W, Y and V; and X13 representing, independently of X9, X10, X11 and X12, an amino acid selected from the list consisting of A, R, D, N, C, E, Q, G, H, I, K, M, F, P , S, T, W, Y and V.
[0012] The inventors have indeed shown, in a completely unexpected manner, that certain mutated specific glucan-saccharases, described hereinafter in the present text, possess the capacity to generate new O-α-glucosyl flavonoids on non-vicinal hydroxyl groups, in particular These mutated enzymes indeed have a higher glucosylation activity, or even much greater than their wild forms, these specific flavonoids, usually considered to be bad receptors because very difficult to glucosylate, especially on the B cycle.
[0013] More particularly, a glucansucrase used in a method of the invention is chosen from the group comprising: a sequence having at least 80% identity with SEQ ID NO: 1, said sequence having an amino acid X1 representing an amino acid selected from the group consisting of H, N or S; a sequence having at least 80% identity with SEQ ID NO: 2, said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of A, C, F, L, M, S or V; a sequence having at least 80% identity with SEQ ID NO: 3, said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A and N; a sequence having at least 80% identity with SEQ ID NO: 4, said sequence having an amino acid X4 representing an amino acid selected from the group consisting of C, I, N, P, V or W; a sequence having at least 80% identity with SEQ ID NO: 5, said sequence having an amino acid X5 representing an amino acid selected from the group consisting of C, K, R or V; a sequence having at least 80% identity with SEQ ID NO: 9, said sequence having an amino acid X8 representing an amino acid selected from the group consisting of C or L; and a sequence having at least 80% identity with SEQ ID NO: 12, said sequence having amino acids X9, X10, X11, X12 and X13, with: (i) X9 representing an amino acid selected from the group consisting of G, V, C and F; X10 representing F; X11 representing A; X12 representing F; and X13 representing L; (ii) X9 represents, independently of X10, X11, X12 and X13, an amino acid selected from the group consisting of S, N, L and I; X10 representing, independently of X9, XII, X12 and X13, an amino acid selected from the group consisting of L, I, H and Y; X11 representing A; Xi2 representing F; and X13 representing L; or (iii) X9 representing W; X10 representing F; XIII representing A or E; X12 representing L; and X13 representing L. The invention also relates to a 0-α-glycosylated flavonoid derivative obtained by the process of the invention. The invention further relates to a compound of the following formula (X): wherein (i) X14 represents a chain consisting of at least two α-glucoside groups, and X15 and X16, which are identical or different, are selected from the group comprising a hydrogen atom; a linear or branched C1-C6 alkyl; a group -C (O) O (C2-C3); and a chain consisting of 1 to 600,000 ci-glucoside moieties, or (ii) X14 is a single ci-glucoside moiety, and X15 and X16, the same or different, are selected from the group consisting of hydrogen; a linear or branched C1-C6 alkyl; a group -C (O) O (C2-C3); and a chain of from 1 to 600,000 α-glucoside moieties, provided that at least one of X15 and X16 is a chain of from 1 to 600,000 α-glucoside moieties. A chain consisting of 1 to 600,000 ci-glucoside groups according to the invention may more particularly consist of 1 to 500,000--glucoside groups, from 1 to 400,000--glucoside groups, from 1 to 300,000--glucoside groups, from 1 to 200,000 α-glucoside groups, from 2 to 100,000 α-glucoside groups, from 5 to 50,000 β-glucoside groups, from 10 to 25,000 β-glucoside groups or from 10 to 10,000 β-glucoside groups.
[0014] The invention also relates to a compound of formula (XI) below: X 17 0 (XI) in which X 17 represents a chain consisting of 1 to 600,000 ci-glucoside groups, and X 18 and X 19, identical or different are selected from the group consisting of a hydrogen atom; a linear or branched C1-C6 alkyl; a group -C (O) O (C2-C3); and a chain consisting of 1 to 600,000 β-glucoside moieties. The present invention also relates to the cosmetic use, as antioxidant agent, of at least one O-α-glycosylated flavonoid derivative according to the invention. The present invention further provides a 0-α-glycosylated flavonoid derivative according to the invention for its pharmaceutical use in the treatment and / or prevention of hepatotoxicity, allergies, inflammation, ulcers, tumors, menopausal disorders, or neurodegenerative diseases. Another aspect of the invention relates to a 0-α-glycosylated flavonoid derivative according to the invention for its pharmaceutical use as a veinotonic.
[0015] Finally, the present invention relates to the use of a 0-α-glycosylated flavonoid derivative according to the invention as a photovoltaic agent, insect repellent agent, bleaching agent, pesticide, fungicide and / or bactericide. In the context of the present invention, and unless otherwise indicated in the text, the following mean: - linear or branched, saturated or unsaturated C1-C10 hydrocarbon group, optionally interrupted by at least one heteroatom selected from O, N or S: an alkyl or an alkylene; alkyl: a saturated, linear or branched, saturated hydrocarbon aliphatic group comprising from 1 to 10, preferably from 1 to 6 carbon atoms; cycloalkyl: a cyclic alkyl group comprising from 3 to 10 ring members, preferably from 3 to 8 ring members. The cycloalkyl group is optionally substituted with one or more halogen atoms and / or alkyl groups; heterocycle: a cyclic alkyl group comprising from 4 to 9 ring members, preferably from 3 to 8 ring members, and consisting of 1 to 3 rings, comprising between 3 and 6 carbon atoms and 1 or more heteroatoms, for example 1, 2 or 3 heteroatoms, preferably 1 or 2, selected from nitrogen, oxygen and sulfur. The heterocycle group is optionally substituted with one or more halogen atoms and / or alkyl groups; partially cyclic alkyl group means an alkyl group of which only one part forms a ring; alkylene: a linear or branched divalent alkylene group comprising from 1 to 10, preferably from 1 to 6, carbon atoms; aryl: a cyclic aromatic group comprising between 5 and 9 carbon atoms, for example a phenyl group; heteroaryl: a cyclic aromatic group comprising between 3 and 10 atoms, including 1 or more heteroatoms, for example between 1 and 4 heteroatoms, such as nitrogen, oxygen or sulfur, this group comprising one or more rings, preferably 1 or 2 cycles. The heterocycles may comprise several fused rings. The heteroaryls are optionally substituted by one or more alkyl groups or an oxygen atom. By way of examples, mention may be made of thienyl, pyridinyl, pyrazolyl, imidazolyl, thiazolyl and triazolyl groups; - halogen: an atom of chlorine, fluorine, bromine or iodine; C1-C3 alcohol: an alcohol chosen from methanol, ethanol, propanol and isopropanol; a (C 1 -C 3) alkoxy: a group chosen from a methoxyl, an ethoxyl, a propyloxyl and an isopropyloxyl; C2-C3 acyl: a group chosen from an acetyl, a propylacetyl and an opyl acetyl; C1-C3 amine: a group chosen from a methylamine, an ethylamine and a propylamine; C1-C3 imine: a group chosen from a methylimine, an ethylimine and a propylimine; In the present application the term "glycoside" is used to designate a glycoside moiety.
[0016] The glycoside units are known to those skilled in the art. As examples of monosaccharide glycosides, the following glycosides may be mentioned: glucose, fructose, sorbose, mannose, galactose, talose, allose, gulose, idose, glucosamine, N-acetylglucosamine, mannoamine, galactosamine, glucuronic acid, rhamnose, arabinose , galacturonic acid, fucose, xylose, lyxose and ribose.
[0017] By way of examples of di- or oligosaccharide glycosides, the following glycosides may be mentioned: - di-saccharides: maltose, gentiobiose, lactose, cellobiose, isomaltose, melibiose, laminaribiose, chitobiose, xylobiose, mannobiose, sophorose, nigerose, kojibiose, rutinose, robinose, - oligo-sacchacaride: panose, galactotriose, 13-glucotriose, 13-glucotetraose, galactotetraose, especially maltodextrins, maltotriose, isomaltotriose, maltotetraose, maltopentaose, maltoheptaose. Examples of glycosides that may be mentioned include: - starch derivatives, especially maltose, maltodextrins, - cellulose derivatives, - pectins and their derivatives, - chitin, chitosan and their derivatives - glucoaminoglucans and their derivatives, - xyloglucan derivatives, - galactomannans and their derivatives. For the purposes of the invention, the term "a chain consisting of 1 to 6 glycoside (s)" a sequence of 1 to 6 glycosides mentioned above. Similarly, within the meaning of the present invention, the term "a chain consisting of 1 to 600,000 ci-glucoside groups a sequence of one to 600,000 glucosyl units linked to each other by a bonds.
[0018] DESCRIPTION OF THE FIGURES FIG. 1 illustrates the glucosylation efficiencies of apigenin (histogram - left-hand ordinate -%), the levels of relative activity on sucrose alone (black spots - right-hand ordinate - AU) and the mass concentrations of glucosylated apigenin (values above the histogram - mg / L), ASNp WT, DSR-S varde144N WT enzymes and their mutants ASNp 1228F, ASNp 1228L, ASNp 1228M, ASNp F229A, ASNp F229N, ASNp A289W, ASNp F290C, ASNp F290K and DSR-S varde144N S512C. Figure 2 illustrates the superposition of the UV chromatograms (k340 nm) for the six mutants representative of the six categories of apigenin glucosylation product profiles. The names of the enzymes corresponding to these reactions are indicated next to each chromatogram: ASNp A289W, DSR-S varde144N S512C, ASNp F290K, ASNp F290C, ASNp F229N and ASNp 1228F. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units).
[0019] Figure 3 illustrates the superposition of UV chromatograms (k340 nm) for the seven representative mutants of the six categories of naringenin glucosylation product profiles. The names of the enzymes corresponding to these reactions are indicated next to each chromatogram: ASNp F290V, ASNp R226N, ASR C-APY del, α-1,2 BrS, ASNp A289C, ASNp A289P / F290C and ASNp 1228A. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). FIG. 4 illustrates the UV chromatography profile obtained after glucosylation of apigenin, for the wild-type ASNp enzyme (ASNp WT), in comparison with the apigenin standard. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). Figure 5 illustrates the UV chromatography profile obtained after glucosylation of apigenin, for the mutant ASNp 1228F enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). The nature of the different peaks is indicated directly on the profile. Figure 6 illustrates the UV chromatography profile obtained after glucosylation of apigenin, for the mutant ASNp 1228L enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units).
[0020] Figure 7 illustrates the UV chromatography profile obtained after glucosylation of apigenin, for the mutant ASNp 1228M enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). The nature of the different peaks is indicated directly on the profile.
[0021] FIG. 8 illustrates the UV chromatography profile obtained after glucosylation of apigenin, for the mutant ASNp F229A enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). The nature of the different peaks is indicated directly on the profile. Figure 9 illustrates the UV chromatography profile obtained after glucosylation of apigenin, for the mutant ASNp F229N enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). The nature of the different peaks is indicated directly on the profile. Figure 10 illustrates the UV chromatography profile obtained after glucosylation of apigenin, for the mutant ASNp A289W enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). The nature of the different peaks is indicated directly on the profile. Figure 11 illustrates the UV chromatography profile obtained after glucosylation of apigenin, for the mutant ASNp F290C enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). The nature of the different peaks is indicated directly on the profile. Figure 12 illustrates the UV chromatography profile obtained after glucosylation of apigenin, for the mutant ASNp F290K enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). The nature of the different peaks is indicated directly on the profile. Figure 13 illustrates the UV chromatography profile obtained after glucosylation of apigenin, for the mutant enzyme DSR-S varde144N S512C. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). The nature of the different peaks is indicated directly on the profile. Figure 14 illustrates the high resolution electrospray-mode mass spectrum positive for the mono-glucosylated form of apigenin obtained with the mutant enzyme DSR-S varde144N S512C. On the abscissa: ratio m / z; On the ordinate: relative abundance.
[0022] Figure 15 illustrates the high resolution MS / MS spectrum in electrospray negative mode for the mono-glucosylated form of apigenin (at m / z 431.11) obtained with the mutant enzyme DSR-S varde144N S512C. On the abscissa: ratio m / z; On the ordinate: relative abundance.
