METHODS FOR THE PRODUCTION OF RHAMNOSYLATED FLAVONOIDS专利检索- 作用于卤键的水解酶专利检索查询-专利查询网

METHODS FOR THE PRODUCTION OF RHAMNOSYLATED FLAVONOIDS

申请号

US16069302

申请日

2017-01-13

公开(公告)号

US20190203240A1

公开(公告)日

2019-07-04

申请人

Universitaet Hamburg;

发明人

Ulrich Rabausch; Henning Rosenfeld; Nele Ilmberger; Tanja Plambeck; Constantin Ruprecht; Friederike Boenisch;

摘要

A method for the production of rhamnosylated flavonoids comprising the steps of contacting/incubating a glycosyl transferase with a flavonoid and obtaining a rhamnosylated flavonoid. In addition, glycosyl transferases suitable for use in such methods and kits comprising said glycosyl transferases.

权利要求

1. A method for the production of rhamnosylated flavonoids, the method comprising(a) contacting/incubating a glycosyl transferase with a flavonoid; and

(b) obtaining a rhamnosylated flavonoid,

wherein the glycosyl transferase

(a) comprises the amino acid sequence of SEQ ID NO: 1;

(b) comprises amino acid sequences having at least 80% sequence identity with SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 56, 58, 61;

(c) is encoded by a polynucleotide comprising the nucleic acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 37, 38, 57, 59, 60, 62, or 63;

(d) is encoded by a polynucleotide having at least 80% sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 37, 38, 57, 59, 60, 62, or 63; or

(e) is encoded by a polynucleotide hybridizable under stringent conditions with a polynucleotide comprising SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 37, 38, 57, 59, 60, 62, or 63,

and

wherein the flavonoid is a compound or a solvate of the following Formula (I)

wherein:

is a double bond or a single bond;

L is

R¹and R²are independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; wherein R²is different from OH;

or R¹and R²are joined together to form, together with the carbon atom(s) that they are attached to, a carbocyclic or heterocyclic ring being optionally substituted with one or more substituents R^e; wherein each R^eis independently selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c;

R⁴, R⁵and R⁶are independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c;

or alternatively, R⁴is selected from hydrogen, C_1-5; alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; and

R⁵and R⁶are joined together to form, together with the carbon atoms that they are attached to, a carbocyclic or heterocyclic ring being optionally substituted with one or more substituents R^c;

or alternatively, R⁴and R⁵are joined together to form, together with the carbon atoms that they are attached to, a carbocyclic or heterocyclic ring being optionally substituted with one or more substituents R^c; and

R⁶is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c;

each R^ais independently selected from a single bond, C_1-5alkylene, C_2-5alkenylene, arylene and heteroarylene; wherein said alkylene, said alkenylene, said arylene and said heteroarylene are each optionally substituted with one or more groups R^c;

each R^bis independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c,

each R^cis independently selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-S-aryl, —(C_0-3alkylene)-S(C_1-5alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, —(C_0-3alkylene)-CHO, alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl), and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl, said alkenyl, said alkynyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R^care each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl;

each R^dis independently selected from a monosaccharide, a disaccharide and an oligosaccharide; and

R³is rhamnoslyated by said method.

2. The method of claim 1, wherein the flavonoid is contacted/incubated with said glycosyl transferase at a final concentration above its solubility in aqueous solutions.

3. The method of claim 1, wherein the method further comprises a step of providing a host cell transformed with said glycosyl transferase.

4. The method of claim 3, wherein said host cell is incubated prior to contacting/incubating said host cell with a flavonoid.

5. The method of claim 3, wherein said host cell is Escherichia coli.

6. The method of claim 1, wherein contacting and/or incubating is/are done at a temperature from about 20° C. to about 37° C., preferably at a temperature from about 24° C. to about 30° C., and more preferably at a temperature of about 28° C.

7. The method of claim 1, wherein contacting/incubating is/are done at a pH of about 6.5 to about 8.5, preferably at a pH of about 7 to about 8, and more preferably at a pH of about 7.4.

8. The method of claim 1, wherein contacting/incubating is/are done at a concentration of dissolved oxygen (DO) of about 30% to about 50%.

9. The method of claim 1, wherein, when the concentration of dissolved oxygen is above about 50%, a nutrient is added, preferably wherein the nutrient is glucose, sucrose, maltose or glycerol.

10. The method of claim 1, wherein contacting/incubating is/are done in a complex nutrient medium.

11. The method of claim 1, wherein contacting/incubating is/are done in minimal medium.

12. The method of claim 3, wherein the method further comprises a step of harvesting said incubated host cell prior to contacting/incubating said host cell with a flavonoid.

13. The method of claim 12, wherein harvesting is done using a membrane filtration method, preferably a hollow fibre membrane device, or centrifugation.

14. The method of claim 12, wherein the method further comprises solubilization of the harvested host cell in a buffer prior to contacting/incubating said host cell with a flavonoid, preferably wherein the buffer is phosphate-buffered saline (PBS), preferably supplemented with a carbon and energy source, preferably glycerol, glucose, maltose, and/or sucrose, and growth additives, preferably vitamins including biotin and/or thiamin.

15. The method of claim 1, wherein the flavonoid is a flavanone, flavone, isoflavone, flavonol, flavanonol, chalcone, flavanol, anthocyanidine, aurone, flavan, chromene, chromone or xanthone.

16. The method of claim 1, wherein rhamnosylating is the addition of —O-(rhamnosyl) at position R³of Formula (I) of claim 1, wherein said rhamnosyl is substituted at one or more of its —OH groups with one or more groups independently selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, a monosaccharide, a disaccharide and an oligosaccharide.

17. The method of claim 1, wherein the flavonoid is contacted/incubated with said glycosyl transferase at a final concentration above about 200 μM.

18. The method of claim 1, wherein the flavonoid is contacted/incubated with said glycosyl transferase at a final concentration above about 500 μM.

19. The method of claim 1, wherein the flavonoid is contacted/incubated with said glycosyl transferase at a final concentration above about 1 mM.

20. The method of claim 1, wherein contacting and/or incubating is/are done at a temperature from about 24° C. to about 30° C., preferably at a temperature of about 28° C.

说明书全文

The present invention relates to methods for the production of rhamnosylated flavonoids comprising the steps of contacting/incubating a glycosyl transferase with a flavonoid and obtaining a rhamnosylated flavonoid. In addition, the invention relates to glycosyl transferases suitable for use in such methods and kits comprising said glycosyl transferases.

Flavonoids are a class of polyphenol compounds which are commonly found in a large variety of plants. Flavonoids comprise a subclass of compounds such as anthoxanthins, flavanones, flavanonols, flavans and anthocyanidins. Flavonoids are known to possess a multitude of beneficial properties which make these compounds suitable for use as antioxidants, anti-inflammatory agents, anti-cancer agents, antibacterials, antivirals, antifungals, antiallergenes, and agents for preventing or treating cardiovascular diseases. Furthermore, some flavonoids have been reported to be useful as flavor enhancing or modulating agents.

Due to this wide variety of possible applications, flavonoids are compounds of high importance as ingredients in cosmetics, food, drinks, nutritional and dietary supplements, pharmaceuticals and animal feed. However, use of these compounds has often been limited due to the low water solubility, low stability and limited availability. A further factor which has severely limited use of these compounds is the fact that only a few flavonoids occur in significant amounts in nature while the abundance of other flavonoids is nearly negligible. As a result, many flavonoids and their derivatives are not available in amounts necessary for large-scale industrial use.

Glycosylation is one of the most abundant modifications of flavonoids, which has been reported to significantly modulate the properties of these compounds. For example, glycosylation may lead to higher solubility and increased stability, such as higher stability against radiation or temperature. Furthermore, glycosylation may modulate pharmacological activity and bioavailability of these compounds.

Glycosylated derivatives of flavonoids occur in nature as O-glycosides or C-glycosides, while the latter are much less abundant. Such derivatives may be formed by the action of glycosyl transferases (GTs) starting from the corresponding aglycones.

Examples of naturally occurring O-glycosides are quercetin-3-O-β-D-glucoside (Isoquercitrin) and genistein-7-O-β-glucoside (Genistin).

However, flavonoids constitute the biggest class of polyphenols in nature (Ververidis (2007) Biotech. J. 2(10):1214-1234). The high variety of flavonoids originates from addition of various functional groups to the ring structure. Herein, glycosylation is the most abundant form and the diversity of sugar moieties even more leads to a plethora of glycones.

But in nature only some flavonoid glycones prevail. As described above, among these are the 3-O-β-D-glucosides, e.g. isoquercitrin, the flavonoid-7-β-D-glucosides, e.g. genistin, and the 3- and 7-rhamnoglucosides, e.g. rutin and naringin. Generally, glucosides are the most frequent glycosidic forms with 3- and 7-O-β-D-glucosides dominating. In contrast, glycosides concerning other sugar moieties, e.g. rhamnose, and other glycosylation positions than C3 and C7 rarely occur and are only present in scarce quantities in specific plant organs. This prevents any industrial uses of such compounds. For example, De Bruyn (2015) Microb Cell Fact 14:138 describes methods for producing rhamnosylated flavonoids at the 3-O position. Also, 3-O rhamnosylated versions of naringenin and quercetin are described by Ohashi (2016) Appl Microbiol Biotechnol 100:687-696. Metabolic engineering of the 3-O rhamnoside pathway in E. coli with kaempferol as an example is described by Yang (2014) J Ind Microbiol Biotech 41:1311-18. Finally, the in vitro production of 3-O rhamnosylated quercetin and kaempferol is described by Jones (2003) J Biol Chem 278:43910-18. None of these documents describes or suggests the production of 5-O rhamnosylated flavonoids.

In fact, very few examples of 5-O rhamnosylated flavonoids are known in the art. The few examples are quercetin-5-O-β-D-glucoside, luteolin-5-O-glucoside, and chrysin-5-O-β-D-xyloside (Hedin (1990) J Agric Food Chem 38(8):1755-7; Hirayama (2008) Photochemistry 69(5):1141-1149; Jung (2012) Food Chem Toxicol 50(6):2171-2179; Chauhan (1984) Phytochemistry 23(10):2404-2405). Up to now, only four flavonoid-5-O-rhamnosides were described. Taxifolin-3,5-di-O-α-L-rhamnoside was extracted from the Indian plant Cordia obliqua which also contains low amounts of Hesperetin-7-O-α-L-rhamnoside (Chauhan (1978) Phytochemistry 17:334; Srivastava (1979) Phytochemistry 18:2058-2059). Eriodictyol-5-rhamnoside was isolated from Cleome viscosa (Srivastava (1979) Indian J Chem Sect B 18:86-87). Another flavanone, Naringenin-5-O-α-L-rhamnoside (N5R) was isolated from Himalayan cherry (Prunus cerasoides) seeds (Shrivastava (1982) Indian J Chem Sect B 21 (6):406-407). Extraction from 2 kg of air dried powdered seeds resulted in 800 mg N5R. The absolute rare occurrence inhibits the commercial use also of other flavanone rhamnosides like naringenin-4′-O-α-L-rhamnoside that was isolated from the stem of a tropical Fabaceae plant (Yadava (1997) J Indian Chem Soc 74(5):426-427).