[0023] Figure 16 illustrates the high resolution electrospray-positive mass spectrum for the mono-glucosylated form of apigenin obtained with the mutant ASNp A289W enzyme. On the abscissa: ratio m / z; On the ordinate: relative abundance. Figure 17 illustrates the high resolution mass spectrum in electrospray positive mode for the di-glucosylated forms of apigenin obtained with the mutant ASNp A289W enzyme. On the abscissa: ratio m / z; On the ordinate: relative abundance. Figure 18 illustrates the high resolution MS / MS spectrum in the electrospray negative mode for one of the two di-glucosyl forms of apigenin (at m / z 593.16) obtained with the mutant ASNp A289W enzyme. On the abscissa: ratio m / z; On the ordinate: relative abundance. Structure of the m / z ion at 353.0667, signature of a glucosylation of each of the two positions 5 and 7 of the A ring of apigenin. Figure 19 illustrates the high resolution MS / MS spectrum in negative electrospray mode for one of the two diglucosylated forms of apigenin (at m / z 593.16) obtained with the mutant ASNp A289W enzyme. On the abscissa: ratio m / z; On the ordinate: 20 relative abundance. Fragmentation of the di-glucosylated form at the 4 'position of the apigenin B cycle leading to the m / z ion at 269.0451. Figure 20 illustrates the UV chromatography profile obtained after glucosylation of naringenin, for the wild ASNp enzyme (ASNp WT), in comparison with the naringenin standard. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). Figure 21 illustrates the UV chromatography profile obtained after glucosylation of naringenin, for the mutant ASNp I228A enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). Figure 22 illustrates the UV chromatography profile obtained after glucosylation of naringenin, for the mutant ASNp A289C enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). FIG. 23 illustrates the UV chromatography profile obtained after glucosylation of naringenin, for the ASR-C-APY-del truncated wild-type enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). Figure 24 illustrates the UV chromatography profile obtained after glucosylation of naringenin, for the mutant ASNp A289P / F290C enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). Figure 25 illustrates the UV chromatography profile obtained after glucosylation of naringenin, for the wild-type α-1,2 BrS enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). Figure 26 illustrates the UV chromatography profile obtained after glucosylation of naringenin, for the mutant ASNp F290V enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). Figure 27 illustrates the UV chromatography profile obtained after glucosylation of naringenin, for the mutant ASNp R226N enzyme. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). Figure 28 illustrates the 1F1 COSY 2D NMR spectrum of 4'-O-α-D-glucopyranosylnaringenin. On the abscissa and ordinate: chemical shift, in part per million (ppm). Figure 29 illustrates the 1D 13C Jmod NMR spectrum of 4'-O-α-D-glucopyranosylnaringenin. On the abscissa and ordinate: chemical shift, in part per million (ppm). Figure 30 illustrates the 2D HMBC NMR spectrum of 4'-O-α-D-glucopyranosylnaringenin. In abscissa and ordinate: chemical shift, in part by million (ppm). Figure 31 illustrates the superposition of the chromatographic profiles obtained by LC-UV-MS for the morin glucosylation products by the enzyme 41 1123-GBD-CD2 WT and three of the most effective mutants to glucosylate this flavonoid. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units). Figure 32 illustrates the superposition of chromatographic profiles obtained by LC-UV-MS for the naringenin glucosylation products by the enzyme 41 1123-GBD-CD2 and three of its mutants effective for glucosylating this flavonoid. On the abscissa: Retention time in minutes; On the ordinate: Absorbance in mAU (Milliabsorbance Units).
[0024] DETAILED DESCRIPTION OF THE INVENTION In order to make available new O-α-glucosyl flavonoids, the Applicant has developed a novel process for the synthesis of novel α-gluco-flavonoid structures specifically glycosylated on non-vicinal hydroxyls, in particular This method uses mutated specific glucan-saccharases, identified by the applicant, capable of performing such a glucosylation. These specific enzymes require only the presence of sucrose, a renewable and cheap agro-resource. As such, a method according to the invention is advantageously inexpensive.
[0025] The present invention relates first of all to a process for producing 0-α-glucosylated flavonoid derivatives comprising at least one step of incubating an enzyme of the invention with a flavonoid of formula (I). ) and at least one sucrose. As indicated above, the enzymes of the invention are advantageously capable of glucosylating flavonoids at the level of non-vicinal hydroxyl function (s), in particular present on the B cycle. These enzymes consist more particularly of glucan. -saccharases belonging to families 13 and 70 of glycoside hydrolases (GH13 and GH70). The glucansaccharases belonging to the family 13 are naturally produced by the bacteria of the genera Deinococcus, Neisseria or Alteromonas. The glucansucrases belonging to the family 70 are naturally produced by lactic acid bacteria of the genera Leuconostoc, Lactobacillus or Streptococcus. Weissela sp .. As stated previously, various wild glucan saccharases of families 13 or 70 of the glycoside hydrolases have already been used for the production of glucosyl flavonoids, but none of them has so far been described as being capable of to glucosylate the flavonoids more particularly targeted in the present invention, namely those monohydroxylated on the ring B or having non-vicinal hydroxyl functions on the ring B.
[0026] However, as shown in the examples, the inventors have determined variants of these enzymes, mutated at their flavonoid binding site, and able to glucosylate such compounds effectively. The set of wild or mutated enzymes described in the present application which were known to those skilled in the art had never been used to date to glucosylate flavonoids according to the invention. The nucleotide sequence of the wild form of the ASNp (Amylosucrase Neisseria polysaccharea) enzyme (GH13 family) is GenBank AJ011781.1, whereas its polypeptide sequence is Uniprot Q9ZEU2. The nucleotide sequence of the wild form of the DSR-S enzyme (from the strain Leuconostoc mesenteroides B-512F) is GenBank 109598. The nucleotide sequence of the wild form of the enzyme DSR-E (from the strain Leuconostoc mesenteroides NRRL B-1299) has the GenBank AJ430204.1 reference and the Uniprot Q8G9Q2 reference. The enzyme AN123-GBD-CD2 (sequence SEQ ID NO: 12) is a truncated form of the aforementioned DSR-E enzyme, as described in Brison et al., J. Biol. Chem., 2012, 287, 7915-24. Bibliographic references describing these mutated enzymes are shown in Tables 1 and 4. In addition, the method for obtaining the mutated enzymes is described in European Patent Application EP 2 100 966 A1.
[0027] The peptide sequences of the different mutated or non-mutated enzymes according to the invention are indicated in the present application. Thus, an enzyme according to the invention can be synthesized by conventional methods of synthetic chemistry, or homogeneous chemical syntheses in solution or in solid phase. By way of illustration, one skilled in the art can use the polypeptide synthesis techniques in solution described by HOUBEN WEIL (1974, In method of Organizational Chemistry, E. Wunsh ed., Volume 154 and 15-II, Thieme, Stuttgart .). An enzyme according to the invention may also be chemically synthesized in the liquid or solid phase by successive couplings of the different amino acid residues (from the N-terminal end to the C-terminal end in the liquid phase, or from the C-terminus towards the N-terminus in solid phase). Those skilled in the art can notably use the solid phase peptide synthesis technique described by Merrifield (MERRIFIELD RB, (1965a), Nature, vol.207 (996): 522-523, MERRIFIELD RB, (1965b), Science , vol.150 (693): 178-185.) In another aspect, an enzyme according to the invention can be synthesized by genetic recombination, for example by a production method comprising the following steps: (a) preparing a vector of expression in which a nucleic acid encoding the peptide sequence of an enzyme of the invention has been inserted, said vector also comprising the regulatory sequences necessary for the expression of said nucleic acid in a selected host cell; (b) transfecting a host cell with the recombinant vector obtained in step (a) (c) culturing the host cell transfected in step b) in a suitable culture medium; (d) recovering the culture supernatant of the transfected cells or the cell lysate of said cells, for example by sonication or osmotic shock; and (e) separating or purifying, from said culture medium, or from the cell lysate pellet, the enzyme of the invention thus obtained. To purify an enzyme according to the invention which has been produced by host cells transfected or infected with a recombinant vector encoding said enzyme, one skilled in the art can advantageously use purification techniques described by Molinier-Frenkel (2002). J. Viral 76, 127-135), by Karayan et al. (1994, Virology 782-795) or Novelli et al. (1991, Virology 185, 365-376).
[0028] Thus, glucan-saccharases that can be used in a process of the invention are chosen from a group comprising: a sequence having at least 80% identity with SEQ ID NO: 1, said sequence having an amino acid X1 representing an acid amine selected from the group consisting of A, C, E, F, G, H, I, K, M, N, P, Q, S, T, V and Y; a sequence having at least 80% identity with SEQ ID NO: 2, said sequence having an amino acid X2 representing an amino acid selected from the group consisting of A, C, D, F, G, H, K, L, M, N, P, S, V and Y; a sequence having at least 80% identity with SEQ ID NO: 3, said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A, C, G, I, K, M, N and W; a sequence having at least 80% identity with the sequence SEQ ID NO: 4, said sequence having an amino acid X4 representing an amino acid selected from the group consisting of C, I, N, P, V and W; a sequence having at least 80% identity with the sequence SEQ ID NO: 5, said sequence having an amino acid X5 representing an amino acid selected from the group consisting of A, C, D, G, I, K, L; , M, R, V and W; a sequence having at least 80% identity with SEQ ID NO: 6, said sequence having an amino acid X6 representing an amino acid selected from the group consisting of C, G, Q, S and T; a sequence having at least 80% identity with SEQ ID NO: 7, said sequence having an amino acid X7 representing an amino acid selected from the group consisting of A and G; a sequence having at least 80% identity with SEQ ID NO: 8; a sequence having at least 80% identity with SEQ ID NO: 9; said amino acid sequence X8 representing an amino acid selected from the group consisting of C, I and L - a sequence having at least 80% identity with SEQ ID NO: 10; a sequence having at least 80% identity with SEQ ID NO: 11; and a sequence having at least 80% identity with SEQ ID NO: 12, said sequence having amino acids X9, X10, X11, X12 and X13, with: (i) X9 representing, independently of X10, Xii, X 2 and X 13, an amino acid selected from the group consisting of G, S, V, C, F, N, I, L and W; X10 representing, independently of X9, XII, X12 and X13, an amino acid selected from the group consisting of L, I, H, Y and F; except where X9 is W and X10 is F; XIII representing A; Xi2 representing F; and X1 representing L; (ii) X9 representing W; X10 representing F; X11 representing, independently of X9, X10, X12 and X13, an amino acid selected from the group consisting of E and A; X12 representing, independently of X9, X10, XII and X13, an amino acid selected from the group consisting of L and F; except where Xii is A and X12 is F; Xi3 representing L; or (iii) X9 representing W; X10 representing F; X11 representing A; X12 represents, independently of X9, X10, X11, and X13, an amino acid selected from the group consisting of A, R, D, N, C, E, Q, G, H, I, L, K, M, P S, T, W, Y and V; and X13 represents, independently of X9, X10, X11, and X12, an amino acid selected from the group consisting of A, R, D, N, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y and V. According to one embodiment of the invention, a sequence having at least 80% identity with the SEQ ID NO: 12 indicated above is preferably such that: i) X9 represents, independently of X10, X11, X12 and X13, an amino acid selected from the group consisting of G, S, V, C, F, N, I, L and W; X10 represents, independently of X9, X11, X12 and X13, an amino acid selected from the group consisting of L, I, H, Y and F; except where X9 is W and X10 is F; Xii is A; X12 is F; and X1 is L; or (ii) X9 is W; Xi0 is F; X11 represents, independently of X9, X10, X12 and X13, an amino acid selected from the group consisting of E and A; X12 represents, independently of X9, X10, X11 and X13, an amino acid selected from the group consisting of L and F; except where X11 is A and X12 is F; X13 represents L. It should be understood from this formulation that the amino acids defined as X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12 and X13 respectively are present and as defined. above in the glucan-saccharases of the invention having at least 80% identity with, respectively, a sequence SEQ ID NO: 1 to 7, 9 and 12, as defined above. As shown in the examples, all the enzymes possessing one of these peptide sequences have a capacity that is statistically greater than that of the wild-type glucosylating enzyme of the invention, having non-vicinal hydroxyl functions, especially on the cycle. B.
[0029] The present invention also encompasses sequences having an amino acid sequence of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of amino acid identity with one of SEQ ID NO: 1 to 12 such as previously defined and a biological activity of the same nature.
[0030] By biological activity of the same nature with regard to the peptide sequences 1 to 12, it is meant the same ability to glucosylate monohydroxylated or hydroxyl flavonoids in a non-vicinal way on the B cycle.
[0031] The "percentage of identity" between two nucleic acid or amino acid sequences, within the meaning of the present invention, is determined by comparing the two optimally aligned sequences, through a comparison window. The part of the nucleotide sequence in the comparison window may thus comprise additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or deletions) so as to obtain a optimal alignment between the two sequences. The percent identity is calculated by determining the number of positions at which an identical nucleotide base (or identical amino acid) is observed for the two compared sequences, and then dividing the number of positions at which there is identity between the two nucleic bases. (or between the two amino acids) by the total number of positions in the comparison window, then multiplying the result by one hundred in order to obtain the percentage of nucleotide (or amino acid) identity of the two sequences between them.
[0032] The optimal alignment of the sequences for the comparison can be performed in a computer manner using known algorithms. Most preferably, the percentage of sequence identity is determined using the CLUSTAL W software (version 1.82) with the parameters set as follows: (1) CPU MODE = ClustalW mp; (2) ALIGNMENT = "full"; (3) OUTPUT FORMAT = "aln w / numbers"; (4) OUTPUT ORDER = "aligned"; (5) COLOR ALIGNMENT = "no"; (6) KTUP (word size) = "default"; (7) WINDOW LENGTH = "default"; (8) SCORE TYPE = "percent"; (9) TOPDIAG = "default"; (10) PAIRGAP = "default"; (11) PHYLOGENETIC TREE / TREE TYPE = "none"; (12) MATRIX = "default"; (13) GAP OPEN = "default"; (14) END GAPS = "default"; (15) GAP EXTENSION = "default"; (16) GAP DISTANCES = "default"; (17) TREE TYPE = "cladogram" and (18) TREE GRAP DISTANCES = "hide". More particularly, the present invention also relates to sequences whose amino acid sequence has 100% amino acid identity with amino acids 225 to 450 of SEQ ID NO: 1 to 9, or 100% identity in amino acids with amino acids 2130 to 2170 of the sequence SEQ ID NO: 12 and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of amino acid identity with the rest of the sequences SEQ ID NO: 1 to 12 as defined above, and a biological activity of the same nature. Among the sequences of interest of the invention, some of them are more particularly interesting in terms of glucosylation activity.
[0033] Thus, according to one embodiment, the glucan-saccharases preferentially used in a method of the invention are chosen from the group comprising: a sequence having at least 80% identity with SEQ ID NO: 1, said sequence having an amino acid X1 representing an amino acid selected from the group consisting of H, N or S; a sequence having at least 80% identity with SEQ ID NO: 2, said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of A, C, F, L, M, S or V; a sequence having at least 80% identity with SEQ ID NO: 3, said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A and N; a sequence having at least 80% identity with SEQ ID NO: 4, said sequence having an amino acid X4 representing an amino acid selected from the group consisting of C, I, N, P, V or W; a sequence having at least 80% identity with SEQ ID NO: 5, said sequence having an amino acid X5 representing an amino acid selected from the group consisting of C, K, R or V; a sequence having at least 80% identity with SEQ ID NO: 9, said sequence having an amino acid X8 representing an amino acid selected from the group consisting of C or L; and a sequence having at least 80% identity with SEQ ID NO: 12, said sequence having amino acids X9, X10, X11, X12 and X13, with: (i) X9 representing an amino acid selected from the group consisting of G, V, C and F; X10 representing F; X11 representing A; X12 representing F; and X13 representing L; (ii) X9 represents, independently of X10, X11, X12 and X13, an amino acid selected from the group consisting of S, N, L and I; X10 representing, independently of X9, XII, X12 and X13, an amino acid selected from the group consisting of L, I, H and Y; X11 representing A; Xi2 representing F; and X13 representing L; or (iii) X9 representing W; X10 representing F; XIII representing A or E; X12 representing L and Xi3 representing L. According to a preferred embodiment, a sequence having at least 80% identity with SEQ ID NO: 12, having amino acids X9, X10, X11, X12 and X13, is such that: i) X9 represents an amino acid selected from the group consisting of G, V, C and F; X10 representing F; X11 representing A; Xi2 representing F; and X13 representing L; (ii) X9 represents, independently of X10, X11, X12 and X13, an amino acid selected from the group consisting of S and I; X10 representing, independently of X9, X11, X12 and X13, an amino acid selected from the group consisting of L, I and Y; X11 representing A; X12 representing F; and X13 representing L; or (iii) X9 representing W; X10 representing F; X11 representing A or E; X12 representing L and X13 representing L. The mutants of particular interest according to the invention of SEQ ID NO: 12 are in particular indicated in Example 11 of the present application. The enzymes whose sequences are SEQ ID Nos. 1 to 11 all have a glucosylation efficiency on the flavonoids of the invention of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% compared with 0.5 +/- 0.5% or 4.7 +/- 1.7% activity respectively for the wild-type 25 enzyme (see especially Tables 2, 3, 5 and 6). The enzymes whose sequence is SEQ ID No. 12 have a glucosylation efficiency on the flavonoids of the invention greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% compared with and respectively at 20.4 +/- 3.2% or 13.9 +/- 4.7% activity for the wild-type enzyme (see especially Tables 7 and 8).
[0034] Flavonoids, derivatives and implementations a) Flavonoids used in a process of the invention The flavonoids specifically used in a process of the invention are of formula (I) as described above.
[0035] According to one embodiment, only one of the groups chosen from R8, R9, R10, R11 and R12 represents a hydroxyl group, the other groups from R8, R9, R10, R11 and R12, which are identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; -N1-12; -00NH2; -CHO; -SH; -C (O) O (C2-C3); a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; and a C 1 -C 3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); Preferably, the group R10 represents a hydroxyl group. Preferably, the groups R8, R9, Ru and R12 represent hydrogen atoms. According to a preferred embodiment, the group R10 represents a hydroxyl group and the groups R8, R9, R11 and R12 represent hydrogen atoms. According to one embodiment, the ring C represents a ring of formula (II) or (IV) as defined above. According to one embodiment of the invention, the group R 1 represents a ring B of formula (VI) as defined above.
[0036] According to one embodiment, the groups R1 ', R2 and R2' represent hydrogen atoms, and R3 and R3 'together form a group = 0. According to a preferred embodiment, the group R 1 represents a ring B of formula (VI), the groups R 1 ', R 2 and R 2' represent hydrogen atoms, and R 3 and R 3 'together form a group = 0.
[0037] According to a preferred embodiment, the ring C represents a ring of formula (II) or (IV), the group R1 represents a ring B of formula (VI), the groups R1 ', R2 and R2' represent atoms of hydrogen, and R3 and R3 'together form a group = O. According to one embodiment, two of the groups R4, R5, R6 and R7 represent a hydroxyl group, the other two groups being as previously defined. Preferably, the two groups representing a hydroxyl group are the groups R4 and R6. According to one embodiment, two of the groups R4, R5, R6 and R7 represent a hydroxyl group, the other two groups representing a hydrogen atom.
[0038] According to one embodiment, the groups R5 and R7 represent hydrogen atoms. According to a preferred embodiment, the groups R4 and R6 represent a hydroxyl group and the groups R5 and R7 represent a hydrogen atom. According to another embodiment, R8 and only one of the groups chosen from R10, R11 and R12 represent a hydroxyl group, R9 and the other groups of R10, R11 and R12, which are identical or different, are chosen from the group comprising a hydrogen atom. hydrogen; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; -N1-12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s). Preferably, the group R10 represents a hydroxyl group. Preferably, the groups R 9, R 11 and R 12 represent hydrogen atoms. According to a preferred embodiment, the groups R 8 and R 10 represent a hydroxyl group and the groups R 9, R 11 and R 12 represent hydrogen atoms. According to one embodiment, the ring C represents a ring of formula (II) or (IV), preferably (II), as defined above.
[0039] According to one embodiment of the invention, the group R 1 represents a ring B of formula (VI) as defined above. According to one embodiment, the groups R 1 'and R 2 represent hydrogen atoms, R 2 represents a hydrogen atom or a group -OH, preferably an -OH group, and R 3 and R 3 together form a group = 0 . According to a preferred embodiment, the group R 1 represents a ring B of formula (VI), the groups R 1 'and R 2 represent hydrogen atoms, R 2 represents a group -OH, and R 3 and R 3 together form a group = O . According to a preferred embodiment, the ring C represents a ring of formula (II), the group R1 represents a ring B of formula (VI), the group R2 represents a group -OH, and R3 and R3 'together form a grouping = 0. According to one embodiment, a flavonoid used in a process of the invention has the following formula (VII), (VIII) or (IX): ## STR2 ## A flavonoid of the invention may be implemented in a process of the invention at a flavonoid sucrose molar ratio of between 1 and 35,000, the reaction mixture comprising at least the enzyme (s), sucrose and flavonoid (s) ( s) receiver (s).
[0040] Preferably, the sucrose to flavonoid molar ratio is between 7 and 292, the reaction mixture comprising at least the enzyme (s), sucrose and the flavonoid (s) receptor (s). b) O-α-Glucosylated Flavonoids Derivatives The present invention also relates to certain O-α-glucosyl flavonoid derivatives. Those are obtainable from a process of the invention. The present invention relates more particularly to compounds of the following formula (X): X 114 in which (i) X 14 represents a chain consisting of at least two α-glucoside groups, and X 15 and X 16, which are identical or different, are chosen from the group comprising a hydrogen atom; a linear or branched C1-C6 alkyl; a group -C (O) O (C2-C3); and a chain consisting of 1 to 600,000--glucoside moieties, or (ii) X14 is a single ci-glucoside moiety, and X15 and X16, the same or different, are selected from the group consisting of hydrogen; a linear or branched C1-C6 alkyl; a group -C (O) O (C2-C3); and a chain of from 1 to 600,000 α-glucoside groups, with the proviso that at least one of X15 and X16 is a chain of 1 to 600,000 β-glucoside moieties. As illustrated in Moulis et al. Understanding the polymerization mechanism of glycoside hydrolase family 70 glucansucrases, J. Biol. Chem. 2006, 281: 31254-31267, a compound according to the invention, and glucosylated using a glucansucrase according to the invention, may in fact comprise a chain consisting of 1 to 600,000 β-glucoside groups. The present invention also provides compounds of the following formula (XI): X (XI) 17 0 in which X 17 represents a chain consisting of 1 to 600,000 α-glucoside groups, and X 18 and X 19, which are identical or different, are chosen from the group comprising a hydrogen atom; a linear or branched C1-C6 alkyl; a group -C (O) O (C2-C3); and a chain consisting of 1 to 600,000 β-glucoside moieties. c) Use of the O-α-Glucosylated Fluconoid Derivatives of the Invention According to one embodiment, the O-α-glucosyl flavonoid derivatives of the invention can be used as an antioxidant (Heim et al. J. Nutr Biochem., 2002, 13: 572-584). According to one embodiment, the O-α-glucosyl flavonoid derivatives of the invention can be used for their pharmaceutical use in the treatment and / or prevention of hepatotoxicity, allergies, inflammation, ulcers, tumors, menopausal disorders, or neurodegenerative diseases (Harborne, J. et al., Phytochemistry, 2000 55: 481-504, Quideau S. et al., Angew Chem Int. End 2011, 50: 586). 621). According to one embodiment, the 0-α-glucosyl flavonoid derivatives of the invention may be used for their pharmaceutical use as veinotonic. (Katsenis K., Curr Vasc, Pharmacol 2005, 3 (1), 1-9) In addition, according to one embodiment of the invention, the O-α-glucosyl flavonoid derivatives of the invention may be implemented as: - Photovoltaic agent (see for this purpose the document Meng et al., Nano Lett 2008, 8 (10), 3266-72, Narayan MR, Renew Sust.Energy Rev. 2012 , 16, 208-215, US 2009/0071534 A1) - insect repellent (see especially for this purpose the documents JP 2002060304 JP 2003104818 Benavente-garcia et al., J. Agric Food Chem., 1997 , 45 (12), 4505-4515, Singh et al., Natural Product Sciences, 1997, 3 (1), 49-54, Diwan and Saxena, Int.J.Chem Sci., 2010, 8 (2), 777-782, Regnault-Roger et al., J. Stored Prod Res, 2004, 40, 395-408); bleaching agent (see in particular for this purpose the document Barkat Ali Khan et al., Asian J. Chem., 2011, 23 (2), pp. 903-906, patent applications WO 2008140440 A1, WO 2005094770 Ai; Zhu W. & Gao J., J. Invest Dermatol, Symposium Proceedings, 2008, 13, 20-24, Kim JH et al., J. Invest Dermatol., 2008, 128, 1227-1235); or - pesticidal, fungicidal and / or bactericidal agent (see especially for this purpose the documents WO 2013043031, CN 102477024 and CN 101002557). The present invention is further illustrated by, but not limited to, the following examples.