WO 2014/191524 relates to enzymes catalyzing the glycosylation of polyphenols, in particular flavonoids, benzoic acid derivatives, stilbenoids, chalconoids, chromones, and coumarin derivatives. In addition, WO 2014/191524 discloses methods for preparing a glycoside of polyphenols. However, glycosylation is limited to C3, C3′, C4′ and C7 of polyphenols. Moreover, the disclosure is silent with regard to the possibility of rhamnosylating polyphenols.

Accordingly, there is an urgent need for reliable methods for the large-scale production of 5-O rhamnosylated flavonoids to allow commercial use.

Thus, the technical problem underlying the present invention is the provision of reliable means and methods for efficient rhamnosylation of flavonoids at C5, corresponding to the R³position of Formula I.

The technical problem is solved by provision of the embodiments characterized in the claims.

Accordingly, the present invention relates to methods for the production of rhamnosylated flavonoids comprising contacting/incubating a glycosyl transferase with a flavonoid and obtaining a rhamnosylated flavonoid. In this regard, it has been surprisingly and unexpectedly found that glycosyl transferases are able to rhamnosylate flavonoids at the C5-OH, i.e. R³position, in particular where the flavonoid is represented by the following formula (I):

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In contrast to what could have been expected based on the prior art, glycosyl transferases are able to rhamnosylate compounds of formula I at the R³position, corresponding to C5 of polyphenols as described in WO 2014/191524. Accordingly, as illustrated in the appended Examples, the methods of the present invention allow the production of 5-O rhamnosides, in particular at large-scale to allow the commercial use of the produced 5-O rhamnosides. In this regard, it was surprisingly found that most efficient production of rhamnosylated flavonoids can be observed in experiments using concentrations of the reactant, i.e. the flavonoid, above its solubility in aqueous solutions. That is, the present invention relates to methods for the production of rhamnosylated flavonoids comprising contacting/incubating a glycosyl transferase with a flavonoid, wherein the flavonoid is contacted/incubated with said glycosyl transferase at a final concentration above its solubility in aqueous solutions, preferably above about 200 μM, more preferably above about 500 μM, and even more preferably above about 1 mM, and subsequently obtaining a rhamnosylated flavonoid. The skilled person will appreciate that the solubility varies depending on the flavonoid used as educt in the methods of the present invention. Thus, the above values can be altered depending on the used flavonoid.

In the methods of the present invention, a glycosyl transferase is used for efficient production of 5-O rhamnosylated flavonoids. In principle, any glycosyltransferase may be used, as is evidenced by the appended Examples; see e.g. Example A3, in particular Tables A7 and A8. However, it is preferred that a glycosyl transferase belonging to family GT1 is used. In this regard, the glycosyl transferases GTC, GTD, GTF, and GTS belong to the glycosyltransferase family GT1 (EC 2.4.1.x) (Coutinho (2003) J Mol Biol 328(2):307-317). This family comprises enzymes that mediate sugar transfer to small lipophilic acceptors. Family GT1 members uniquely possess a GT-B fold. They catalyze an inverting reaction mechanism concerning the glycosidic linkage in the sugar donor and the formed one in the acceptor conjugate, creating natural β-D- or α-L-glycosides.

Within the GT-B fold the enzymes form two major domains, one N-terminal and a C-terminal, with a linker region in between. Generally, the N-terminus constitutes the AA-residues responsible for acceptor binding and the residues determining donor binding are mainly located in the C-terminus. In family GT1 the C-terminus contains a highly conserved motif possessing the AA residues that take part in nucleoside-diphosphate (NDP)-sugar binding. This motif was also termed the plant secondary product glycosyltransferase (PSPG) box (Hughes (1994) Mit DNA 5(1):41-49.

Flavonoid-GTs belong to family GT1. Due to the natural biosynthesis of flavonoids in plants most of the enzymes are also known from plants. However, several enzymes from the other eukaryotic kingdoms fungi and animals and also from the domain of bacteria are described. In eucarya, sugar donors of GT1 enzymes are generally uridinyl-diphosphate (UDP)-activated. Of these so called UGTs or UDPGTs, most enzymes transfer glucose residues from UDP-glucose to the flavonoid acceptors. Other biological relevant sugars from UDP-galactose, -rhamnose, -xylose, -arabinose, and -glucuronic acid are less often transferred.

Also several bacterial GT1s were discovered that are able to glycosylate also flavonoid acceptors. These enzymes all belong to the GT1 subfamily of antibiotic macrolide GTs (MGT). In bacteria GT1 enzymes use UDP-glucose or galactose but also deoxythymidinyl-diphosphate (dfDP)-activated sugars as donor substrates. However, all the bacterial flavonoid active GT1 enzymes have UDP-glucose as the native donor. There is only one known exception with the metagenome derived enzyme GtfC that was the first bacterial GT1 reported to transfer rhamnose to flavonoids (Rabausch (2013) Appl Environ Microbiol 79(15):4551-4563). However, until the present invention was made, it was established that this activity is limited to C3-OH or the C7-OH groups of flavonoids. Transfer to the C3′-OH and the C4′-OH of the flavonoid C-ring was already less commonly observed. Other positions are rarely glycosylated, if at all. Specifically, there are only few examples concerning the glycosylation of the C5-OH group, which is based on the fact that this group is sterically protected if a keto group at C4 is present. Therefore, the only examples relate to anthocyanidins (Janvary (2009) J Agric Food Chem 57(9):3512-3518; Lorenc-Kukala (2005) J Agric Food Chem 53(2):272-281; Tohge (2005) The Plant J 42(2):218-235). This class of flavonoids lacks the C4 keto group which facilitates nucleophilic attack. The C5-OH group of (iso)flavones and (iso)flavanones is protected through hydrogen bridges with the neighbored carbonyl group at C4. This was thought to even hinder chemical glycosylation approaches at C5 of these classes.

Today, there are only three GT1 enzymes characterized that create 5-O-β-D-glucosides of flavones. One is UGT71G1 from Medicago truncatula which was proven to be not regio-selective and showed a slight side activity in glucosylation of C5-OH on quercetin (He (2006) JBC 281(45):34441-7. An exceptional UGT was identified in the silkworm Bombyx mori capable of specifically forming quercetin-5-O-β-D-glucoside (Daimon (2010) PNAS 107(25):11471-11476; Xu (2013) Mol Biol Rep 40(5):3631-3639) Finally, a mutated variant of MGT from Streptomyces lividans presented low activity at C5-OH of 5-hydroxyflavone after single AA exchange (Xie (2013) Biochemistry (Mosc) 78(5):536-541). However, the wild type MGT did not possess this ability nor did other MGTs.

Flavanol-5-O-α-D-glucosides were synthesized through transglucosylation activity of hydrolases, i.e. α-amylases (EC 3.2.1.x) (Noguchi (2008) J Agric Food Chem 56(24):12016-12024; Shimoda (2010) Nutrients 2(2):171-180). However, the flavanols also lack the C4=O-group and the enzymes create a “non-natural” α-D-glucosidic linkage.

It is noteworthy that all so far known 5-O-GTs mediated only glucosylation. The prior art is entirely silent with regard to rhamnosylation of flavonoids, much less using the method of the present invention.

Thus, GTC from Elbe river sediment metagenome, GTD from Dyadobacter fermentans, GTF from Fibrosoma limi, and GTS from Segetibacter koreensis and chimeras 1, 3, and 4 are the first experimentally proved flavonoid-5-O-rhamnosyltransferases (FRTs). This is evidenced by the appended Examples. In particular, Example A3 provides results for all chimeras in Tables A7 and A8. Further production examples are shown in the further Examples, in particular using GTC. Furthermore, related enzymes from, Flavihumibacter solisilvae, Cesiribacter andamanensis, Niabella aurantiaca, Spirosoma radiotolerans, Fibrella aestuarina, Flavisolibacter sp. LCS9 and Aquimarina macrocephali, present the same functionality as they share important amino acid sequence features. In contrast to all other GT1 enzymes that use NDP-sugars FRTs possess several unique amino acid patterns.

Accordingly, the present invention relates to a method for the production of 5-O, i.e. R³in formula I, rhamnosylated flavonoids using a glycosyl transferase comprising said conserved amino acids. These conserved amino acid sequences, which were surprisingly and unexpectedly identified by the present inventors, comprise the following motifs (all amino acid positions are given with respect to the wild-type GTC amino acid sequence): (1) strictly conserved amino acids Asp (D³⁰) and aromatic Phe (F³³) in the motif ²¹K/R ILFAXXPXDGHF N/S PLTX L/I A⁴⁰both located around His³², i.e. the active site residue of GT1 enzymes, wherein the amino acid at position 30 is preferably a polar amino acid; (2) the motif ⁴⁷GXDVRW Y/F⁵³comprising the loop before Nβ2 and strand Nβ2; (3) strictly conserved amino acid Arg (R⁸⁸) of motif ⁸⁵F/Y/L P E/D R⁸⁸where Pro⁸⁶and Glu⁸⁷are reported for substrate binding in GT1 enzymes and neighboring Arg (R⁸⁸) is unique to Rhamnosyl-GTs; (4) strictly conserved amino acids Phe (F¹⁰⁰), Asp (D¹⁰¹), Phe (F¹⁰⁶), Arg (R¹⁰⁹) and Asp (D¹¹⁶) of the motif ¹⁰⁰FDXXXXFXXRXXE Y/F XXD¹¹⁶forming the long N-terminal helix Nα3, wherein the amino acids at positions 103 and 108 preferably are non-polar amino acids; (5) the motif ¹²⁴F/W PFXXXXX D/E XXFXXXXF¹⁴⁰comprising the loop before Nβ4, strand Nβ4, and the loop to the downstream N-α-helix, wherein amino acids at positions 128 to 130 are preferably non-polar amino acids; (6) the motif ¹⁵⁶PLXEXXXXL P/A PXGXGXXPXXXXXG K/R¹⁸⁰comprising conserved amino acid Gly (G¹⁷⁰); (7) the motif ²³⁰LQXGXXGFEYXR²⁴¹before the linker region of the N-terminal domain with the C-terminal domain; (8) the motif ²⁸¹TQGTXE K/R XXXKXXXPTLEAF R/K³⁰¹comprising the loop before Cα1 and helix Cα1 and strictly conserved amino acids Thr (T²⁸⁴) and Glu (G²⁸⁶) where Thr is involved in substrate binding and wherein the amino acid at position 285 preferably is a non-polar amino acid and amino acids at positions 292 to 294 preferably are non-polar amino acids; (9) the motif ³⁰⁶LVXXTTGG³¹³forming strand Cβ2, wherein amino acids at positions 308 and 309 preferably are non-polar amino acids; and (10) the motif ³³⁰I E/D DFIPFXX V/I MPXXDV Y/F I/V T/S NGG Y/F GGV M/L LXIX N/H XLPXVXAGXH EGKNE³⁷⁶comprising conserved acidic amino acids Glu/Asp (E/D³³¹), Asp (D³³²), conserved aromatic amino acid Phe (F³³⁶) instead of Gln (Q) in other GT1 enzymes at start of helix Cα2, strictly conserved amino acid Asn (N³⁴⁹) involved in substrate binding, and strictly conserved amino acid Gly (G³⁶⁹) instead of Pro (P) in other GT1 enzymes, wherein the motif forms the conserved donor binding region of GT1 enzymes, wherein the amino acids at positions 367 and 372 preferably are non-polar amino acids and where the ³⁷¹HEGKNE³⁷⁶motif is absolutely unique to the 5-O-FRTs, as GT 1 enzymes usually show a D/E Q/N/K/R motif responsible for hexose sugar binding and catalytic activity.

The following alignment of said 5-O-FRTs illustrates the homologous AAs positions and shows consensus SEQ ID NO:1.

....|....| ....|....|.... |....| ....|....|

5 15 25 35

GTC

---------M SNLFSSQTNL ASVKPLKGRK ILFANFPADG

GTD

---------M TKYKN----- ----ELTGKR ILFGTVPGDG

GTF

---------M TTK------- ---------K ILFATMPMDG

GTS

---------- MKYIS----- ---SIQPGTK ILFANFPADG

GT from S. radiotolerans

--------MI TPQ------- ---------R ILFATMPMDG

GT from N. aurantiaca

--------MY TKTANTTNAA APLHGGEKKK ILFANIPADG

GT from F. solisilvae

---------M NHKHS----- --------RK ILMANVPADG

GT from F. aestuarina

---------M NPQ------- ---------R ILFATMPFDG

GT from C. andamanensis

METSQKGGTQ SPKPF----- --------RR ILFANCPADG

GT from A. macrocephali

---------M TRMSQ----- --------KK ILFACIPADG

GT from F. sp. LCS9

MNNTLSTVID HTIAS----- ---QIKPGTK ILFATFPADG

Chimera 1

---------M TKYKN----- ----ELTGKR ILFGTVPGDG

Chimera 3

---------M TKYKN----- ----ELTGKR ILFGTVPGDG

Chimera 4

---------M TKYKN----- ----ELTGKR ILFGTVPGDG

SEQ ID NO. 1

---------- ---------- ---------K ILFAXXPXDG

alternate aa SEQ ID NO. 1

....|....| ....|....|.... |....| ....|....|

45 55 65 75

GTC

HFNPLTGLAV HLQWLGCDVR WYTSNKYADK LRRLNIPHFP

GTD

HFNPLTGLAK YLQELGCDVR WYASDVFKCK LEKLSIPHYG

GTF

HFNPLTGLAV HLHNQGHDVR WYVGGHYGAK VKKLGLIHYP

GTS

HFNPLTGLAV HLKNIGCDVR WYTSKTYAEK IARLDIPFYG

GT from S. radiotolerans

HFSPLTGLAV HLSNLGHDVR WYVGGEYGEK VRKLKLHHYP

GT from N. aurantiaca

HFNPLTGLAV RLKKAGHDVR WYTGASYAPR IEQLGIPFYL

GT from F. solisilvae

HFNPLTGIAV HLKQQGYDVR WYGSDVYSKK AAKLGIPYFP

GT from F. aestuarina

HFSPLTNLAV HLSQLGHDVR WFVGGHYGQK VTQLGLHHYP

GT from C. andamanensis

HFNPLIPLAE FLKQQGHDVR WYSSRLYADK ISRMGIPHYP

GT from A. macrocephali

HFNPMTAIAI HLKTKGYDVR WYTGEGYKNT LHRIGIPYLP

GT from F. sp. LCS9

HFNPLTGLAM HLKQIGCDVR WYTAKKYANK LQQLDIPHYD

Chimera 1

HFNPLTGLAK YLQELGCDVR WYASDVFKCK LEKLSIPHYG

Chimera 3

HFNPLTGLAK YLQELGCDVR WYASDVFKCK LEKLSIPHYG

Chimera 4

HFNPLTGLAK YLQELGCDVR WYASDVFKCK LEKLSIPHYG

SEQ ID NO. 1

HFNPLTXLA- -----GXDVR WY-------- ----------

alternate aa SEQ ID NO. 1

S I F

....|....| ....|....|.... |....| ....|....|

85 95 105 115

GTC

FRKAMDIA-- -DLENMFPER DAIKGQVAKL KFDIINAFIL

GTD

FKKAWDVNG- VNVNEILPER QKLTDPAEKL SFDLIHIFGN

GTF

YHKAQVINQ- ENLDEVFPER QKIKGTVPRL RFDLNNVFLL

GTS

LQRAVDVSAH AEINDVFPER KKYKGQVSKL KFDMINAFIL

GT from S. radiotolerans

FVNARTINQ- ENLEREFPER AALKGSIARL RFDIKQVFLL

GT from N. aurantiaca

FNKAKEVTV- HNIDEVFPER KTIRNHVKKV IFDICTYFIE

GT from F. solisilvae

FSKALEVNS- ENAEEVFPER KRINSKIGKL NFDLQNFFVR

GT from F. aestuarina

YVKTRTVNQ- ENLDQLFPER ATIKGAIARI RFDLGQIFLL

GT from C. andamanensis

FKKALEFDT- HDWEGSFPER SKHKSQVGKL RFDLEHVFIR

GT from A. macrocephali

FQNAQELKI- EEIDKMYPDR KMLKG-IAHI KFDIINLFIN

GT from F. sp. LCS9

LVRALDFAS- GEPDEIFPER KQHKSQLAKL KFDIINVFIK

Chimera 1

FKKAWDVNG- VNVNEILPER QKLTDPAEKL SFDLIHIFGN

Chimera 3

FKKAWDVNG- VNVNEILPER QKLTDPAEKL SFDLIHIFGN

Chimera 4

FKKAWDVNG- VNVNEILPER QKLTDPAEKL SFDLIHIFGN

SEQ ID NO. 1

---------- ------FPER ---------- -FDXXXXFXX

alternate aa SEQ ID NO. 1

Y D

alternate aa SEQ ID NO. 1

....|....| ....|....|.... |....| ....|....|

125 135 145 155

GTC

RGPEYYVDLQ EIHKSFPFDV MVADCAFTGI PFVTDKMDIP

GTD

RAPEYYEDIL EIHESFPFDV FIADSCFSAI PLVSKLMSIP

GTF

RAPEFITDVT AIHKSFPFDL LICDTMFSAA PMLRHILNVP

GTS

RSTEYYEDIL EIYEEFPFQL MIADITFGAI PFVEEKMNIP

GT from S. radiotolerans

RAPEFVEDMK DIYQTWPFTL VVHDVAFIGG SFIKQLLPVK

GT from N. aurantiaca

RGTEFYED1K DINKSFDFDV LICDSAFTGM SFVKEKLNKH

GT from F. solisilvae

RAPEYYADLI DIHREFPFDL LIADCMFTAI PFVKELMQIP

GT from F. aestuarina

RVPEQIDDLR AIYDEWPFDL IVQDLGFVGG TFLRELLPVK

GT from C. andamanensis

RGPEYFEDIR DLHQEFPFDV LVAEISFTGI AFIRHLMHKP

GT from A. macrocephali

RMKGYYEDIA EIHQVFPFDI LVCDNTFPGS -IVKKKLNIP

GT from F. sp. LCS9

RGPEFYDDIK EIHQTFPFEV MIADVAFTGT PMVKEKMNIP

Chimera 1

RAPEYYEDIL EIHESFPFDV FIADSCFSAI PLVSKLMSIP

Chimera 3

RAPEYYEDIL EIHESFPFDV FIADSCFSAI PLVSKLMSIP

Chimera 4

RAPEYYEDIL EIHESFPFDV FIADSCFSAI PLVSKLMSIP

SEQ ID NO. 1

RXXEYXXD-- -----FPFXX XXXDXXFXXX XF--------

alternate aa SEQ ID NO. 1

F W E

....|....| ....|....|.... |....| ....|....|

165 175 185 195

GTC

VVSVGVFPLT ETSKDLPPAG LGITPSFSLP GKFKQSILRS

GTD

VVAVGVIPLA EESVDLAPYG TGLPPAATEE QRAMYFGMKD

GTF

VAAVGIVPLS ETSKELPPAG LGMEPATGFF GRLKQDFLRF

GTS

VISISVVPLP ETSKDLAPSG LGITPSYSFF GKIKQSFLRF

GT from S. radiotolerans

TVAVGVVPLT ESDDYLPPSG LGRQPMRGIA GRWIQHLMRY

GT from N. aurantlaca

AVAIGILPLC ASSKQLPPPI MGLTPAKTLA GKAVHSFLRF

GT from F. solisilvae

VLSIGIAPLL ESSRDLAPYG LGLHPARSWA GKFRQAGLRW

GT from F. aestuarina

VVGVGVVPLT ESDDWVPPTS LGMKPQSGRV GRLVSRLLNY

GT from C. andamanensis

VIAVGIFPNI ASSRDLPPYG LGMRPASGFL GRKKQDLLRF

GT from A. macrocephali

IASIGVVPLA LSAPDLPLYG IGHQPATTFF GKRKQNFIKL

GT from F. sp. LCS9

VITVGILPLP ETSKDLAPYG LAITPNYSFW GKKKQTFLRF

Chimera 1

VVAVGVIPLA EESVDLAPYG TGLPPAATEE QRAMYFGMKD

Chimera 3

VVAVGVIPLA EESVDLAPYG TGLPPAATEE QRAMYFGMKD

Chimera 4

VVAVGVIPLA EESVDLAPYG TGLPPAATEE QRAMYFGMKD

SEQ ID NO. 1

-------PLX ESXXXLPPXG XFXXPXXXXX GK--------

alternate aa SEQ ID NO. 1

A R

....|....| ....|....|.... |....| ....|....|

205 215 225 235

GTC

VADLVLFRES NKVMRKMLTE HGIDHLYTN- VFDLMVKKST

GTD

ALANVVFKTA IDSFSAILDR YQVPHEKAI- LFDTLIRQSD

GTF

MTTRILFKPC DDLYNEIRQR YNMEPARDF- VFDSFIRTAD

GTS

IADELLFAQP TKVMWGLLAQ HGIDAGKAN- IFDILIQKST

GT from S. radiotolerans

MVQQVMFKPI NVLHNQLRQV YGLPPEPDS- VFDSIVRSAD

GT from N. aurantiaca

LTNKVLFKKP HALINEQYRR AGMLTNGKN- LFDLQIDKAT

GT from F. solisilvae

VADNILFRKS INVMYDLFEE YNIPHNGEN- FFDMGVRKAS

GT from F. aestuarina

LVQDVMLKPA NDLHNELRAQ YGLRPVPGF- IFDATVRQAD

GT from C. andamanensis

LTDKLVFGKQ NELNRQILRS WGIEAPGHLN LFDLQTQHAS

GT from A. macrocephali

MADKLIFDET KVVYNQLLRS LDLSEEENLT IFDIAPLQSD

GT from F. sp. LCS9

VADQVLFRKP YLVMKEMLAD YGIKP-DGN- LFSTLIRKSS

Chimera 1

ALANVVFKTA IDSFSAILDR YQVPHEKAI- LFDTLIRQSD

Chimera 3

ALANVVFKTA IDSFSAILDR YQVPHEKAI- LFDTLIRQSD

Chimera 4

ALANVVFKTA IDSFSAILDR YQVPHEKAI- LFDTLIRQSD

SEQ ID NO. 1

---------- ---------- ---------- ----------

....|....| ....|....|.... |....| ....|....|

245 255 265 275

GTC

LLLQSGTPGF EYYRSDLGKN IRFIGSLLPY QSKKQTT---

GTD

LFLQIGAKAF EYDRSDLGEN VRFVGALLPY SESKSRQ---

GTF

LYLQSGVPGF EYKRSKMSAN VRFVGPLLPY SSGIKPN---

GTS

LVLQSGTPGF EYKRSDLSSH VHFIGPLLPY TKKKERE---

GT from S. radiotolerans

VYLQSGVPSF EYPRKRISAN VQFVGPLLPY AKGQKHP---

GT from N. aurantiaca

LFLQSCTPGF EYQRAHMSRH IHFIGPLLPS HSDAPAP---

GT from F. solisilvae

LFLQSGTPGF EYNRSDLSEH IRFIGALLPY AGERKEE---

GT from F. aestuarina

LYLQSGVPGF EFPRKRISPN VRFIGPMLPY SRANRQP---

GT from C. andamanensis

VVLQNGTPGF EYTRSDLSPN LVFAGPLLPL VKKVRED---

GT from A. macrocephali

VFLQNGIPEI DYPRYSLPES IKYVGALQVQ TNNNNNQKLK

GT from F. sp. LCS9

LVLQSGTPGF EYFRSDLGHN IRFAGALLPY TTQKQTT---

Chimera 1

LFLQIGAKAF EYDRSDLGKN IRFIGSLLPY QSKKQTT---

Chimera 3

LFLQIGAKAF EYDRSDLGEN VRFVGALLPY SESKSRQ---

Chimera 4

LFLQIGAKAF EYDRSDLGEN VRFVGALLPY SESKSRQ---

SEQ ID NO. 1

--LQXGXPGF EYXR------ ---------- ----------

alternate aa SEQ ID NO. 1

C K D

....|....| ....|....|.... |....| ....|....|

285 295 305 315

GTC

AWSDERLNRY EKIVVVTQGT VEKNIEKILV PTLEAFR-DT

GTD

PWFDQKLLQY GRIVLVTQGT VEHDINKILV PTLEAFK-NS

GTF

FAHAAKLKQY KKVILATQGT VERDPEKILV PTLEAFK-DT

GTS

SWYNEKLSHY DKVILVTQGT IEKDIEKLIV PTLEAFK-NS

GT from S. radiotolerans

FIQAKKALQY KKVILVTQGT IERDVQKIIV PTLEAFKNEP

GT from N. aurantlaca

FHFEDKLHQY AKVLLVTQGT FEGDVRKLIV PAIEAFK-NS

GT from F. solisilvae

PWFDSRLNKF DRVILVTQGT VERDVTKIIV PVLKAFR-DS

GT from F. aestuarina

FEQAIKTLAY KRVVLVTQGT VERNVEKIIV PTLEAYKKDP

GT from C. andamanensis

LPLQEKLRKY KNVILVTQGT AEQNTEKILA PTLEAFK-DS

GT from A. macrocephali

KDWSAILDTS KKIILVSQGT VEKNLDKLII PSLEAFK-DS