[0041] EXAMPLES Example 1 Production and Implementation of Recombinant Glucan Saccharases for the Glucosylation of Apigenin and Naringenin A library of 183 variants including 174 mutants, single or double, constructed from the amylosaccharase of N. polysaccharea ( GH13 family of glycoside hydrolases) and 10 variants, constructed from the glucansucrases DSR-S, ASR and ci-1,2 BrS (belonging to the GH70 family) were tested for their ability to glucosylate apigenin and naringenin. The origin of the glucan-sucrases selected for the study is reported in Tables 1 and 4. Tables 1 and 4 illustrate indeed a certain number of the glucanesaccharases tested in the examples of this text and specifies: column 1: the organism whose enzyme originates; column 2: the various wild-type enzymes tested as well as the mutated positions of the active site of these wild-type enzymes in the mutated glucan-saccharases also tested; column 3: the specificities of majority bonds during the synthesis of the natural polymer; column 4: the bibliographical references in which these enzymes, both in wild and mutated forms, have been described in the state of the art. These enzymes have been used in recombinant form and are expressed in Escherichia neck. 1.1. Production of Enzymes in Microplates All Escherichia neck strains overexpressing the heterologous glucansaccharases of the wild-type GH13 and GH70 families or their mutants are maintained in 96-well microplate format to facilitate future flavonoid glucosylation screening steps. From the source microplates, a pre-culture of these E. coli strains. This is done for 22 hours at 30 ° C, 700 rpm in 96-well microplates, in 200 μl of LB culture medium supplemented with 100 μg / ml of ampicillin. These precultures are in turn used to seed the "deep-well" microplates, each well containing 1 ml per well of self-inducing medium ZYM5052 containing in particular 0.2% (w / v) α-lactose, 0.05%. % (w / v) D-glucose, 0.5% (w / v) glycerol and 0.05% (w / v) L-arabinose (Studier et al., 2005). After 22 hours of culture at 30 ° C. and 700 rpm, the cell suspension is centrifuged for 20 minutes at 3000 g at 4 ° C. The cell pellets are resuspended in 96 well deep well microplates, with 300 μl of phosphate buffered saline (24 mM sodium / potassium phosphate and 274 mM NaCl) containing 0.5 g / L lysozyme and 5 mg / L Bovine pancreatic RNAse. Then incubation is carried out for 30 minutes at 30 ° C. with stirring, these microplates then being stored overnight at -80 ° C. After thawing, the microplates are vigorously stirred and then centrifuged for 20 minutes at 3000 g at 4 ° C. The centrifuged cell lysates containing the recombinant enzymes are transferred into clean deep-well 96-well microplates. 1.2. Implementation of the acceptor reactions The enzymatic extracts obtained are used to conduct the enzymatic flavonoid glucosylation screening reactions. The enzymatic activity of each centrifuged cell lysate is evaluated in microplate format, in final weight after 30 minutes of incubation in the presence of 146 mM final sucrose, by determination of reducing sugars by 3,5-dinitrosalicylic acid (DNS). . Finally, after dilution in ultrapure water, the absorbance is read at 540 nm.
[0042] The flavonoid acceptor reactions are then carried out in "deep-well" microplates, in a volume of 300 μl, at final concentrations, of sucrose of 146 mM, of flavonoid of 2.5 mM (apigenin) or 5 mM (naringenin) (initially dissolved in 100% DMSO), and 140 μl of centrifuged cell lysate. The final concentration of DMSO in the reaction medium is 3% (v / v).
[0043] Incubation is conducted at 30 ° C and 700 rpm. After 24 hours, the enzymes are denatured at 95 ° C for 15 minutes. These microplates are stored at -80 ° C for rapid evaluation of flavonoid glucosylation by mass spectrometry-coupled liquid chromatography (HPLC-MS or LC-MS). 1.3. Analytical Techniques For their HPLC-MS analyzes, extensively homogenized reaction media are diluted 1: 30 in DMSO. The separation of the flavonoids and their glucosylated forms is carried out in inverse phase with a ProntoSIL Eurobond® 53x3.0 mm 120-3-C18-AQ column (120 A porosity, 3 μm particle size, C18 grafting, Bischoff Chromatography, Germany). This column is maintained at 40 ° C on a Dionex Ultimate 3000 HPLC system equipped with a UV / Vis detector. This system is coupled to a single quadrupole mass spectrometer (Thermo Scientific, MSQ Plus).
[0044] The mobile phase is composed of an ultrapure water mixture (Solvent A) / LC-MS grade acetonitrile (solvent B) each containing 0.05% (v / v) of formic acid. The separation is ensured in 10 minutes by a solvent gradient B defined as follows: 0 min, 15% (v / v); 3 min, 25% (v / v); 6.5 min, 49.5% (v / v); 6.6 min, 80% (v / v); 6.8 min, 15% (v / v); and 10 min, 15% (v / v). Ionization in mass spectrometry on the MSQ Plus equipment is performed in electrospray positive (ESI +) mode for apigenin and negative (EST-) for naringenin. The voltage of the capillary is set at 3000 V, that of the cone at 75 V. The temperature of the source block is fixed at 450 ° C. The LC-MS / MS system used for high-resolution mass spectrometry or MS / MS fragmentation analysis includes an Ultimate 3000 (Dionex) chromatographic separation system coupled to a linear trap / Orbitrap hybrid mass spectrometer (LQT Orbitrap). Thermo Fischer Scientific). The ionization in mass spectrometry on the equipment LQT Orbitrap is this time carried out either in electrospray mode positive (ESI +) or in negative mode (EST-). EXAMPLE 2 Determination of the Glucosylation Efficiencies of Apigenin by the Recombinant Amylosaccharase of N Polysaccharea and its Variants The reactions in the presence of acceptor were carried out by applying the conditions described in Example 1. The effectiveness Flavonoid glucosylation was determined from the following formula: Glucosylation efficiency = ((peak area of (s) flavonoid (s) glucosylate (s))) / ((peak area of (s) flavonoid (s) Glucosylate (s)) + residual flavonoid peak aglycone area) x 100 The flavonoid glucosylation efficiencies, expressed as a percentage, were calculated from the peak areas of the various products analyzed, as described in Example 1, by HPLC equipped with a UV detector (X.340 nm), after 24 hours of reaction. The values obtained are reported in Table 2. Table 2 illustrates the glucosylation efficiency, during microplate screening, of apigenin for the wild form of ASNp (N. polysaccharea recombinant amylosaccharase) as well as for his 174 mutants from his active site. On the ordinate: the mutation positions of the wild-type enzyme (ASNp WT); on the abscissa: the amino acid substituting the one present in the sequence of the wild-type enzyme.
[0045] Thus, by way of illustration, the percentage of 1.7% indicated in line 2, column 2 was obtained using an enzyme mutated at position 226 by the substitution of the amino acid R (Arginine) with the acid amine A (Alanine). Each cell represents a single mutation at positions R226, 1228, F229, A289, F290, 1330, V331, D394 and R446 or a double mutation, that is, two simple mutations at two of these positions. The gray bar in each box shows the level of glucosylation efficiency compared to the most effective mutant. The results obtained for the wild-type enzyme are shown above Table 2 as well as at the R226R, 12281, F229F, A289A, F290F, 13301, V331V, D394D and R446R intersections. The 3 double mutant variants are shown in Table 2. For the wild form of the ASNp (Amylosucrase Neisseria polysaccharea) enzyme, the glucosylation efficiency is very low (0.5 ± 0.5, n = 16). The glucosylation efficiencies obtained for R226R, 12281, F229F, A289A, F290F, 13301, V331V, D394D and R446R given in Table 2 are also in the range of 0.5 ± 0.5. With a higher glucosylation efficiency of apigenin than that of the wild-type enzyme (greater than 1%), a large number of mutant enzymes emerge from this screening. More particularly, with a glucosylation efficiency of apigenin greater than 5%, eight enzymes stand out more particularly from the screening. The glucosylation efficiencies for these eight mutated enzymes are as follows: ASNp 1228F: 9.9%; ASNp 1228L: 11.1%; ASNp 1228M: 5.4%; ASNp F229A: 5.6%; ASNp F229N: 5.7%; ASNp A289W: 22.1%; ASNp F290C: 5.4%; and ASNp F290K: 8.9%. This illustrates the advantage of using directed engineering enzymes for the glucosylation of weakly recognized acceptors such as monohydroxy or hydroxyl flavonoids in a non-vicinal manner, in particular on cycle B.
[0046] Example 3: Determination of Glucosylation Efficacy of Apigenin by Glucan Saccharases of the GH70 Family GH70 family glucansucrases tested for their activity of glucosylation of apigenin are listed in Table 4.
[0047] Table 4 illustrates the glucan saccharases of the GH70 family (glycoside hydrolase 70) tested in the examples of this text. Thus, ASR C-APY-del WT represents the truncated form of ASR (alternansucrase), DSR-S varde144N WT represents the wild truncated form DSR-S (dextransucrase) and, for example, DSR-S varde144N F353T represents the truncated form of the DSR-S mutated at position 353 by the substitution of the amino acid F (phenylalanine) with the amino acid T (Threonine). Glucosylation results of apigenin by glucan saccharases of the GH70 family are reported in Table 5.
[0048] Table 5 illustrates the glucosylation efficiency of apigenin for the wild form of the truncated DSR-S variant (varde144N WT), for the truncated wild form of ASR (ASR C-APY-del WT), for the wild form of the enzyme α-1,2 BrS, and for seven mutants of DSR-S varde144N. The gray bar in each box shows the level of glucosylation efficiency compared to the most effective mutant. While the wild form of the truncated DSR-S variant (DSR-S varde144N WT) shows only a very low glucosylation activity (0.5%), the S512C mutant has a higher glucosylation efficiency of apigenin. (13.9%). Example 4: Comparison of the glucosylation efficiencies of apigenin for the most effective enzymes Among the tested enzymes of the GH13 and GH70 families, nine mutants have apigenin glucosylation efficiencies greater than 5%, namely ASNp 1228F, ASNp 1228L, ASNp 1228M, ASNp F229A, ASNp F229N, ASNp A289W, ASNp F290C, ASNp F290K and DSR-S (varde144N S512C). These efficiencies are compared for these nine most effective mutants, with their relative activities in the presence of sucrose alone (see Figure 1). The saccharose hydrolysis activities of the wild-type enzymes, ASNp WT 5 (GH13) or DSR-S varde144N WT (GH70), were taken as references for the calculation of the relative activities of sucrose hydrolysis of their respective mutants. Although the mutants show lower sucrose activity alone than wild-type enzymes, the glucosylation efficiencies of these same mutants are 10- to 44-fold higher than for wild-type enzymes. More generally, the correlation coefficient between the glucosylation efficiencies of apigenin and the sucrose hydrolysis activities, calculated for all mutant enzymes of N. polysaccharea amylosucrase, is 0.08. This illustrates the interest of the method for identifying enzymes that are not very active on sucrose alone but capable of glucosylating the flavonoids of the invention. In the case of the ASNp A289W mutant enzyme, a mass concentration of 149 mg / ml of glucosylated apigenin is reached. This is the minimum concentration obtained in microplate. Thus, a factor of improvement can be expected after optimization of the medium. EXAMPLE 5 LC-MS Analysis of Glucosylation Products of Apigenin The nine mutants mentioned in Example 4 can be classified into 6 categories according to the profile of glucosylation products obtained in LC-MS. The superposition of the UV chromatograms (k340 nm) for a representative of each of these 6 categories of profiles (ASNp A289W, DSR-S (varde144N S512C), ASNp 25 F290K, ASNp F290C, ASNp F229N and ASNp I228F respectively) is presented in FIG. 2. The superposition of these chromatograms demonstrates the diversity of forms of glucosylated apigenin that can be achieved. The LC-MS profiles obtained for ASNp WT and the nine most effective mutants mentioned in Example 4 are shown in Figures 4 to 13. The molar masses as determined by LC-MS in Example 1 of the peak of The most intense glucosylated apigenin for each of the nine mutants are as follows: Figure 5: 432.7 g / mol Figure 6: 432.7 g / mol Figure 7: Not determined Figure 8: 432.8 g / mol Figure 9: 432 , 7 g / mol Figure 10: 594.8 g / mol Figure 11: no data available Figure 12: no data available Figure 13: 432.7 g / mol.
[0049] The wild-type enzyme has a very low glucosylation efficiency on apigenin (0.5%). In fact, if the apigenin standard is compared with the final products of the glucosylation reaction, the appearance on the UV chromatogram of several peaks with very low intensities of glucosylated apigenin is detected (FIG. 4). Mutants I228F (Figure 5), I228L (Figure 6) and I228M (Figure 7) have similar product profiles to each other. However, variations in the proportions of the different forms of glucosylated apigenin are observed with the I228M mutant (Figure 7). Mutant group F229A (Figure 8) and F229N (Figure 9) also show similar product profiles. Finally, the F290K mutant has a more complex product profile than that of the F290C mutant. Example 6 LC-MS High-Resolution LC-MS / MS Analysis of Apigenin Glucosylation Products A study was performed by high-resolution LC-MS and LC-MS / MS (results obtained from Imagif), on the glucosylation products of apigenin obtained with the mutant enzymes ASNp A289W and DSR-S varde144N S512C. The glucosylation product of apigenin produced by the enzyme mutant DSR-S varde144N S512C is a monoglucosylated form (Figure 13) whose retention time is 4.23 min, and the m / z ratio in electrospray mode Positive was determined at 433.1199 (Figure 14). LC-MS / MS analysis in the negative electrospray mode of this monoglucosylated form produced by DSR-S varde144N S512C led to the identification of two major ions whose m / z ratios were determined at 269.0451 and 268.9360 (Figure 15). , thus making it possible to support obtaining 0-glucosylation at the 4 'position of the B-ring of apigenin. The ASNp A289W enzyme glucosylated apigenin to give a monoglucosylated product whose retention time was 4.25 min (FIG. 10) and whose m / z ratio in electrospray positive mode was determined at 433.1110 (FIG. 16). ). This analysis also showed that the peak of glucosylation product eluting at 3.68 min (FIG. 10) corresponds to a diglucosylation of apigenin. The m / z ratio in electrospray positive mode was determined at 595.1645 (Figure 17). LC-MS / MS analysis in negative electrospray mode reveals that two diglucosylated forms of apigenin co-elute at 3.68 min. The LC-MS / MS negative electrospray analysis of the first diglucosylated form produced by ASNp A289W led to the identification of three major ions whose m / z ratios were determined at 353.0660, 311.0555 and 269. , 0451 (Figure 18). This thus makes it possible to support obtaining a diglucosylated form for which each of positions 5 and 7 of ring A is 0-glucosylated. LC-MS / MS analysis in the negative electrospray mode of the second diglucosylated form produced by ASNp A289W led to the identification of a single major ion whose m / z ratio was determined to be 269.0451 (Figure 19), thus allowing to support obtaining di-O-glucosylation at the 4 'position of ring B, only apigenin.