GT from F. sp. LCS9

PWYNKKLEQY DKVILVTQGT VEKDVEKIIV PTLEAFK-DS

Chimera 1

AWSDERLNRY EKIVVVTQGT VEKNIEKILV PTLEAFR-DT

Chimera 3

PWFDQKLLQY GRIVLVTQGT VEHDINKILV PTLEAFK-NS

Chimera 4

PWFDQKLLQY GQIVVVTQGT VEKNIEKILV PTLEAFR-DT

SEQ ID NO. 1

---------- ------TQGT XEKXXXKXXX PTLEAFR---

alternate aa SEQ ID NO. 1

R K

....|....| ....|....|.... |....| ....|....|

325 335 345 355

GTC

DLLVIATTGG SGTAELKKRY PQ-GNLIIED FIPFGDIMPY

GTD

ETLVIATTGG NGTAELRARF PF-ENLIIED FIPFDDVMPR

GTF

DHLVVITTGG SKTAELRARY PQ-KNVIIED FIDFNLIMPH

GTS

DCLVIATTGG AYTEELRKRY PE-ENIIIED FIPFDDVMPY

GT from S. radiotolerans

TTLVIVTIGG SQTSELRARF PQ-ENFIIDD FIDFNAVMPY

GT from N. aurantiaca

RHLVVVTTAG WHTHKLRQRY KAFANVVIED FIPFSQIMPF

GT from F. solisilvae

NYLVVATTGG NGTKLLREQY KA-DNIIIED FIPFTDIMPY

GT from F. aestuarina

DILVIVTIGG SGTLALRKRY PQ-ANFIIED FIDFNAVMPY

GT from C. andamanensis

TWLVVATTGG AGTEALRARY PQ-ENFLIED YIPFDQIMPN

GT from A. macrocephali

DYIVLVATGY TDTKGLQKRY PQ-QHFYIED FIAYDAVMPH

GT from F. sp. LCS9

DCLVVVTTGG SRTLELRLRY PQ-NNIIIED FIPFGDVMPY

Chimera 1

DLLVIATTGG SGTAELKKRY PQ-GNLIIED FIPFGDIMPY

Chimera 3

ETLVIATTGG NGTAELRARF PQ-GNLIIED FIPFGDIMPY

Chimera 4

DLLVIATTGG SGTAELKKRY PQ-GNLIIED FIPFGDIMPY

SEQ ID NO. 1

--LVXXTTGG ---------- -------IED FIPFXXVMPX

alternate aa SEQ ID NO. 1

D I

....|....| ....|....|.... |....| ....|....|

365 375 385 395

GTC

ADVYITNGGY GGVMLGIENQ LPLVVAGIHE GKNEINARIG

GTD

ADVYVTNGGY GGTLLSIHNQ LPMVAAGVHE GKNEVCSRIG

GTF

ADVYVTNSGF GGVMLSIQHG LPMVAAGVHE GKNEIAARIG

GTS

ADVYVSNGGY GGVLLSIQHQ LPMVVAGVHE GKNEINARVG

GT from S. radiotolerans

ASVYVTNGGY GGVMLALQHN LPIVVAGIHE GKNEIAARID

GT from N. aurantiaca

ADVFISNGGY GGVMQSISNK LPMVVAGIHE GKNEICARVG

GT from F. solisilvae

TDVYVTNGGY GGVMLGIENQ LPLVVAGVHE GKNEINARIG

GT from F. aestuarina

VSVYVTNGGY GGVMLALQHK LPIVAAGVHE GKNEIAARIG

GT from C. andamanensis

ADVYVSNGGF GGVLQAISHQ LPMVVAGVHE GKNEICARVG

GT from A. macrocephali

IDVFIMNGGY GSALLSIKHG VPMITAGVNE GKNEICSRMD

GT from F. sp. LCS9

ADVYITNGGY GGVMLGIENQ LPMVVAGVHE GKNEICARVG

Chimera 1

ADVYITNGGY GGVMLGIENQ LPLVVAGIHE GKNEINARIG

Chimera 3

ADVYITNGGY GGVMLGIENQ LPLVVAGIHE GKNEINARIG

Chimera 4

ADVYITNGGY GGVMLGIENQ LPLVVAGIHE GKNEINARIG

SEQ ID NO. 1

XDVYITNGGY GGVMLXIXNX LPXVXAGXHE GKNE------

alternate aa SEQ ID NO. 1

FVS F L H

....|....| ....|....|.... |....| ....|....|

405 415 425 435

GTC

YFELGINLKT EWPKPEQMKK AIDEVIGNKK YKENITKLAK

GTD

HFGCGINLET ETPTPDQIRE SVHKILSNDI FKKNVFRIST

GTF

YFKLGMNLKT ETPTPDQIRT SVETVLTDQT YRRNLARLRT

GTS

YFDLGINLKT ERPTVLQLRK SVDAVLQSDS YAKNVKRLGK

GT from S. radiotolerans

YCKVGIDLKT ETPSPTRIRH AVETVLTNDM YRQNVRQMGQ

GT from N. aurantiaca

YFKTGINMRT EHPKPEKIKT AVNEILSNPL YRKSVERLSK

GT from F. solisilvae

YFRLGIDLRN ERPTPEQMRN AIEKVIANGE YRRNVQALAR

GT from F. aestuarina

YCQVGVDLRT ETPTPDQIRR AVATILGDET YRRQVRRLSD

GT from C. andamanensis

YFKLGLDLKT ETPKPAQIRA AVEQVLQDPQ YRHKVQALSA

GT from A. macrocephali

YSGVGIDLKT EKPRAVTIQN ATERILGTDK YLDTIQKIQQ

GT from F. sp. LCS9

YFQLGINLKT EQPIPAQIRN SVEEILSNVV YKKNVVKLSK

Chimera 1

YFELGINLKT EWPKPEQMKK AIDEVIGNKK YKENITKLAK

Chimera 3

YFELGINLKT EWPKPEQMKK AIDEVIGNKK YKENITKLAK

Chimera 4

YFELGINLKT EWPKPEQMKK AIDEVIGNKK YKENITKLAK

SEQ ID NO. 1

---------- ---------- ---------- ----------

....|....| ....|....|.... |....| ....|....|

445 455 465 475

GTC

EFSNYHPNEL CAQYISEVLQ KTGRLYISSK KEEEKIY---

GTD

HLD-VDANEK SAGHILDLLE ERVVCG---- ----------

GTF

EFAQYDPMAL SERYINELLA KQPRKQHEAV EAI-------

GTS

EFKQYDPNEI CEKYVAQLLE NQISYKEKAN SYQAEVLV--

GT from S. radiotolerans

EFSQYQPTEL AEQYINALLI QEKSSRLAVV A---------

GT from N. aurantiaca

EFSEYDPLAL CEKFVNALPV LQKP------ ----------

GT from F. solisilvae

EFKTYAPLEL TERFVTELLL SRRHKLVPVN DDALIY----

GT from F. aestuarina

EFGRYNPNQL AEQYINELLA QSVGEPVAAL S---------

GT from C. andamanensis

EFRQYNPQQL CEHWVQRLTG GRRAAAPAPQ SAGGQLLSLT

GT from A. macrocepha1i

RMNSYNTLDI CEQHISRLIS E--------- ----------

GT from F. sp. LCS9

EFAQYKPNEL CAKYVAQLVQ -QESSSQKVN VAAVEAVLEA

Chimera 1

EFSNYHPNEL CAQYISEVLQ KQAG-FISAV KRKKKRYTKD

Chimera 3

EFSNYHPNEL CAQYISEVLQ KTGRLYISSK KEEEKIY---

Chimera 4

EFSNYHPNEL CAQYISEVLQ KTGRLYISSK KEEEKIY---

SEQ ID NO. 1

---------- ---------- ---------- ----------

....|....|

485

GTC

----------

GTD

----------

GTF

----------

GTS

----------

GT from S. radiotolerans

----------

GT from N. aurantiaca

----------

GT from F. solisilvae

----------

GT from F. aestuarina

----------

GT from C. andamanensis

LN--------

GT from A. macrocephali

----------

GT from F. sp. LCS9

----------

Chimera 1

PAANKARKEA

Chimera 3

----------

Chimera 4

----------

SEQ ID NO. 1

----------

Accordingly, in the methods of the present invention, it is preferred that a glycosyl transferase comprising some or preferably all of the above conserved amino acids/sequence motifs is used as long as the glycosyl transferase maintains its desired function of rhamnosylating flavonoids at position R3 of formula (I). These amino acids/sequence motifs are comprised in SEQ ID NO:1. Thus, in one preferred embodiment of the present invention, a glycosyl transferase is used, which comprises the amino acid sequence of SEQ ID NO:1 and which shows the desired activity of rhamnosylating flavonoids at position R3 of Formula (I) as shown above, corresponding to 5-O rhamnosylation of flavonoids. The invention furthermore relates to a method for rhamnosylation of flavonoids using a glycosyl transferase comprising an amino acid sequence of the known glycosyl transferases GTC, GTD, GTF or related enzymes from Segetibacter koreensis, Flavihumibacter solisilvae, Cesiribacter andamanensis, Niabella aurantiaca, Spirosoma radiotolerans, Fibrella aestuarina, or Aquimarina macrocephali. Accordingly, in one embodiment, a glycosyl transferase having the amino acid sequence as shown in any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 56, 58, or 61 is used in the methods of the present invention. In this regard, the skilled person is well-aware that these sequences may be altered without altering the function of the polypeptide. For example, it is known that enzymes such as glycosyl transferases generally possess an active site responsible for the enzymatic activity. Amino acids outside of the active site or even within the active site may be altered while the enzyme in its entirety maintains a similar or identical activity. It is known that enzymatic activity may even be increased by alterations to the amino acid sequence. Therefore, in the methods of the present invention, glycosyl transferases may be used comprising an amino acid sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 56, 58, or 61, respectively, as long as the function of rhamnosylating flavonoids at position R3 of Formula (I) is maintained. Methods how to test this activity are described herein and/or are known to the person skilled in the art.

In the methods of the present invention, glycosyl transferases may be used that are encoded by a polynucleotide comprising the nucleic acid sequences encoding the above glycosyl transferases. In particular, a glycosyl transferase encoded by a polynucleotide comprising any of the nucleic acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 57, 59, 60, 62, or 63 may be used. As is known in the art, the genetic code is degenerated, which allows alterations to the sequence of nucleic acids comprised in a polynucleotide without altering the polypeptide encoded by the polynucleotide. Accordingly, in the methods of the present invention, glycosyl transferases may be used that are encoded by a polynucleotide having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 57, 59, 60, 62, or 63. Because further alterations to the polynucleotide may be made without altering the structure/function of the encoded polypeptide, glycosyl transferases may be used in the methods of the present invention that are encoded by a polynucleotide hybridizable under stringent conditions with a polynucleotide comprising SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 57, 59, 60, 62, or 63.

Within the meaning of the present invention, the term “polypeptide” or “enzyme” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide may have many types of modifications. Modifications can include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and/or transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins Structure and Molecular Properties 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).

While the glycosyl transferase used in the methods of the present invention may be contacted/incubated with a flavonoid directly, it is preferred that the method further comprises a step of providing a host cell transformed with a gene encoding said glycosyl transferase. As such, the glycosyl transferase is recombinantly expressed by the host cell and provided by the host cell for being contacted/incubated with the flavonoid. It is preferred that the host cell is incubated prior to contacting/incubating said host cell with a flavonoid. That is, it is preferred that the host cell is allowed to recombinantly express the glycosyl transferase prior to addition of a flavonoid for production of a rhamnosylated version thereof.

The type of host cell is not particularly limited. In principle, any cell may be used as host cell to recombinantly express a glycosyl transferase. For example, the organism may be used from which the glycosyl transferase gene is derived. However, it is preferred in the methods of the present invention that the host cell is a prokaryotic host cell.

As used herein, “prokaryote” and “prokaryotic host cell” refer to cells which do not contain a nucleus and whose chromosomal material is thus not separated from the cytoplasm. Prokaryotes include, for example, bacteria. Prokaryotic host cells particularly embraced by the present invention include those amenable to genetic manipulation and growth in culture. Exemplary prokaryotes routinely used in recombinant protein expression include, but are not limited to, E. coli, Bacillus lichenifauuis (van Leen, et al. (1991) Bio/Technology 9:47-52), Ralstonia eutropha (Srinivasan, et al. (2002) Appl. Environ. Microbiol. 68:5925-5932), Methylobacterium extorquens (Belanger, et al. (2004) FEMS Microbiol Lett. 231 (2): 197-204), Lactococcus lactic (Oddone, et al. (2009) Plasmid 62(2): 108-18) and Pseudomonas sp. (e.g., P. aerugenosa, P. fluorescens and P. syringae). Prokaryotic host cells can be obtained from commercial sources (e.g., Clontech, Invitrogen, Stratagene and the like) or repositories such as American Type Culture Collection (Manassas, Va.).

In the methods of the present invention, it is preferred that the prokaryotic host cell, in particular the bacterial host cell, is E. coli. The expression of recombinant proteins in E. coli is well-known in the art. Protocols for E. coli-based expression systems are found in Sambrook “Molecular Cloning” Cold Spring Harbor Laboratory Press 2012.

The host cells of the invention are recombinant in the sense that they have been genetically modified for the purposes of harboring polynucleotides encoding a glycosyl transferase. Generally, this is achieved by isolating nucleic acid molecules encoding the protein or peptide of interest and introducing the isolated nucleic acid molecules into a prokaryotic cell.

Nucleic acid molecules encoding the proteins of interest, i.e. a glycosyl transferase, can be isolated using any conventional method. For example, the nucleic acid molecules encoding the glycosyl transferase may be obtained as restriction fragments or, alternatively, obtained as polymerase chain reaction amplification products. Techniques for isolating nucleic acid molecules encoding proteins such as glycosyl transferases are routinely practiced in the art and discussed in conventional laboratory manuals such as Sambrook and Russell (Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory press (2012)) and Ausubel et al. (Short Protocols in Molecular Biology, 52nd edition, Current Protocols (2002)).

To facilitate the expression of proteins (including enzymes) or peptides in the prokaryotic host cell, in particular the glycosyl transferase, the isolated nucleic acid molecules encoding the proteins or peptides of interest are incorporated into one or more expression vectors. Expression vectors compatible with various prokaryotic host cells are well-known and described in the art cited herein. Expression vectors typically contain suitable elements for cloning, transcription and translation of nucleic acids. Such elements include, e.g., in the 5′ to 3′ direction, a promoter (unidirectional or bidirectional), a multiple cloning site to operatively associate the nucleic acid molecule of interest with the promoter, and, optionally, a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylase. In addition to regulatory control sequences discussed herein, the expression vector can contain additional nucleotide sequences. For example, the expression vector can encode a selectable marker gene to identify host cells that have incorporated the vector. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that containing the nucleic acid of interest or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). Expression vectors can be obtained from commercial sources or be produced from plasmids routinely used in recombinant protein expression in prokaryotic host cells. Exemplary expression vectors include, but are not limited to pBR322, which is the basic plasmid modified for expression of heterologous DNA in E. coli; RSF1010 (Wood, et al. (1981) J. Bacteriol. 14:1448); pET3 (Agilent Technologies); pALEX2 vectors (Dualsystems Biotech AG); and pET100 (Invitrogen).

The regulatory sequences employed in the expression vector may be dependent upon a number of factors including whether the protein of interest, i.e. the glycosyl transferases, is to be constitutively expressed or expressed under inducible conditions (e.g., by an external stimulus such as IPTG). In addition, proteins expressed by the prokaryotic host cell may be tagged {e.g., his6-, FLAG- or GST-tagged) to facilitate detection, isolation and/or purification.

Vectors can be introduced into prokaryotic host cells via conventional transformation techniques. Such methods include, but are not limited to, calcium chloride (Cohen, et al. (1972) Proc. Natl. Acad. Sci. USA 69:2110-2114; Hanahan (1983) J. Mol. Biol. 166:557-580; Mandel & Higa (1970) J. Mol. Biol. 53:159-162), electroporation (Shigekawa & Dower (1988) Biotechniques 6:742-751), and those described in Sambrook et al. (2012), supra. For a review of laboratory protocols on microbial transformation and expression systems, see Saunders & Saunders (1987) Microbial Genetics Applied to Biotechnology Principles and Techniques of Gene Transfer and Manipulation, Croom Helm, London; Puhler (1993) Genetic Engineering of Microorganisms, Weinheim, N.Y.; Lee, et al. (1999) Metabolic Engineering, Marcel Dekker, NY; Adolph (1996) Microbial Genome Methods, CRC Press, Boca Raton; and Birren & Lai (1996) Nonmammalian Genomic Analysis: A Practical Guide, Academic Press, San Diego.

As an alternative to expression vectors, it is also contemplated that nucleic acids encoding the proteins (including enzymes) and peptides disclosed herein can be introduced by gene targeting or homologous recombination into a particular genomic site of the prokaryotic host cell so that said nucleic acids are stably integrated into the host genome.