[0050] EXAMPLE 7 Determination of the Glucosylation Efficiencies of Naringenin by the Recombinant Amylosaccharase of N Polysaccharea and its Variants The reactions in the presence of acceptor were carried out by applying the conditions described in Example 1.
[0051] The glucosylation efficiency of the flavonoid was determined from the formula stated in Example 2. The flavonoid glucosylation efficiencies, expressed as a percentage, were calculated from the peak areas of the various products analyzed, as described in FIG. Example 1, by HPLC equipped with a UV detector (k340 nm), after 24 hours of reaction.
[0052] The values obtained are reported in Table 3.
[0053] Table 3 illustrates the glucosylation efficiency, during microplate screening, of naringenin for the wild-type form of ASNp (N. polysaccharea recombinant amylosaccharase) as well as for the 174 mutants of its active site. On the y-axis: mutation positions of the wild-type enzyme (ASNp WT); on the abscissa: the amino acid substituting the one present in the sequence of the wild-type enzyme. Thus, by way of illustration, the percentage of 2.4% indicated in line 2, column 2 was obtained using an enzyme mutated at position 226 by the substitution of the amino acid R (Arginine) with the acid amine A (Alanine). Each cell represents a single mutation at positions R226, 1228, F229, A289, F290, 1330, V331, D394 and R446 or a double mutation, ie, two simple mutations at two of these positions. The gray bar in each box shows the level of glucosylation efficiency compared to the most effective mutant. The results obtained for the wild-type enzyme are shown at the top of Table 3 as well as at the R226R, 12281, F229F, A289A, F290F, 13301, V331V, D394D and R446R intersections. The results obtained for the doubly mutated enzymes at positions 289 and 290 are shown at the bottom of Table 3. For the wild form of the ASNp enzyme the glucosylation efficiency is reduced (4.7 ± 1.7, n = 16). With a higher glucosylation efficiency of naringenin than that of the wild-type enzyme (greater than 6.4%), a large number of mutant enzymes emerge from this screening. More particularly, with a glucosylation efficiency of naringenin greater than 10%, sixteen mutant enzymes are more particularly apparent from the screening. Seven of these mutant enzymes in particular have a naringenin glucosylation efficiency greater than 20% and two of them have an efficiency greater than 50%. The glucosylation efficiencies for these sixteen mutated enzymes are respectively: ASNp R226H: 13.5%; ASNp R226N: 16.0%; ASNp 30 R2265: 14.1%; ASNp 1228A: 70.2%; ASNp 1228C: 30.9%; ASNp I228S: 16.4%; ASNp 1228V: 12.3%; and ASNp A289C: 27.8%; ASNp A2891: 11.2%; ASNp A289N: 14.5%; ASNp A289P: 10.3%; ASNp A289V: 21.8%; ASNp F29OR: 11.2%; ASNp F290V: 21.1%; ASNp A289P / F290C: 50.9%; ASNp A289P / F290L: 22.9 ° A. The glucosylation of naringenin illustrates the interest of using directed engineering enzymes for the glucosylation of weakly recognized acceptors such as flavonoids. Example 8: Determination of glucosylation efficiencies of naringenin by glucansucrases of the GH70 family The glucansucrases of the GH70 family tested for their activity of glucosylation of apigenin are listed in Table 4. The glucosylation results of naringenin by GH70 family glucansucrases are reported in Table 6. Table 6 illustrates the glucosylation efficiency of naringenin for the wild form of the truncated DSR-S variant (varde144N WT), in the form of ASR truncated wild-type (ASR C-APY-del WT), for the wild form of the enzyme α-1,2 BrS and for seven mutants of DSR-S varde144N. The gray bar in each box shows the level of glucosylation efficiency compared to the most effective mutant. The wild form of the ASR truncated variant (ASR C-APY-del WT) has a glucosylation efficiency of 27.1 ° A. The wild-α-1,2 BrS enzyme has a naringenin glucosylation efficiency of 26.8 ° A. Example 9: LC-MS analysis of naringenin glucosylation products The eighteen mutants with a naringenin glucosylation efficiency greater than 10% discussed in Examples 7 and 8 can be classified into seven categories according to the product profile of glucosylation, obtained in LC-MS. The superposition of the UV (X.340 nm) chromatograms for a representative of each of these seven profile categories is shown in Figure 3.
[0054] The superposition of these chromatograms reveals the diversity of forms of glucosylated naringenin that can be achieved. The LC-MS profiles obtained for ASNp WT, the five mutant enzymes of ASNp and the two most effective GH70 family glucan-saccharases are shown in Figures 20 to 27. Molar masses, as determined by LC-MS in Example 1, the peak of the most intense glucosylated naringenin for each of these profiles are as follows: Figure 20: Not determined Figure 21: 433.6 g / mol Figure 22: 595.5 g / mol Figure 23: 758 , 4 g / mol Figure 24: 595.5 g / mol Figure 25: 433.8 g / mol Figure 26: 433.8 g / mol Figure 27: 758.0 g / mol The wild-type enzyme (Figure 20) reduced glucosylation efficiency on naringenin (4.7%). Indeed, if one compares the naringenin standard with the final products of the glucosylation reaction, the appearance on the UV chromatogram of several peaks, low intensities, of glucosylated naringenin (Figure 20) is detected.
[0055] The glucosylation profiles of naringenin obtained with the ASNp R226N, ASNp 1228A, ASNp A289C, ASNp F290V, ASNp A289P / F290C, ASR-C-APYdel or α-1,2 BrS enzymes are all distinct (Figures 21 to 27). EXAMPLE 10 Production, purification and structural determination by R1VIN of 4'-O-α-D-glucopyranosylnaringenin by ASNA mutant 1228A Production of 4'-O-α-D-glucopyranosylnaringenin The production of the glucosylation products is carried out with the enzyme ASNp 1228A on 204 mg of naringenin. The reaction conditions are as follows: final concentration of 146 mM sucrose, 5 mM naringenin (initially dissolved in 150 mM DMSO), PBS buffer pH 7.2, ASNp 1228A 0.5 U / mL and ultrapure water qs 145 mL. The reaction is conducted with stirring at 30 ° C for 24 h. At the end of the reaction, the enzyme is thermally inactivated. The reaction mixture is stored at -20 ° C. Purification of 4'-O-α-D-glucopyranosylnaringenin A pre-purification step is carried out by solid phase extraction (SPE) on a cartridge containing 5 g of C18 stationary phase. After conditioning the column, the centrifuged reaction mixture is deposited on the column and percole by gravity. After the washing steps with ultrapure water, the elution is carried out with methanol. The eluate is dried under a stream of nitrogen gas before being taken up in 100% DMSO at a concentration of 100 g / l.
[0056] The different glucosylated forms of naringenin are separated at room temperature by semi-preparative HPLC-UV on a Waters equipment. A 250 × 10 mm C18 column provided with a precolumn makes it possible to separate the different glucosyl forms of naringenin with an aqueous mobile phase at 0.05% (v / v) of formic acid with an acetonitrile (B) gradient. . The different steps of the gradient are as follows: 0 min, 22% B; 1 min, 22% B; 17 min, 25% B; 21 min, 29% B; 21.5 min, 95% B; 24.5 min, 95% B; 25 min, 22% B; 27.5 min, 22% B. Based on the UV signal, the elution fractions are collected in an automated manner. The purity of the elution fractions is evaluated by LC-UV-MS with a C18 250 × 4.6 mm analytical column (gradient described above). The elution fractions containing a monoglucosyl form of 96% pure naringenin, eluent at a retention time of 18.4 min in semi-preparative HPLC-UV, are collected and dried using GeneVac equipment. The product is then solubilized in 300 μl of deuterated methanol, dried under a stream of nitrogen gas and then lyophilized for 48 hours.
[0057] Structural Characterization of 4'-O-α-D-glucopyranosylnaringenin The structural determination of this mono-glucosylation product was carried out by NMR. The 1H, 1H-1H COZY, JMod, 1H-13C HMBC spectra were recorded on a 500 MHz Bruker Avance equipment at 298 K (500 MHz for 1E1 and 125 MHz for 13C) with a 5 mm z-gradient TBI probe. The data was acquired and processed using the TopSpin 3 software. The sample was analyzed in deuterated methanol.
[0058] The assignment of the various NMR signals is shown in FIGS. 28, 29 and 30. The identified compound is 4'-O-α-D-glucopyranosylnaringenin. Example 11: Glucosylation of naringenin and morine by the enzyme AN123-GBD-CD2 and its mutants Single or double variants constructed from glucan-saccharase 4N123-GBD-CD2 (belonging to the GH70 family of glycosides hydrolases) were tested for their ability to glucosylate naringenin and morine. The glucosylation results of these two flavonoids, by variants of 4N123-GBD-CD2, are reported in Tables 7 and 8. Regarding the morine, the wild-type enzyme glucosyl with a glucosylation efficiency of 20.4 ± 3 , 2%. Sixteen mutants glucosylate this flavonol more efficiently than the wild-type enzyme, namely W403G, W4035-F404L, W403V, W403C, W403F, F431L, A430E-F431L, W403F-F4041, W403C-F4041, W403N-F404Y, W403N-F404H, W4031-F404Y, W403L-F404L, as well as three mutants with indeterminate mutations. The glucosylation efficiencies obtained for these mutants are shown in Table 7. Among them, ten mutants glucosylate the morine with a glucosylation efficiency greater than or equal to 30% (mutants W403G, W4035-5404L, W403V, W403C, W403F, W403F -54041, F43 1L, A430E-F431L and two mutants with indeterminate mutations). Two mutants even have a morin glucosylation efficiency greater than or equal to 40% or even 45% (mutants W4035-5404L and W403G). The best glucosylation efficiencies were obtained with mutants W4035-5404L (49.5%) and W403G (66.7%). Glucosylation products from the morine were detected by LC-UV-MS (Figure 31). A mono-glucosyl compound, two diglucosyl compounds and a triglucosyl compound have been identified. The best mutant for the glucosylation of the morine is the W403G variant which synthesizes four times more di-glucosylated morine than the wild-type enzyme. The naringenin is glucosylated by 4N123-GBD-CD2 WT with a glucosylation yield of 13.9 ± 4.7% (Table 8).
[0059] Ten variants have a higher glucosylation efficiency of 20%, namely W4031-F404Y, W403V, W403G, W403F, W403S-F404L, W403C, F431L, A430EF431L as well as two variants with indeterminate mutations. The glucosylation efficiencies obtained are shown in Table 8.
[0060] Eight of them have a glucosylation efficiency greater than or equal to 25% (W403S-S404L, W403G, W403V, W403C, W403F, W4031-F404Y and two mutants with unknown mutation). More particularly, four variants have a glucosylation efficiency greater than or equal to 30% or even 35% (W403G, W403V, W403F, W4031-F404Y). The best conversion rate of 59.3% was obtained with the W4031-F404Y variant. With respect to the glucosylation products of naringenin, reaction products were detected by LC-UV-MS (Figure 32). A monoglucosyl compound, two diglucosyl compounds and a triglucosyl compound were identified by mass spectrometry. Naringenin is low in glucosylated by the wild enzyme (14%) and the essential is monoglucosylated (13%). In particular, a variant of the W403-F404 library shows an increase in the production of the mono-glucosylated product, up to 49% with the mutant W4031-F404Y. Finally, a variant (W403 S-F404L) converts 10% of the naringenin into triglucosyl compound (against only 1% for the wild-type enzyme).
[0061] Organism Glucan-saccharase Liaisons Majority references in the natural polymer Albenne C. et al., J. Biol. Chem., 2004 279 (1) 726-734 Neisseria ASNp WT and 152 Champion mutants E., 2008. Ph.D. dissertation, INSA, single and 3 mutants Toulouse polysaccharea double active site (EC 2.4.1.4) (Positions 228, 229 , 289, Champion C. et al., J. Am Chem Soc., 2009, 290, 330, 331, 394, 446) 131, 7379-7389 Champion C. et al., J. Am. Soc., 2012, 134, 18677-18688 Table 1 ASNp VVT (n = 16) 0.5 A C D E F G H 1 K L M N P CI R 5 T V W Y R226 k 1.7, 1.4, 1.4, 0.7 1.4 2.7 1.4 1.5 1 1.7 1.1 0.9 1.7 1.3 2.2 1 24 I 0.3 1.6 g 1.9! 1.6 nd 1.1, 1228 0.0 0.0 I 2.2 и 0.5 9.9 2.6 3.3 i 0.8 3.6 111 5.4 1.9 2.4 0 , 0.0, 1.7, 0.2, 0.9, 0.2, 2.0, F229, 5.6, 4.7, 0.7, 0.6, 0.8, 1.8, 0, 5 I 23 1.1 0.5, 1.2 _ 5.7 1 02 0.1 0.0 0.0 0.0 0.1 i 1.2 I 0.2 A289, 0.6 1.0 0.3 0.0 0.7 0.3 0.0 1 0.9 0.4 0.6 0.1 i 0.7 0.0 0.2 0.3 0.4 1 0.4 0, 0 22.1 0.8 F290 1 19 54 1 1.6 0.5 1 0.8 1 0.5 1 0.3 3.2 8.9 2.4 1 2.0 i 0.5 0.0 I 0.4 3.5 0.5 0.6 3.8 2.2 I 0.3 1330 04 0.4 i 0.5 li 0.6 1 1 0.6 I 0.6 0.8 0.2 0.0 0.0 0.1 1 0.2 1 0.2 0.0 0.0 0.0 0.2 0.0 0.0 'V331 0.5 1 1.2!, 0 , 9 0.7 1 0.4 1 ::. 1.9 0.1 0.3 0.2 0.3 1 0.1 i'i 1.0 0.0 I 0.2 0.2 1 0.4 0.0 1 0.2 0.2 i 0, 4 D394 3.2 i 0.4 0.1 0.6 0.0 1 1.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0 , 1 0.0 0.0 0.6 1 0.8 R446 0.1 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0 , 1 0.0 I 1.9 nd 1 0.3 0.0 nd 0.0 0.0 A289P / A289P / A289P / F290C F2901 F290L 2.1 K 3.0 ',. .> ,. ' 2.9 na: no data available ASNp WT (n = 16) 4.7 ACDEFGH 1 KLMNPQRSTVWY R226 2.4 3.9 3.9 1.0 I 4.6 I 4.8 13.5 4.9 1 5 , 1 3.7 3.5 16.0 3.1 4.7 2.5 14.1 8.7 5.9 id 2.3 1228 70.2 30.9 0.0 0.0 0.0 0 , 0.0 0.0 0.0 0.0 3.3 2.2 6.1 1.4 0.0 16.4 6.0 112.3 0.0 0.0 F229 0.0 0, 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6 , 9 0.0 A289 3.4 27.8 0.0 0.0 0.0 0.0 0.0 0 e 11.2 0.0 0.0 0.0 14.5 10.3 0.0 0.0 0.0 2.2 g 0.0 0.0 '21.8 F290 0.0 0.0 0.0 0.0 I: 7.4 1 3.4 0.0 0.0 i 4.5 R 6.400 3.4 0.0 Li 11.2 2.4 0.0, -a, 21.1 0.0 0.0 1330 0.0 0.0 0.0 0, 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 V331 0.0 5.4 0.0 0.0 0.0 6.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6.8 0.0 1 6.7 7 2 4.7 0.0 3.8 D394 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 R446 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0 0.0 0.0 0.0 0.0 6.0 0.0 0.0 0.0 0.0 0.0 A289P / A289P / A289P / F290C F290I F290L 50.9 1.6 22.9 na: no data available Glu Organism cane-saccharase Liaisons Majority references in the natural polymer Leuconostoc DSR-S vardel.A4N WT a- (1-> 6) Moulis. C., 2006. PhD thesis, INSA, Toulouse and mesenteroides B-512F Moulis C. et al., FEMS (EC 2.4.1.5) Microbiol. Lett., 2006 261 203-210 Leuconostoc 7 mutants of DSR-S Irague R. et al., Anal. Chem. mesenteroides B-512F vardel.A4N: F3531 or S512C 2011 83 (4) 1202-1206 (EC 2.4.1.5) or F3 53W or H463R / T464D / S512T or H463R / T464V / S512T or D460A / H463S / 1464L or D460M / H463Y / T464M / S512C Leuconostoc mesenteroides NRRL B- / a- (1 -> 6) Joucla. G., 2003. Thesis of 1355 ASR C-APY-doctorate, INSA, Toulouse and (EC 2.4.1.140) Joucla G et al., FEBS Lett. 2006 580 (3) 763-768 Leuconostoc DSR-E mutant a- (1 -> 2) Brison et al., J. Biol. Chem., Mesenteroides NRRL B-A Ni23-GBD-CD2 2012, 287, 7915-24 1299 Table 4 55 0, s 2.5 I 1 1 1.3.9 Table 5 The number indicated in each box is the percentage of effectiveness glucosylation.