Recombinant prokaryotic host cells harboring nucleic acids encoding a glycosyl transferase can be identified by conventional methods such as selectable marker expression, PCR amplification of said nucleic acids, and/or activity assays for detecting the expression of the glycosyl transferase. Once identified, recombinant prokaryotic host cells can be cultured and/or stored according to routine practices.

With regards to culture methods of recombinant host cells, the person skilled in the art is well-aware how to select and optimize suitable methods for efficient culturing of such cells.

As used herein, the terms “culturing” and the like refer to methods and techniques employed to generate and maintain a population of host cells capable of producing a recombinant protein of interest, in particular the glycosyl transferase, as well as the methods and techniques for optimizing the production of the protein of interest, i.e. the glycosyl transferase. For example, once an expression vector has been incorporated into an appropriate host, preferably E. coli, the host can be maintained under conditions suitable for high level expression of the relevant polynucleotide. When using the methods of the present invention, the protein of interest, i.e. the glycosyl transferase, may be secreted into the medium. Where the protein of interest is secreted into the medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, e.g., an Amicon™ or Millipore Pellicon™ ultrafiltration unit, which can then be subjected to one or more additional purification techniques, including but not limited to affinity chromatography, including protein A affinity chromatography, ion exchange chromatography, such as anion or cation exchange chromatography, and hydrophobic interaction chromatography.

Culture media used for various recombinant host cells are well known in the art. Generally, a growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells. There are different types of media for growing different types of cells.

Culture media used to culture recombinant bacterial cells will depend on the identity of the bacteria. Culture media generally comprise inorganic salts and compounds, amino acids, carbohydrates, vitamins and other compounds that are either necessary for the growth of the host cells or improve health or growth or both of the host cells. In particular, culture media typically comprise manganese (Mn²⁺) and magnesium (Mg²⁺) ions, which are co-factors for many, but not all, glycosyltransferases. The most common growth/culture media for microorganisms is LB medium (Lysogeny Broth). LB is a nutrient medium.

Nutrient media contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections.

In this regard, an undefined medium (also known as a basal or complex medium) is a medium that contains: a carbon source such as glucose for bacterial growth, water, various salts needed for bacterial growth, a source of amino acids and nitrogen (e.g., beef, yeast extract). In contrast, a defined medium (also known as chemically defined medium or synthetic medium) is a medium in which all the chemicals used are known and no yeast, animal or plant tissue is present. In the methods of the present invention, either defined or undefined nutrient media may be used. However, it is preferred that lysogeny broth (LB) medium, terrific broth (TB) medium, Rich Medium (RM), Standard I medium or a mixture thereof be used in the methods of the present invention.

Alternatively, minimal media may be used in the methods of the present invention. Minimal media are those that contain the minimum nutrients possible for colony growth, generally without the presence of amino acids. Minimal medium typically contains a carbon source for bacterial growth, which may be a sugar such as glucose, or a less energy-rich source like succinate, various salts, which may vary among bacteria species and growing conditions; these generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic acid and water. Supplementary minimal media are a type of minimal media that also contains a single selected agent, usually an amino acid or a sugar. This supplementation allows for the culturing of specific lines of auxotrophic recombinants. Accordingly, in one embodiment the methods of the present invention are done in minimal medium. Preferably, the minimal medium is a mineral salt medium (MSM) or M9 medium supplemented with a carbon source and an energy source, preferably wherein said carbon and energy sources are glycerol, glucose, maltose, sucrose, starch and/or molasses.

Media used in the methods of the present invention are prepared following methods well-known in the art. In this regard, a method for preparing culture medium generally comprises the preparation of a “base medium”. The term “base medium” or broth refers to a partial broth comprising certain basic required components readily recognized by those skilled in the art, and whose detailed composition may be varied while still permitting the growth of the microorganisms to be cultured. Thus in embodiments and without limitation, base medium may comprise salts, buffer, and protein extract, and in embodiments may comprise sodium chloride, monobasic and dibasic sodium phosphate, magnesium sulphate and calcium chloride. In embodiments a liter of core medium may have the general recipe known in the art for the respective medium, but in alternative embodiments core media will or may comprise one or more of water, agar, proteins, amino acids, caesein hydrolysate, salts, lipids, carbohydrates, salts, minerals, and pH buffers and may contain extracts such as meat extract, yeast extract, tryptone, phytone, peptone, and malt extract, and in embodiments medium may be or may comprise luria bertani (LB) medium; low salt LB medium (1% peptone, 0.5% yeast extract, and 0.5% NaCl), SOB medium (2% peptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄), SOC medium (2% peptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM Glucose), Superbroth (3.2% peptone, 2% yeast extract, and 0.5% NaCl), 2×TY medium (1.6% peptone, 1% yeast extract, and 0.5% NaCl), TerrificBroth (TB) (1.2% peptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, and 0.4% glycerol), LB Miller broth or LB Lennox broth (1% peptone, 0.5% yeast extract, and 1% NaCl). It will be understood that in particular embodiments one or more components may be omitted from the base medium.

In the methods of the present invention, the host cell may be cultured in the medium prior to incubating/contacting the host cell with an agent for inducing expression of the foreign gene, i.e. the glycosyl transferase, and prior to addition of the flavonoid to be bioconverted. Alternatively, the flavonoid may be added to the culture together with the host cell, thus, prior to amplifying the number of host cells in the culture medium.

The person skilled in the art will readily understand that the growth of a desired microorganism, in particular E. coli, will be best promoted at selected temperatures suited to the microorganism in question. In particular embodiments culturing may be carried out at about 28° C. and the broth to be used may be pre-warmed to this temperature preparatory to inoculation with a sample for testing. However, in the methods of the present invention culturing may be carried out at any temperature suitable for the desired purpose, i.e. the production of a rhamnosylated flavonoid. However, it is preferred that culturing is done at a temperature between about 20° C. and about 37° C. That is, culturing is preferably done at a temperature of about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C. or about 37° C. More preferably, culturing may be carried out at a temperature between about 24° C. to about 30° C. Most preferably, culturing in the methods of the present invention is done at a temperature of about 28° C.

Similarly, contacting/incubating the cultured host cell with a flavonoid may be done at any temperature suitable for efficient production of a rhamnosylated flavonoid. Preferably, the temperature for culturing the host cell and the temperature for contacting/incubating the host cell and the glycosyl transferase with a flavonoid are about identical. That is, it is preferred that contacting/incubating the host cell and the expressed glycosyl transferase with a flavonoid is done at a temperature between about 20° C. and about 37° C. Contacting/incubating the host cell and the expressed glycosyl transferase with a flavonoid is preferably done at a temperature of about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C. or about 37° C. More preferably, contacting/incubating the host cell and the expressed glycosyl transferase with a flavonoid may be carried out at a temperature between about 24° C. to about 30° C. Most preferably, contacting/incubating the host cell and the expressed glycosyl transferase with a flavonoid in the methods of the present invention is done at a temperature of about 28° C.

In the methods of the present invention, the pH of culture medium is generally set at between about 6.5 and about 8.5 and for example in particular embodiments is or is about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5 or may be in ranges delimited by any two of the foregoing values. Thus, in particular embodiments the pH of culture medium is in ranges with lower limits of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, or 8.4 and with upper limits of about 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5. In a preferred embodiment the culture medium has a pH between about 7.0 and 8.0. In a more preferred embodiment of the present invention, the medium has a pH of about 7.4. However, it will be understood that a pH outside of the range pH 6.5-8.5 may still be useable in the methods of the present invention, but that the efficiency and selectivity of the culture may be adversely affected.

A culture may be grown for any desired period following inoculation with a recombinant host cell, but it has been found that a 3 hour culture period above 20° C. and starting from an optical density (OD) of 0.1 at 600 nm is sufficient to enrich the content of E. coli sufficiently to permit efficient expression of the glycosyl transferase and subsequent contacting/incubating with the flavonoid for successful bioconversion. However, the culture period may be longer or shorter and may be up to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more hours. Those skilled in the art will readily select a suitable culture period to satisfy particular requirements.

In the methods of the present invention, the culture medium may be further enriched/supplemented. That is, it is preferred that during culturing of the host cell and/or during contacting/incubating the host cell and the expressed glycosyl transferase with a flavonoid, the concentration of dissolved oxygen (DO) is monitored and maintained at a desired value. Preferably, in the methods of the present invention, the concentration of dissolved oxygen (DO) is maintained at about 30% to about 50%. Moreover, when the concentration of dissolved oxygen is above about 50%, a nutrient may be added, preferably wherein the nutrient is glucose, sucrose, maltose or glycerol. That is, the medium may be supplemented/enriched during culturing/contacting/incubating to maintain conditions that allow efficient production of the glycosyl transferase and/or efficient bioconversion of the flavonoid.

In one embodiment, the methods of the present invention may be done as fed-batch culture or semi-batch culture. These terms are used interchangeably to refer to an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run. In some embodiments, all the nutrients are fed into the bioreactor.

In the methods of the present invention, a step of harvesting the incubated host cell prior to contacting/incubating said host cell with a flavonoid may be added. That is, the methods of the present invention may comprise culturing the host cell in a culture medium until a desired optical density (OD) and harvesting the host cell when the desired OD is reached. The OD may be between about 0.6 and 1.0, preferably about 0.8. Expression of the glycosyl transferase may either be induced prior to harvesting or subsequently to harvesting, for example together with addition of the flavonoid. The culture medium may be changed subsequently to harvesting or the host cell may be resuspended in culture medium used for growth of the host cell. That is, in one embodiment, methods of the present invention further comprise solubilization of the harvested host cell in a buffer prior to contacting/incubating said host cell with a flavonoid, preferably wherein the buffer is phosphate-buffered saline (PBS), preferably supplemented with a carbon and energy source, preferably glycerol, glucose, maltose, and/or sucrose, and growth additives, preferably vitamins including biotin and/or thiamin.

In the methods of the present invention, harvesting may be done using any method suitable for that purpose. It is preferred that harvesting is done using a membrane filtration method, preferably a hollow fibre membrane device, or centrifugation.

In the methods of the present invention, the flavonoid to be rhamnosylated is not particularly limited as long as the flavonoid belongs to the class of flavonoids as known in the art and, as such, is a member of a group of compounds widely distributed in plants, fulfilling many functions. Flavonoids are the most important plant pigments for flower coloration, producing yellow or red/blue pigmentation in petals designed to attract pollinator animals. In higher plants, flavonoids are involved in UV filtration, symbiotic nitrogen fixation and floral pigmentation.

As such, the flavonoid preferably is a flavanone, flavone, isoflavone, flavonol, flavanonol, chalcone, flavanol, anthocyanidine, aurone, flavan, chromene, chromone or xanthone. Within the meaning of the present invention, the latter three are comprised in this class. As such, the term “flavonoid” refers to any compounds falling under the general formula (I) and is thus not limited to compounds which are generally considered flavonoid-type compounds.

It is preferred that the flavonoid used in the methods of the present invention is a compound or a solvate of the following Formula (I)

embedded image

wherein:

custom-character is a double bond or a single bond;

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or alternatively, R⁴is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; and

R⁵and R⁶are joined together to form, together with the carbon atoms that they are attached to, a carbocyclic or heterocyclic ring being optionally substituted with one or more substituents R^c;

each R^cis independently selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_1-3alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-S-aryl, —(C_0-3alkylene)-S(C_1-5alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, alkylene)-CHO, —(C_0-3alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl), and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl, said alkenyl, said alkynyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R^care each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl;

each R^dis independently selected from a monosaccharide, a disaccharide and an oligosaccharide; and

R³is rhamnoslyated by the method of the present invention.

In this regard, rhamnosylating/rhamnosylation preferably is the addition of —O-(rhamnosyl) at position R³of Formula (I) as shown above, wherein said rhamnosyl is substituted at one or more of its —OH groups with one or more groups independently selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, a monosaccharide, a disaccharide and an oligosaccharide.

As used herein, the term “hydrocarbon group” refers to a group consisting of carbon atoms and hydrogen atoms. Examples of this group are alkyl, alkenyl, alkynyl, alkylene, carbocyl and aryl. Both monovalent and divalent groups are encompassed.

As used herein, the term “alkyl” refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an “alkyl” group does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond. A “C_1-5alkyl” denotes an alkyl group having 1 to 5 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). Unless defined otherwise, the term “alkyl” preferably refers to C_1-4alkyl, more preferably to methyl or ethyl, and even more preferably to methyl.

As used herein, the term “alkenyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond. The term “C_2-5alkenyl” denotes an alkenyl group having 2 to 5 carbon atoms. Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-1-en-1-yl, prop-1-en-2-yl, or prop-2-en-1-yl), butenyl, butadienyl (e.g., buta-1,3-dien-1-yl or buta-1,3-dien-2-yl), pentenyl, or pentadienyl (e.g., isoprenyl). Unless defined otherwise, the term “alkenyl” preferably refers to C_2-4alkenyl.

As used herein, the term “alkynyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more carbon-to-carbon double bonds. The teen “C_2-5alkynyl” denotes an alkynyl group having 2 to 5 carbon atoms. Preferred exemplary alkynyl groups are ethynyl, propynyl, or butynyl. Unless defined otherwise, the term “alkynyl” preferably refers to C_2-4alkynyl.

As used herein, the term “alkylene” refers to an alkanediyl group, i.e. a divalent saturated acyclic hydrocarbon group which may be linear or branched. A “C_1-5alkylene” denotes an alkylene group having 1 to 5 carbon atoms, and the teen “C_0-3alkylene” indicates that a covalent bond (corresponding to the option “Co alkylene”) or a C_1-3alkylene is present. Preferred exemplary alkylene groups are methylene (—CH₂—), ethylene (e.g., —CH₂—CH₂— or —CH(—CH₃)—), propylene (e.g., —CH₂—CH₂—CH₂—, —CH(—CH₂—CH₃)—, —CH₂—CH(—CH₃)—, or —CH(—CH₃)—CH₂—), or butylene (e.g., —CH₂—CH₂—CH₂—CH₂—). Unless defined otherwise, the term “alkylene” preferably refers to C_1-4alkylene (including, in particular, linear C_1-4alkylene), more preferably to methylene or ethylene, and even more preferably to methylene.

As used herein, the term “carbocyclyl” refers to a hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. Unless defined otherwise, “carbocyclyl” preferably refers to aryl, cycloalkyl or cycloalkenyl.

As used herein, the term “heterocyclyl” refers to a ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. Unless defined otherwise, “heterocyclyl” preferably refers to heteroaryl, heterocycloalkyl or heterocycloalkenyl.

As used herein, the term “heterocyclic ring” refers to saturated or unsaturated rings containing one or more heteroatoms, preferably selected from oxygen, nitrogen and sulfur. Examples include heteroaryl and heterocycloalkyl as defined herein. Preferred examples contain, 5 or 6 atoms, particular examples, are 1,4-dioxane, pyrrole and pyridine.

The term “carbocyclic ring” means saturated or unsaturated carbon rings such as aryl or cycloalkyl, preferably containing 5 or 6 carbon atoms. Examples include aryl and cycloalkyl as defined herein.

As used herein, the term “aryl” refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic). “Aryl” may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1,2-dihydronaphthyl), tetralinyl (i.e., 1,2,3,4-tetrahydronaphthyl), anthracenyl, or phenanthrenyl. Unless defined otherwise, an “aryl” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, and most preferably refers to phenyl.

As used herein, the term “heteroaryl” refers to an aromatic ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). “Heteroaryl” may, e.g., refer to thienyl (i.e., thiophenyl), benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl, pyrrolyl (e.g., 2H-pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl; e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, indolyl (e.g., 3H-indolyl), indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (e.g., [1,10]phenanthrolinyl, [1,7]phenanthrolinyl, or [4,7]phenanthrolinyl), phenazinyl, thiazolyl, isothiazolyl, phenothiazinyl, oxazolyl, isoxazolyl, furazanyl, phenoxazinyl, pyrazolo[1,5-a]pyrimidinyl (e.g., pyrazolo[1,5-a]pyrimidin-3-yl), 1,2-benzisoxazol-3-yl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, 1H-tetrazolyl, 2H-tetrazolyl, coumarinyl, or chromonyl. Unless defined otherwise, a “heteroaryl” preferably refers to a 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a “heteroaryl” refers to a 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized.

The term “heteroalkyl” refers to saturated linear or branched-chain monovalent hydrocarbon radical of one to twelve carbon atoms, including from one to six carbon atoms and from one to four carbon atoms, wherein at least one of the carbon atoms is replaced with a heteroatom selected from N, O, or S, and wherein the radical may be a carbon radical or heteroatom radical (i.e., the heteroatom may appear in the middle or at the end of the radical). The heteroalkyl radical may be optionally substituted independently with one or more substituents described herein. The term “heteroalkyl” encompasses alkoxy and heteroalkoxy radicals.

As used herein, the term “cycloalkyl” refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings). “Cycloalkyl” may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or adamantyl. Unless defined otherwise, “cycloalkyl” preferably refers to a C_3-11cycloalkyl, and more preferably refers to a C_3-7cycloalkyl. A particularly preferred “cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members.

As used herein, the term “heterocycloalkyl” refers to a saturated ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). “Heterocycloalkyl” may, e.g., refer to oxetanyl, tetrahydrofuranyl, piperidinyl, piperazinyl, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, morpholinyl (e.g., morpholin-4-yl), pyrazolidinyl, tetrahydrothienyl, octahydroquinolinyl, octahydroisoquinolinyl, oxazolidinyl, isoxazolidinyl, azepanyl, diazepanyl, oxazepanyl or 2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl. Unless defined otherwise, “heterocycloalkyl” preferably refers to a 3 to 11 membered saturated ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, “heterocycloalkyl” refers to a 5 to 7 membered saturated monocyclic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized.

As used herein, the term “halogen” refers to fluoro (—F), chloro (—Cl), bromo (—Br), or iodo (—I).

As used herein, the term “haloalkyl” refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) halogen atoms which are selected independently from fluoro, chloro, bromo and iodo, and are preferably all fluoro atoms. It will be understood that the maximum number of halogen atoms is limited by the number of available attachment sites and, thus, depends on the number of carbon atoms comprised in the alkyl moiety of the haloalkyl group. “Haloalkyl” may, e.g., refer to —CF₃, —CHF₂, —CH₂F, —CF₂—CH₃, —CH₂—CF₃, —CH₂—CHF₂, —CH₂—CF₂—CH₃, —CH₂—CF₂—CF₃, or —CH(CF₃)₂.

As used herein, the term “rhamnosyl” refers to a substituted or unsubstituted rhamnose residue which is preferably connected via the C1-OH group of the same.

The term “monosaccharide” as used herein refers to sugars which consist of only a single sugar unit. These include all compounds which are commonly referred to as sugars and includes sugar alcohols and amino sugars. Examples include tetroses, pentoses, hexoses and heptoses, in particular aldotetroses, aldopentoses, aldohexoses and aldoheptoses.

Aldotetroses include erythrose and threose and the ketotetroses include erythrulose.

Aldopentoses include apiose, ribose, arabinose, lyxose, and xylose and the ketopentoses include ribulose and xylulose. The sugar alcohols which originate in pentoses are called pentitols and include arabitol, xylitol, and adonitol. The saccharic acids include xylosaccharic acid, ribosaccharic acid, and arabosaccharic acid.

Aldohexoses include galactose, talose, altrose, allose, glucose, idose, mannose, rhamnose, fucose, olivose, rhodinose, and gulose and the ketohexoses include tagatose, psicose, sorbose, and fructose. The hexitols which are sugar alcohols of hexose include talitol, sorbitol, mannitol, iditol, allodulcitol, and dulcitol. The saccharic acids of hexose include mannosaccharic acid, glucosaccharic acid, idosaccharic acid, talomucic acid, alomucic acid, and mucic acid.

Examples of aldoheptoses are idoheptose, galactoheptose, mannoheptose, glucoheptose, and taloheptose. The ketoheptoses include alloheptulose, mannoheptulose, sedoheptulose, and taloheptulose.

Examples of amino sugars are fucosamine, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, and N-acetylgalactosamine.

As used herein, the term “disaccharide” refers to a group which consists of two monosaccharide units. Disaccharides may be formed by reacting two monosaccharides in a condensation reaction which involves the elimination of a small molecule, such as water.

Examples of disaccharides are maltose, isomaltose, lactose, nigerose, sambubiose, sophorose, trehalose, saccharose, rutinose, and neohesperidose.

As used herein, the term “oligosaccharide” refers to a group which consists of three to eight monosaccharide units. Oligosaccharide may be formed by reacting three to eight monosaccharides in a condensation reaction which involves the elimination of a small molecule, such as water. The oligosaccharides may be linear or branched.

Examples are dextrins as maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and maltooctaose, fructo-oligosaccharides as kestose, nystose, fructosylnystose, bifurcose, inulobiose, inulotriose, and inulotetraose, galacto-oligosaccharides, or mannan-oligosaccharides.

As used herein, the expression “the compound contains at least one OH group in addition to any OH groups in R³” indicates that there is at least one OH group in the compound at a position other than residue R³. Examples of the OH groups in R³are OH groups of the rhamnosyl group or of any substituents thereof. Consequently, for the purpose of determining whether the above expression is fulfilled, the residue R³is disregarded and the number of the remaining OH groups in the compound is determined.