[0062] Table 6 The number indicated in each box is the percentage of glucosylation efficiency 2.7.1 2E, 8 0.0 0.0 0.0 OSR-S viectel_14N5 Mount W4036 56.7 V453 SF 404L 49 5 W403 -F404 32.6 W405V 35.5 \ -403C 354 W-403F 33.4 F431 -L434 32.4 F431L,, A430E-F431L 1/4 -40.11-F4041 30.4 W403C-F4041 294 70: 403N-F404Y 28.0 W403N-F404H. 25.7 W4031-F404V -) E-_), - ,, W.403 -F404 25 W-403L-F404L 24.8 123-G8D-CD2 WT 20.4 Table 7 Naiinnii W403I-F404Y E} c37 40, 38-403.72: Ir), W-403S-1: 404L 29.4 W403 -F404 Y, T4, C3, C6, F131, 25.0 F4311, 24.8 430E-F4311, 20.9 N1.23-G6D-CD2 WT 13.9 Table 8 Glucosylation efficiencies of morine (Table 7) and naringenin (Table 8) by glucan wild-type AN123-GBD-CD2 saccharase and the best mutants derived from secondary screening.
[0063] 10 SEQUENCES: Series SEQ ID NO: 1: (Protein = mutated sequences of glucansucrase Asnp (amylosucrase Neisseria polysaccharea) R226X1) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQF SRRMDTHFPKLMNELD SV YGNNEALLPMLEMLLAQAWQ SYS QRNS SLKD ID IARENNPDWIL SNKQVGGVCYV DLFAGDLKGLKDKIPYF QELGLTYLHLMPLFKCPEGK SDGGYAVS S YRDVNPAL GT IGDLREVIAALHEAGISAVVDFIFNHT SNEHEWAQRCAAGDPLFDNFYYIFPDRRIVIP D QYDRTLX1EIFPD QHP GGF SQLEDGRWVWTTFNSFQWDLNYSNPWVFRAMAGEM LFL ANL GVD ILRMD AVAF IWK QMGT SCENLPQAHALIRAFNAVM RIAAPAVFFK SE AIVHPDQVVQYIGQDECQIGYNPLQMALLWNTLATREVNLLHQALTYRHNLPEHT AWVNYVRSHDDIGWTFADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPF QY NPSTGDCRVSGTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLND DDW SQD SNK SDS SRWAHRPRYNEALYAQRNDP S TAAGQIYQDLRHMIAVRQ SNP RFDGGRLVTFNTNNKHIIGYIRNNALLAF GNF SEYPQTVTAHTLQAMPFKAHDLIGG KTVSLNQDLTLQPYQVMWLEIA Series SEQ ID NO: 2: (protein = mutated sequences of glucansucrase Asnp (amylosucrase Neisseria polysaccharea) 1228X2) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQF SRRMD THFPKLMNELD SV YGNNEALLPMLEMLLAQAWQ SYS QRNS SLKD ID IARENNPDWIL SNKQVGGVCYV DLFAGDLKGLKDKIPYF QELGLTYLHLMPLFKCPEGK SDGGYAVS S YRDVNPAL GT IGDLREVIAALHEAGISAVVDFIFNHT SNEHEWAQRCAAGDPLFDNFYYIFPDRRIVIP D QYDRTLREX2FPD QHP GGF SQLEDGRWVWTTFNSFQWDLNYSNPWVFRAMAGE MLFLANLGVDILRMDAVAFIWKQMGT SCENLPQAHALIRAFNAVM RIAAPAVFFK S EAIVHPDQVVQYIGQDECQIGYNPLQMALLWNTLATREVNLLHQALTYRHNLPEH TAWVNYVRSHDDIGWTFADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPF Q YNPSTGDCRVSGTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLN DDDW SQD SNK SDS SRWAHRPRYNEALYAQRNDP STAAGQIYQDLRHMIAVRQ SN PRFDGGRLVTFNTNNKHIIGYIRNNALLAFGNF SEYPQTVTAHTLQAMPFKAHDLIG GKTVSLNQDLTLQPYQVMWLEIA Series SEQ ID NO: 3: (Protein = mutated sequences of glucansucrase Asnp (amylosucrase Neisseria polysaccharea) F229X3) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQF SRRMDTHFPKLMNELD SV YGNNEALLPMLEMLLAQAWQ SYS QRNS SLKDIDIARENNPDWIL SNKQVGGVCYV DLFAGDLKGLKDKIPYF QELGLTYLHLMPLFKCPEGK SDGGYAVS S YRDVNPAL GT IGDLREVIAALHEAGISAVVDFIFNHT SNEHEWAQRCAAGDPLFDNFYYIFPD RRIVIP D QYDRTLREIX3PD QHP GGF SQLEDGRWVWTTFNSF QWDLNYSNPWVFRAMAGE MLFLANLGVDILRMDAVAFIWKQMGT SCENLPQAHALIRAFNAVM RIAAPAVFFK S EAIVHPDQVVQYIGQDECQIGYNPLQMALLWNTLATREVNLLHQALTYRHNLPEH TAWVNYVRSHDDIGWTFADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPF Q YNPSTGDCRVSGTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLN DDDW SQD SNK SDS SRWAHRPRYNEALYAQRNDP S TAAGQIYQDLRHMIAVRQ SN PRFDGGRLVTFNTNNKHIIGYIRNNALLAF GNF SEYPQTVTAHTLQAMPFKAHDLIG GKTVSLNQDLTLQPYQVMWLEIA SEQ ID NO: 4: (Protein = mutated sequence of glucansucrase Asnp 20 (amylosucrase Neisseria polysaccharea) A289X4) SPNSQYLKTRILDIYTPEQRAGIEK SEDWRQF SRRMDTHFPKLMNELD SV YGNNEALLPMLEMLLAQAWQ SYSQRNS SLKDIDIARENNPDWIL SNKQVGGVCYV DLFAGDLKGLKDKIPYF QELGLTYLHLMPLFKCPEGK SDGGYAVS S YRDVNPAL GT IGDLREVIAALHEAGISAVVDFIFNHT SNEHEWAQRCAAGDPLFDNFYYIFPDRRIVIP 25 D QYDRTLREIFPD QHP GGF SQLEDGRWVWTTFNSFQWDLNYSNPWVFRAMAGEM LFLANLGVDILRMDAVX4FIWKQMGT SCENLPQAHALIRAFNAVMRIAAPAVFFK S EAIVHPDQVVQYIGQDECQIGYNPLQMALLWNTLATREVNLLHQALTYRHNLPEH TAWVNYVRSHDDIGWTFAD EDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPF Q YNPSTGDCRVSGTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLN 30 DDDW SQD SNK SDS SRWAHRPRYNEALYAQRNDP STAAGQIYQDLRHMIAVRQ SN PRFDGGRLVTFNTNNKHIIGYIRNNALLAF GNF SEYPQ TVTAHTLQAMPFKAHDLIG GKTVSLNQDLTLQPYQVMWLEIA Series SEQ ID NO: 5: (Protein = mutated sequences of glucansucrase Asnp (amylosucrase Neisseria polysaccharea) F290X5) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQF SRRMDTHFPKLMNELD SV YGNNEALLPMLEMLLAQAWQ SYSQRNS SLKDIDIARENNPDWILSNKQVGGVCYV DLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS S YRDVNPAL GT IGDLREVIAALHEAGISAVVDFIFNHT SNEHEWAQRCAAGDPLFDNFYYIFPDRRIVIP D QYDRTLREIFPD QHP GGF SQLEDGRWVWTTFNSFQWDLNYSNPWVFRAMAGEM LFLANLGVDILRIVIDAVAX5IWKQMGTSCENLPQAHALIRAFNAVMRIAAPAVFFKS EAIVHPDQVVQYIGQDECQIGYNPLQMALLWNTLATREVNLLHQALTYRHNLPEH TAWVNYVRSHDDIGWTFADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPFQ YNP STGDCRVSGTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLN DDDWSQD SNKSDD SRWAHRPRYNEALYAQRNDP STAAGQIYQDLRHMIAVRQ SN PRFDGGRLVTFNTNNKHIIGYIRNNALLAFGNF SEYPQTVT AHTLQAMPFKAHDLIG GKTVSLNQDLTLQPYQVMWLEIA Series SEQ ID NO: 6: (Protein = mutated sequences of glucansucrase Asnp (amylosucrase Neisseria polysaccharea) V331X6) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQF SRRMDTHFPKLMNELD SV YGNNEALLPMLEMLLAQAWQ SYSQRNS SLKDIDIARENNPDWILSNKQVGGVCYV DLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS SYRD VNP G GT IGDLREVIAALHEAGISAVVDFIFNHT SNEHEWAQRCAAGDPLFDNFYYIFPDRRIVIP DQ YDRTLREIFPD QHP GGF SQLEDGRWVWTTFNSFQWDLNYSNPWVFRAMAGEM LFL ANL GVDILRIVIDAVAF IWK QMGT S CENLPQAHAL IRAFNAVM RIAAP AVFFK SE AIX6HPDQVVQYIGQDECQIGYNPLQMALLWNTLATREVNLLHQALTYRHNLPEHT AWVNYVRSHDDIGW TF ADEDAAYL GISGYDHRQFLNRFFVNRFDGSFARGVPF QY NP STGDCRVSGTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLND DDWSQD SNKSDD SRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQ SNP RFDGGRLVTFNTNNKHIIGYIRNNALLAFGNF SEYPQTVTAHTLQAMPFKAHDLIGG KTVSLNQDLTLQPYQVMWLEIA Series SEQ ID NO: 7: (= Proteins mutated sequences of glucansucrase Asnp (amylosucrase Neisseria polysaccharea) D394X7) SPNSQYLKTRILDIYTP EQRAGIEKSEDWRQF SRRMDTHFPKLMNELD SV YGNNEALLPMLEMLLAQAWQ SYSQRNS SLKDIDIARENNPDWILSNKQVGGVCYV DLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS S YRDVNPAL GT IGDLREVIAALHEAGISAVVDFIFNHT SNEHEWAQRCAAGDPLFDNFYYIFPDRRIVIP D QYDRTLREIFPD QHP GGF SQLEDGRWVWTTFNSFQWDLNYSNPWVFRAMAGEM LFLANLGVDILRIVIDAVAFIWKQMGT SCENLPQAHALIRAFNAVM RIAAPAVFFK SE AIVHPDQVVQYIGQDECQIGYNPLQMALLWNTLATREVNLLHQALTYRHNLPEHT AWVNYVRSHDX7IGWTFADEDAAYLGI S GYDHRQFLNRFF VNRFD G SF ARGVPF QY NP STGDCRVSGTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLND DDWSQD SNKSDD SRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQ SNP RFDGGRLVTFNTNNKHIIGYIRNNALLAFGNF SEYPQTVTAHTLQAMPFKAHDLIGG KTVSLNQDLTLQPYQVMWLEIA SEQ ID NO: 8: (protein = mutated sequence the glucansucrase Asnp (amylosucrase Neisseria polysaccharea) R446Q) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQF SRRMDTHFPKLMNELD SV YGNNEALLPMLEMLLAQAWQ SYSQRNS SLKDIDIARENNPDWIL SNKQVGGVCYV 25 DLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS SYRD VNP G GT IGDLREVIAALHEAGISAVVDFIFNHT SNEHEWAQRC AAGDPLFD NF YYIF PDRRIVIP DQ YDRTLREIF PD QHP GGF SQLEDGRWVWTTFNSFQWDLNYSNPWVFRAMAGEM LFL ANL GVDILRIVID AVAF IWK QMGT SCENLPQAHALIRAFNAVM RIAAP AVFFK SE AIVHPDQVVQYIGQDECQIGYNPLQMALLWNTLATREVNLLHQALTYRHNLPEHT 30 AWVNYVRSHDDIGW TF ADED AAYL GI SGYDHRQF LNRFFVNRFDGSFARGVPF QY NP STGDCQVSGTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLND DDWSQD SNKSDD SRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQ SNP RFDGGRLVTFNTNNKHIIGYIRNNALLAFGNF SEYPQTVTAHTLQAMPFKAHDLIGG KTV SLNQDL TL QPYQVMWLEIA Series SEQ ID NO: 9: (Protein doubly mutated sequences = 1 (1 glucansucrase Asnp (amylosucrase Neisseria polysaccharea) A289P / F290X8) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQF SRRMD THFPKLMNELD SV YGNNEALLPMLEMLLAQAWQ SYSQRNS SLKDIDIARENNPDWIL SNKQVGGVCYV DLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS SYRD VNP G GT IGDLREVIAALHEAGISAVVDFIFNHT SNEHEWAQRCAAGDPLFDNFYYIFPDRRIVIP DQ YDRTLREIFPD QHP GGF SQLEDGRWVWTTFNSFQWDLNYSNPWVFRAMAGEM LFLANLGVDILRMDAVPX8IWKQMGT SCENLPQAHALIRAFNAVMRIAAPAVFFKS EAIVHPDQVVQYIGQDECQIGYNPL QMALLWNTLATREVNLLHQALTYRHNLPEH TAWVNYVRSHDDIGW TF ADEDAAYL GISGYDHRQFLNRFF VNRFDGSF ARGVPF Q YNP STGDCRVSGTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLN DDDWSQDSNKSDDSRWAHRPRYNEALYAQRNDP STAAGQIYQDLRHMIAVRQ SN PRFDGGRLVTFNTNNKHIIGYIRNNALLAFGNF SEYPQ TV TAHTLQAMPFKAHDLIG GKTVSLNQDLTLQPYQVMWLEIA SEQ ID NO: 10: (protein = mutated sequence of glucansucrase truncated DSR-S vardel44N - S512C) TQQVSGKYVEKDGSWYYYFDD GKNAK GL S TIDNNIQ YF YE SGK Qak GQ Yvt IDNQ TYYF DK GS GDEL T GL QS ID GNIVAFNDEGQ QIFNQ YYQ SENGT T YYF D DKGHAATGIKNIEGKNYYFDNLGQLKKGF SGVIDGQIMTFDQETGQEVSNTT Seike GL TTQNTDYSEHNAAHGTD AEDF ENID GYL TAS SWYRPTGILRNGTDWEPSTDTDF RP IT VWWPDKNT Q VNYLNYMADL GF I SNAD SFET GD SQ SLLNEASNYVQKSIEMK I SAQQ STEWLKD AMAAF IVAQP QWNET SEDMSNDHL QNGALTYVNSPL TPD AN SN FRLLNRTPTNQTGEQAYNLDNSKGGFELLLANQEDNSNVVVEAEQLNWLYYLMNF GTITANDADANFD GIRVDAVDNVDADLL QIAADYFKLAYGVD QNDATANQHL S IL EDWSHNDPLYVTDQGSNQLTMDDYVHTQLIWSLTKS SDIRGTMQRFVDYYMVDR SND STENEAIPNYSFVRAHDCEVQTVIAQIVSDLYP DVENSLAPTTEQLAAAFKVYN EDEKLADKKYTQYNMASAYAMLLTNKDTVPRVYYGDLYTDDGQYMATKSPYYD AINTLLKARVQYVAGGQ SM S VD SNDVLT S VRYGKD AMTASDT GT SETRTEGIGVIV SNNAELQLEDGHTVTLHMGAAHKNQAYRALL S TTAD GL AYYD TDENAP VAYTD A NGDL IF TNESIYGVQNPQVS GYLAVWVP VGAQ QD QD ARTASDT TTNT SDKVFH SN AALD SQVIYEGF SNFQAFATD S SEYTNVVIAQNADQFKQWGVT SFQLAPQYRS STD T SFLD SIIQNGYAF TDRYDL GYGTP TKYGTAD Q LRD AIKALHA S IQM AIADW VPD IQ YNLPEQEL AT VTRTN SF GDDD TD SDIDNALYVVQ SRGGGQ YQEMYGGAF LEEL QA LYPSLFKVNQISTGVPIDGSVKITEWAAKYFNGSNIQGKGAGYVLKDMGSNKYFKV V SNTED GD YLPK QL TNDL SET GF THDDK GIIYYTL S GYRAQNAF IQDDDNNYYYF D KT GHLVT GL QKINNHTYFFLPNGIELVK SFL QNED GTIVYFDKKGHQVFD QYITD QN GNAYYF DD AGVMLK S GL AT ID GHQ Q YFD QNGVQ VKDKF VIGTD GYKYYF EP GS G NL AILRYVQN SKNQWF YF D GNGHAV T GF Q TINGKK Q YF YN GHQ SK GEF ID AD GD TF YT SATDGRLVTGVQKINGITYAFDNTGNLITNQYYQLADGKYMLLDD SGRAKT GF VL QD GVLRYFD QNGEQ VKD AIIVDPD TNL S. ITXIMOASXYNSIHAÀllOMYSIIUOYMNVOSJAIXdVMTWIOUIIÙS S Naxin GCNIAIVÔINIIISIVXS GAMA oni IIIIMIMÔVDÀV CEÔ9 ÔAHÔ NIA-91 iA GVI MID S O-M1ÀdV1 11119 CMIGNIS NakiO Oni ÔIAIÔVIVHNICEAMA Ô9DIAA S 0 MA HQ ID dÀÔÔ S AIAIÔNNANDIVIDVINAD dVNFITTO GINS LINA dANÔM JAJGI {IINLtM'TVùS CRAUÔ IGNId SS WORLD S al IVADVIIA O9 ons IAIDVIÔÀ9 NA Adv1 11119 CMINVÀ dS S IF GA MINÔ9 Ô'ICIDDV JILINM S AD NS GEX ÔAÔNNNÀS dISNNIUGÔÀOEINAÔINCHdll S ISÀINNÀVS-Vdad IINNOELA dADIÔAIICKS S dNIANÔMS ANL1À O9 I MCDFIAÀDIAID IDIANLID CZ 1 S 11AUNV MUNINI IQ CFIUVUL NS GAIÀÀA I91 199A1 INA CUUÀI IN SJI G: IÀVV NINCE1 JUDDÀ MM: UN-999A INAAMAIÔATI S 11 INGNIMGAXI II S VAUND CREA IÔKVAAGVINAÔIAIDS SITIÔ NIV ffifICHC [1 19ANI DUNU 'DAMADMCIIIIS QI INGÔS PIMMVIAlaiSIIDMS NJICIVNCIVIAANLIÀNLIdINdÔ dl IS dVUXIII IS CF1V VNISHIAND CNA SS OO MY ITHAMV S IAD S SMINHANIMIÔ GNI OZ ÔNS diVICEGNICKLL) 11AdVI INS S SÀS GIGNICEIITTIVXÀMÔMfflVVOINIMANS CENS S KINIVIAA DID IXIT IID I (11 IVIINV9 N9 DIA O S-99 VA Dō IJAFTVIIIOETKIES ÔNÔIAIÀ O9 (Flô XINCIDÀÀAXdAI CDIMITTIVÀVS dINÀÔNÀNNU INÙUWS'IONÙ'IÙIdLiNLLMGVOIYUIIYYOANUÙIUNGHVHIISXMdIYAMII SNI IVX (IVIINCIDUNCEÔIT IS 1S IMS S) 1) 11 / S WUXI ITIOVÇ I KINGDOM S 11-1VI IVUNCEI DICEADÀVVUIÀ CULDIÔLICKS A Keav CRUI SUllSNNHiNOùIIIS ^ dl ITIÀXIMI FICHVÔAIdl ISMGACKVTIIITISMCWINSNANdrICESMÔÀNIXD -OO IHÀUS US NAVIÀÔ Oni TIANNIMOELI dS IDIÔSINNUIDIÔVÔÔVV UKITIÔCLINFICUAVS ÔNIADUI Io IAIMMI IA I CDINdMMAI KDICEI S VAMG IDHS IIIÔVcRIÀMIUV end GAT DI dl IS INNAANHÔ IX CINDINS dVIAIIIÀÔÀSGNI 01 ^ I CDICLIÀÀDID GINDIVÔDNIÔID IV'ICLIÀMT IDMIDXTDNIVUNNNCFIÀÀANICIDMI NÀÔDIdVINAASKII 19) INDUJÀÀIIDOELIVIRDNIÔUDIHUCLIMMUDNACKION IVI 19ÔNITIÀVAUS IT 19 CDIGNI ÔUVÀXGNLIAV S (IV dÀÀIAD GA IÔHNV O1 S S OT 19 19 CEXID NA dō NUE S ceaa UNION MIAID IN S GNIS INI MID SI DICLIÀÀVNICKEII INIUD OR ÔÀ9 INKE IHD SA N9 S ÙUIX ADÙOU NUAÀÔGDMIS IMIUdIASNS OF QI INA dVI S UNAD NU S MI NI 19 GcrIÔ VNLI of ÔICEÔNIUGVNIINVCWSICIOVNIÔVSMITAdUNNIMAUNIIdNIUÔMIÔDEICEÔÔS Oi IDA dIÔIGV S AIIAVS A-OKAY JV IVVVA MS OUR NKINNXIIIUNÔUIN (SWI `/ -73 asmumpans-aunang nj ap = amanbas aumeodd) II ONI IQ OUS 179 ZZ8810E NLNGQ STWIDKRAF TATFDQVVALNATIVARQRPDGMFKTAPYGEAGAQFVDYVT ## STR2 ## SEQ ID NO: 12 series: Protein = mutant sequence of glucan-saccharase AN123-GBD-CD2 (W403X9; F404X10; A430X11; F431 (12; and L434X13.) MAHHIHIHIHHVT SLYKKAGSAAAPF TMAQAGHYITKNGNDWQYDTNGE LAKGLRQD SNGKLRYFDLTTGIQAKGQFVTIGQETYYF SKDHGDAQLLPMVTEGH YGTITLKQ GQD TKTAWVYRD QNNTILKGLQNINGTLQFFDPYTGEQLKGGVAKYD DKLFYFESGKGNLVSTVAGDYQDGHYISQDGQTRYADKQNQLVKGLVTVNGALQ YFDNATGNQIKNQQVIVDGKTYYFDDKGNGEYLF TNTLDMSTNAF STKNVAFNHD SS SFDHTVDGFLTADTWYRPKSILANGTTWRD STDKDMRPLITVWWPNKNVQVNY LNFMKANGLLT TAAQYTLH SD QYDLNQAAQD VQVAIERRIA SEHGTDWL QKLLFE SQNNNP SFVKQQFIWNKD SEYHGGGDAX9XioQ GGYLKYGNNPLTP TTN SDYRQP G NX11X12DFX13LANDVDNSNPVVQAENLNWLHYLMNFGTITAGQDDANFD SIRIDAV DFIHNDTIQRTYDYLRDAYQVQQ SEAKANQHISLVEAGLDAGTSTIHNDALIESNLR EAATL SLTNEPGKNKPLTNMLQDVDGGTLITDHTQNSTENQATPNYSIIHAHDKGV QEKVGAAITDATGADWTNF TDEQLKAGLELFYKDQRATNKKYNSYNIP SIYALML TNKD TVPRMYYGDMYQDD GQYMANK IYYDALV SLMTARK S S S YV GGQ TM S VDN HGLLKSVRFGKDAMTANDLGT SATRTEGLGVIIGNDPKLQLND SDKVTLDMGAAH KNQKYRAVILTTRDGLATFNSDQAPTAWTNDQGTLTF SNQEINGQDNTQIRGVANP QVSGYLAVWVPVGASDNQDARTAATTTENHDGKVLHSNAALD SNLIYEGF SNFQP KAT THDEL TNVVIAKNADVFNNWGIT SFEMAP Q YRS SGDHTFLD STIDNGYAF TDR YDL GFNTP TKYGTD GDLRATIQALHHANMQVMADVVDNQVYNLP GKEVV SATRA GVYGNDDATGF GT QLYVTN S VGGGQYQEKYAGQYLEALKAKYPDLFEGKAYDY WYKNYANDGSNPYYTL SHGDRESIPADVAIKQWSAKYMNGTNVLGNGMGYVLK DWHNGQYFKLDGDKSTLPKGGRADPAFLYKVVSAWSHPQFEK

权利要求:
Claims (19)
[0001]
REVENDICATIONS1. A process for producing 0-α-glucosyl flavonoid derivatives comprising at least one step of incubating a glucan-saccharase with a flavonoid and at least one sucrose, wherein: (A) said flavonoid is of the following formula (I) R7 wherein ring C represents a ring selected from the group consisting of the following rings of formula (II), (III), (IV) or (V): R3 R3 R3 'R3 (III) 25 (IV) ( V) in which: one of the groups R1, R2 or R3 represents a ring B of the following formula (VI): embedded image in which: (a) only one of the groups chosen from R8, R0, R10, R11 and R12 represents a hydroxyl group, the other groups of R8, R9, R10, R11 and R12, identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C 5 -C 9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; } -N 12; -00NH2; -CHO; -SH; -C (O) O (C2-C3); a C1-C3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); Or (b) R8 and only one of the groups selected from R10, R11 and R12 represent a hydroxyl group, R9 and the other groups of R10, R11 and R12, which may be identical or different, are chosen from the group comprising a hydrogen atom ; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C 1 -C 3 alcohol; a -COOH group; } -N 12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); or (e) R9 and only one of the groups selected from R11 and R12 represent a hydroxyl group, the groups R8, R10 and the other group from R11 and R12, which are identical or different, being chosen from the group comprising an atom of hydrogen; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; } -N 12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C1-C3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); or (d) Rio and R12 represent a hydroxyl group, the groups R8, R9, and Ru, identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a group - COOH; } -N 12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C1-C3 amine; a C1-C3 imine; a nitrile group; a C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); or (e) R8, R10 and R12 represent a hydroxyl group, the groups R9 and R11, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; } -N 12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); the groups R1, R2 and R3 which do not represent a ring B of formula (VI), which are identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched C1-C6 alkyl; an -OH group; a C 1 -C 3 amine; a -COOH group; -C (O) O (C2-C3); a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); R1 ', R2' and R3 ', which are identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated, C1-C1 hydrocarbon-based group optionally interrupted by at least one heteroatom selected from O, N or 5; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; -NH2; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C2-C3 amine; a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); or the groups R 1 and R 1 'when R 1 is not a ring B of formula (VI), or R 2 and R 2' when R 2 is not a ring B of formula (VI), or R 3 and R 3 when R 3 is not a cycle B of formula (VI) together form a group = 0; R4, R5, R6 and R7, identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C 1 -C hydrocarbon-based group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; an -OH group; -COOH; } -N 12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C 1 -C 3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); and (B) said glucansucrase being selected from the group consisting of: - a sequence having at least 80% identity with SEQ ID NO: 1, said sequence having an amino acid X1 representing an amino acid selected from the group consisting A, C, E, F, G, H, I, K, M, N, P, Q, S, T, V and Y; a sequence having at least 80% identity with SEQ ID NO: 2, said sequence having an amino acid X2 representing an amino acid selected from the group consisting of A, C, D, F, G, H, K, L, M, N, P, S, V and Y; a sequence having at least 80% identity with SEQ ID NO: 3, said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A, C, G, I, K, M, N and W; a sequence having at least 80% identity with the sequence SEQ ID NO: 4, said sequence having an amino acid X4 representing an amino acid selected from the group consisting of C, I, N, P, V and W; a sequence having at least 80% identity with the sequence SEQ ID NO: 5, said sequence having an amino acid X5 representing an amino acid selected from the group consisting of A, C, D, G, I, K, L; , M, R, V and W; a sequence having at least 80% identity with SEQ ID NO: 6, said sequence having an amino acid X6 representing an amino acid selected from the group consisting of C, G, Q, S and T; a sequence having at least 80% identity with SEQ ID NO: 7, said sequence having an amino acid X7 representing an amino acid selected from the group consisting of A and G; a sequence having at least 80% identity with SEQ ID NO: 8; a sequence having at least 80% identity with SEQ ID NO: 9, said sequence having an amino acid X8 representing an amino acid selected from the group consisting of C, I and L; a sequence having at least 80% identity with SEQ ID NO: 10; a sequence having at least 80% identity with SEQ ID NO: 11; and a sequence having at least 80% identity with SEQ ID NO: 12, said sequence having amino acids X9, X10, X11, X12 and X13, with: (i) X9 representing, independently of X10, X11, X12 and X13, an amino acid selected from the group consisting of G, S, V, C, F, N, I, L and W; X10 representing, independently of X9, X11, X12 and X13, an amino acid selected from the group consisting of L, I, H, Y and F; except where X9 is W and X10 is F; XIII representing A; Xi2 representing F; and X13 representing L; (ii) X9 representing W; X10 representing F; X11 representing, independently of X9, X10, X12 and X13, an amino acid selected from the group consisting of E and A; X12 represents, independently of X9, X10, X11 and X13, an amino acid selected from the group consisting of L and F; and X13 representing L; except where X11 is A and X12 is F; or (iii) X9 representing W; X10 representing F; X11 representing A; X12 represents, independently of X9, X10, X11 and X13, an amino acid selected from the list consisting of A, R, D, N, C, E, Q, G, H, I, L, K, M, P, S, T, W, Y and V; and X13 representing, independently of X9, X10, X11 and X12, an amino acid selected from the list consisting of A, R, D, N, C, E, Q, G, H, I, K, M, F, P , S, T, W, Y and V.
[0002]
2. Method according to claim 1, in which only one of the groups chosen from R8, R9, R10, Ru and R12, preferably R10, represents a hydroxyl group, the other groups from R8, R9, R10, R11 and R12, which are identical. or different, being selected from the group consisting of a hydrogen atom; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; } -N 12; -00NH2; -CHO; -SH; -C (O) O (C2-C3); a C1-C3 amine; a C1-C3 imine; a nitrile group; C1-C3 haloalkyl; and a C 1 -C 3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s); the groups Rg, R9, R11 and R12 preferably represent hydrogen atoms.
[0003]
3. Process according to claim 2, wherein R10 represents a hydroxyl group and Rg, R9, R11 and R12 represent hydrogen atoms.
[0004]
4. Process according to claim 1, in which: Rg and only one of the groups chosen from R10, R11 and R12, preferably R10, represent a hydroxyl group, R9 and the other groups of R10, R11 and R12, which are identical or different, being selected from the group consisting of a hydrogen atom; a linear or branched, saturated or unsaturated C1-C10 hydrocarbon group optionally interrupted by at least one heteroatom selected from O, N or S; a halogen atom; a C5-C9 aryl; a C4-C9 heterocycle; a (C 1 -C 3) alkoxy group; C2-C3 acyl; a C1-C3 alcohol; a -COOH group; } -N 12; -CONH2; -CHO; -SH; -C (O) O (C2-C3); a C1-C3 amine; a C1-C3 imine; a nitrile group; a C1-C3 haloalkyl; a C1-C3 thioalkyl; a group -C (W); and a group -0 (W); W represents a chain consisting of 1 to 6 glycoside (s).
[0005]
5. Process according to claim 4, wherein R10 represents a hydroxyl group and R9, R11 and R12 represent hydrogen atoms.
[0006]
6. Process according to any one of the preceding claims, in which ring C represents a ring of formula (II) or (IV) as defined in claim 1.
[0007]
7. Process according to any one of the preceding claims, in which R1 represents a ring B of formula (VI) as defined in claim 1.
[0008]
8. A process according to any one of the preceding claims wherein R 1 'and R 2' are hydrogen, R 2 is hydrogen or -OH, and R 3 and R 3 'together are a group = 0.
[0009]
9. Method according to any one of the preceding claims, wherein two of the groups R4, R5, R6 and R7, preferably R4 and R6, represent a hydroxyl group, the two other groups being as defined in claim 1.
[0010]
The process of any one of the preceding claims, wherein R5 and R7 are hydrogen atoms.
[0011]
11. Process according to any one of the preceding claims, in which the said flavonoid is of formula (VII), (VIII) or (IX): H HO, H (IX)
[0012]
The method according to any of the preceding claims, wherein the glucansucrase is selected from the group consisting of: - a sequence having at least 80% identity with SEQ ID NO: 1, said sequence having an amino acid X1 represents an amino acid selected from the group consisting of H, N or S; a sequence having at least 80% identity with SEQ ID NO: 2, said sequence having an amino acid X 2 representing an amino acid selected from the group consisting of A, C, F, L, M, S or V; a sequence having at least 80% identity with SEQ ID NO: 3, said sequence having an amino acid X3 representing an amino acid selected from the group consisting of A and N; a sequence having at least 80% of identity with SEQ ID NO: 4, said X4 amino acid sequence being an amino acid selected from the group consisting of C, I, N, P, V or W; a sequence having at least 80% identity with SEQ ID NO: 5, said sequence having an amino acid X5 representing an amino acid selected from the group consisting of C, K, R or V; a sequence having at least 80% identity with SEQ ID NO: 9, said sequence having an amino acid X8 representing an amino acid selected from the group consisting of C or L; and a sequence having at least 80% identity with SEQ ID NO: 12, said sequence having amino acids X9, X10, X11, X12 and X13, with: (i) X9 representing an amino acid selected from the group consisting of G, V, C and F; X10 representing F; X11 representing A; X12 representing F; and X13 representing L; (il) X9 represents, independently of X10, X11, X12 and X13, an amino acid selected from the group consisting of S, N, L and I; X10 representing, independently of X9, X11, X12 and X13, an amino acid selected from the group consisting of L, I, H and Y; X11 representing A; X12 representing F; and X13 representing L; or (iii) X9 representing W; X10 representing F; X11 representing A or E; X12 representing L; and X13 representing L.
[0013]
13. O-α-glycosylated flavonoid derivative obtained by the process as defined in any one of claims 1 to 12.
[0014]
14. A compound of the following formula (X): X 14 o (X) in which (i) X14 represents a chain consisting of at least two α-glucoside groups, and X15 and X16, which are identical or different, are chosen from the group comprising a hydrogen atom; a linear or branched C1-C6 alkyl; a group -C (O) O (C2-C3); and a chain consisting of 1 to 600,000--glucoside moieties, or (ii) X14 is a single ci-glucoside moiety, and X15 and X16, the same or different, are selected from the group consisting of hydrogen; a linear or branched C1-C6 alkyl; a group -C (O) O (C2-C3); and a chain of 1 to 600,000 β-glucoside moieties, with the proviso that at least one of X15 and Xl6 represents a chain of 1 to 600,000 β-glucoside moieties.
[0015]
15. Compound of formula (XI) below: X 17 0 (XI) in which X 17 represents a chain consisting of 1 to 600,000 ci-glucoside groups, and X 1 and X 19, identical or different, are chosen from the group comprising a hydrogen atom ; a linear or branched C1-C6 alkyl; a group -C (O) O (C2-C3); and a chain consisting of 1 to 600,000 β-glucoside moieties.
[0016]
16. Cosmetic use, as antioxidant, of at least one O-α-glycosylated flavonoid derivative as defined in one of Claims 14 and 15.
[0017]
17. O-α-glycosylated flavonoid derivative as defined in one of claims 14 and 15, for its pharmaceutical use in the treatment and / or prevention of hepatotoxicity, allergies, inflammation, ulcers , tumors, menopausal disorders, or neurodegenerative diseases.
[0018]
18. O-α-glycosylated flavonoid derivative as defined in one of claims 14 and 15, for its pharmaceutical use as veinotonic.
[0019]
19. Use of a 0-α-glycosylated flavonoid derivative as defined in one of claims 14 and 15, as photovoltaic agent, insect repellent agent, bleaching agent, agent pesticide, fungicide and / or bactericide.

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同族专利:

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公开号 | 公开日

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FR3018822B1|2018-03-23|

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US20170107242A1|2017-04-20|

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EP3122890A1|2017-02-01|

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US10647739B2|2020-05-12|

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WO2015144731A1|2015-10-01|

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FR3018821A1|2015-09-25|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

JP2001046096A|1999-08-09|2001-02-20|Lotte Co Ltd|PRODUCTION OF GLYCOSIDE WITH alpha-GLUCOSIDASE AND NEW alpha- GLUCOSIDASE AND ITS PRODUCTION|

---

KR20060063703A|2004-12-03|2006-06-12|전남대학교산학협력단|Method for preparing derivatives of glyco-compounds by using glycosyltransferases and the derivatives thereof|

---

EP2145615A1|2007-04-09|2010-01-20|Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo|Skin-lightening agent containing equol compound as active ingredient|

---

JPH11225840A|1998-02-12|1999-08-24|Miyagen:Kk|Pipe leg non-slip cover and manufacture thereof|

---

JP4947608B2|2000-06-05|2012-06-06|株式会社コシイプレザービング|Extraction method of flavonoid derivatives|

---

JP2003104818A|2001-07-26|2003-04-09|Nippon Fine Chem Co Ltd|Exterminator containing flavonoid glycoside for noxious insect|

---

WO2005094770A1|2004-03-30|2005-10-13|Unilever Plc|Skin lightening compositions comprising vitamines and flavonoids|

---

WO2008140440A1|2005-12-07|2008-11-20|Mmp International Development And Manufacturing|Stable flavonoid solutions|

---

EP1867729A1|2006-06-14|2007-12-19|Libragen|Water soluble phenolics derivatives with dermocosmetic and therapeutic applications|

---

CN100569077C|2007-01-24|2009-12-16|四川大学|A kind of mixed pesticide of radix euphorbiae lantu chromocor and preparation method thereof|

---

TW200915583A|2007-09-17|2009-04-01|Univ Nat Taiwan Science Tech|Photoelectric electrodes capable of absorbing solar energy, fabrication methods, and applications thereof|

---

EP2100966A1|2008-03-12|2009-09-16|Institut Pasteur|Mutants of glycoside hydrolases and uses thereof for synthesizing complex oligosaccharides and disaccharide intermediates|

---

CN102477024A|2010-11-26|2012-05-30|西北农林科技大学农药研究所|Flavone compound for preventing and controlling plant diseases|

---

MX2011010032A|2011-09-23|2013-03-25|Promotora Tecnica Ind S A De C V|Pesticide made of isoquinoline alkaloids, flavonoids and vegetable and/or essential oils.|CN106565654B|2016-10-14|2018-08-31|云南中烟工业有限责任公司|A kind of novel flavone compound, Its Preparation Method And Use extracted from Bai Yun Shen|

---

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KR102105412B1|2018-06-20|2020-04-29|한국원자력연구원|A chroman compound having anti-cancer effect and pharmaceutical composition for treating or preventing cancer comprising the same|

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优先权:

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申请号 | 申请日 | 专利标题

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FR1452461A|FR3018821A1|2014-03-24|2014-03-24|NOVEL O-ALPHA-GLUCOSYL FLAVONOIDS ON CYCLE B, PROCESS FOR OBTAINING AND USES|

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FR1452461|2014-03-24|

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FR1456417A|FR3018822B1|2014-03-24|2014-07-03|NOVEL O-ALPHA-GLUCOSYL FLAVONOIDS ON CYCLE B, PROCESS FOR OBTAINING AND USES|FR1456417A| FR3018822B1|2014-03-24|2014-07-03|NOVEL O-ALPHA-GLUCOSYL FLAVONOIDS ON CYCLE B, PROCESS FOR OBTAINING AND USES|

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EP15714775.2A| EP3122890A1|2014-03-24|2015-03-24|Novel flavonoids o-alpha-glucosylated on the b cycle, method for the production thereof and uses|

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PCT/EP2015/056307| WO2015144731A1|2014-03-24|2015-03-24|Novel flavonoids o-α-glucosylated on the b cycle, method for the production thereof and uses|

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US15/128,673| US10647739B2|2014-03-24|2015-03-24|Flavonoids O-A-glucosylated on the B cycle, method for the production thereof and uses|