As used herein, the expression “an OH group directly linked to a carbon atom being linked to a neighboring carbon or nitrogen atom via a double bond” indicates a group of the following partial structure:

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in which Q is N or C which may be further substituted. The double bond between C and Q may be part of a larger aromatic system and may thus be delocalized. Examples of such OH groups include OH groups which are directly attached to aromatic moieties, such as, aryl or heteroaryl groups. One specific example is a phenolic OH group.

As used herein, the term “substituted at one or more of its —OH groups” indicates that a substituent may be attached to one or more of the “—OH” groups in such a manner that the resulting group may be represented by “—O-substituent”.

Various groups are referred to as being “optionally substituted” in this specification. Generally, these groups may carry one or more substituents, such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety. Unless defined otherwise, the “optionally substituted” groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent. Moreover, unless defined otherwise, it is preferred that the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.

As used herein, the terms “optional”, “optionally” and “may” denote that the indicated feature may be present but can also be absent. Whenever the term “optional”, “optionally” or “may” is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. For example, the expression “X is optionally substituted with Y” (or “X may be substituted with Y”) means that X is either substituted with Y or is unsubstituted. Likewise, if a component of a composition is indicated to be “optional”, the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.

When specific positions in the compounds of formula (I) or formula (II) are referred to, the positions are designated as follows:

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A skilled person will appreciate that the substituent groups comprised in the compounds of formula (I) may be attached to the remainder of the respective compound via a number of different positions of the corresponding specific substituent group. Unless defined otherwise, the preferred attachment positions for the various specific substituent groups are as illustrated in the examples.

As used herein, the term “about” preferably refers to ±10% of the indicated numerical value, more preferably to ±5% of the indicated numerical value, and in particular to the exact numerical value indicated.

Accordingly, it is preferred that a compound of the following formula (I) or a solvate thereof is used in the methods of the present invention as starting compound

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Many specific examples of the compound of following formula (I) are disclosed herein, such as, compounds of formulae (II), (IIa), (IIb), (IIc), (IId), (III) and (IV). It is to be understood that, if reference is made to the compound of formula (I), this reference also includes any of the compounds of formulae (II), (IIa), (IIb), (IIc), (IId), (III), (IV) etc.

In the present invention, the sign custom-character represents a double bond or a single bond. In some examples, the sign represents a single bond. In other examples, the sign represents a double bond.

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- It is preferred that L be

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In preferred compounds of formula (I), R¹and R²are independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; wherein R²is different from —OH.

In preferred compounds of formula (I), R¹is selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. In more preferred compounds of formula (I), R¹is selected from cycloalkyl, heterocycloalkyl, aryl and heteroaryl; wherein said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. In even more preferred compounds of formula (I), R¹is selected from aryl and heteroaryl; wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. In still more preferred compounds of formula (I), R¹is selected from aryl and heteroaryl; wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. In still more preferred compounds of formula (I), R¹is aryl which is optionally substituted with one or more groups R^c. In one compound of formula (I), R¹is aryl which is optionally substituted with one, two or three groups independently selected from —OH, —O—R^dand —O—C_1-4alkyl. Still more preferably, R¹is phenyl, optionally substituted with one, two or three groups independently selected from —OH, —O—R^dand —O—C_1-4alkyl.

In other preferred compounds of formula (I), R²is selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c, and wherein R²is different from —OH. In more preferred compounds of formula (I), R²is selected from cycloalkyl, heterocycloalkyl, aryl and heteroaryl; wherein said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. In even more preferred compounds of formula (I), R²is selected from aryl and heteroaryl; wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. In still more preferred compounds of formula (I), R²is selected from aryl and heteroaryl; wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. Still more preferably, R²is aryl which is optionally substituted with one or more groups R^c. In some compounds of formula (I), R²is aryl which is optionally substituted with one, two or three groups independently selected from —OH, —O—R^dand —O—C_1-4alkyl. Still more preferably, R²is phenyl, optionally substituted with one, two or three groups independently selected from —OH, —O—R^dand —O—C_1-4alkyl.

Alternatively, R¹and R²are joined together to form, together with the carbon atom(s) that they are attached to, a carbocyclic or heterocyclic ring being optionally substituted with one or more substituents R^e; wherein each R^eis independently selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c.

Preferably, each R^eis independently selected from C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^band —R^a—OR^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. More preferably, each R^eis independently selected from C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—OR^band —R^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. Even more preferably, each R^eis independently selected from C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, —R^a—OR^band —R^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl and said heterocycloalkyl are each optionally substituted with one or more groups R^c. Still more preferably, each R^eis independently selected from C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, —OR^band —OR^d; wherein said alkyl, said alkenyl, said heteroalkyl and said heterocycloalkyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN —OH and —O—R^d. Still more preferably, each R^eis independently selected from —OH, —O—C_1-5alkyl, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl and —OR^d; wherein said alkyl, said alkenyl, said heteroalkyl, said heterocycloalkyl and the alkyl in said —O—C_1-5alkyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN —OH and —O—R^d. Still more preferably, each R^eis independently selected from —OH, —O—R^d, C_1-5alkyl, C_2-5alkenyl and —O—C_1-5alkyl; wherein said alkyl, said alkenyl, and the alkyl in said —O—C_1-5alkyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN —OH and —O—R^d. Most preferably, each R^eis independently selected from —OH, —O—R^d, —O—C_1-5alkyl and C_2-5alkenyl wherein the alkyl in said —O—C_1-5alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d.

R⁴, R⁵and R⁶can independently be selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c.

Alternatively, R⁴is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; and R⁵and R⁶are joined together to form, together with the carbon atoms that they are attached to, a carbocyclic or heterocyclic ring being optionally substituted with one or more substituents R^c.

In a further alternative, R⁴and R⁵are joined together to form, together with the carbon atoms that they are attached to, a carbocyclic or heterocyclic ring being optionally substituted with one or more substituents R^c; and R⁶is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c.

R⁴is preferably selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^band —R^a—OR^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. More preferably, R⁴is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—OR^band —R^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. Even more preferably, R⁴is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, —R^a—OR^band —R^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl and said heterocycloalkyl are each optionally substituted with one or more groups R^c. Still more preferably, R⁴is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, —OR^band —OR^d; wherein said alkyl, said alkenyl, said heteroalkyl and said heterocycloalkyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN —OH and —O—R^d. Still more preferably, R⁴is selected from hydrogen, —OH, —O—C_1-5alkyl, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl and —OR^d; wherein said alkyl, said alkenyl, said heteroalkyl, said heterocycloalkyl and the alkyl in said —O—C_1-5alkyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN —OH and —O—R^d. Still more preferably, R⁴is selected from hydrogen, —OH, —O—R^d, C_1-5alkyl, C_2-5alkenyl and —O—C_1-5alkyl; wherein said alkyl, said alkenyl, and the alkyl in said —O—C_1-5alkyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN —OH and —O—R^d. Most preferably, R⁴is selected from hydrogen, —OH, —O—R^d, —O—C_1-5alkyl and C_2-5alkenyl wherein the alkyl in said —O—C_1-5alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d.

R⁵is preferably selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^band —R^a—OR^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. More preferably, R⁵is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—OR^band —R^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. Even more preferably, R⁵is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, —R^a—OR^band —R^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl and said heterocycloalkyl are each optionally substituted with one or more groups R^c. Still more preferably, R⁵is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, —R^a—OR^band —R^a—OR^d; wherein said alkyl and said alkenyl are each optionally substituted with one or more groups R^c. Still more preferably, R⁵is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, —OR^band —OR^d; wherein said alkyl and said alkenyl are each optionally substituted with one or more groups R^c. Still more preferably, R⁵is selected from hydrogen, —OH, —O—R^d, C_1-5alkyl, C_2-5alkenyl, —O—C_1-5alkyl and —O-aryl; wherein said alkyl, said alkenyl, the alkyl in said —O—C_1-5alkyl and the aryl in said —O-aryl are each optionally substituted with one or more groups R^c; Most preferably, R⁵is selected from hydrogen, —OH, —O—R^d, —O—C_1-5alkyl and C_2-5alkenyl, wherein the alkyl in said —O—C_1-5alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d;

R⁶is preferably selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^band —R^a—OR^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. More preferably, R⁶is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—OR^band —R^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. Even more preferably, R⁶is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heteroalkyl, heterocycloalkyl, —R^a—OR^band —R^a—OR^d; wherein said alkyl, said alkenyl, said heteroalkyl and said heterocycloalkyl are each optionally substituted with one or more groups R^c. Still more preferably, R⁶is selected from hydrogen, —OH, C_1-5alkyl, C_2-5alkenyl, heterocycloalkyl and —R^a—OR^d; wherein said alkyl, said alkenyl and said heterocycloalkyl are each optionally substituted with one or more groups R^c. Still more preferably, R⁶is selected from hydrogen, —OH, C_1-5alkyl, C_2-5alkenyl and —R^a—OR^d; wherein said alkyl and said alkenyl and said heterocycloalkyl are each optionally substituted with one or more groups R^c. Still more preferably, R⁶is selected from hydrogen, —OH, —O—R^d, C_1-5alkyl and C_2-5alkenyl, wherein said alkyl and said alkenyl are each optionally substituted with one or more groups R^c. Still more preferably, R⁶is selected from hydrogen, —OH, —O—R^d, —C_1-5alkyl and C_2-5alkenyl, wherein said alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN —OH and —O—R^d. Most preferably, R⁶is selected from hydrogen, —OH, —O—R^d, —C_1-5alkyl and C_2-5alkenyl, wherein said alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d;

In all compounds of the present invention, each R³is —O-(rhamnosyl), i.e. the residue to be rhamnosylated by the methods of the present invention, wherein said rhamnosyl is optionally substituted at one or more of its —OH groups with one or more groups independently selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, a monosaccharide, a disaccharide and an oligosaccharide. The rhamnosyl group in —O—R³may be attached to the —O— group via any position. Preferably, the rhamnosyl group is attached to the —O— group via position C1. The optional substituents may be attached to the rhamnosyl group at any of the remaining hydroxyl groups.

In preferred embodiments of the present invention, R³is —O-α-L-rhamnopyranosyl, —O-α-D-rhamnopyranosyl, —O-β-L-rhamnopyranosyl or —O-β-D-rhamnopyranosyl.

In the present invention, each R^ais independently selected from a single bond, C_1-5alkylene, C_2-5alkenylene, arylene and heteroarylene; wherein said alkylene, said alkenylene, said arylene and said heteroarylene are each optionally substituted with one or more groups R^c. Preferably, each R^ais independently selected from a single bond, C_1-5alkylene and C_2-5alkenylene; wherein said alkylene and said alkenylene are each optionally substituted with one or more groups R^c. More preferably, each R^ais independently selected from a single bond, C_1-5alkylene and C_2-5alkenylene; wherein said alkylene and said alkenylene are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH and —O—C_1-4alkyl. Even more preferably, each R^ais independently selected from a single bond, C_1-5alkylene and C_2-5alkenylene; wherein said alkylene and said alkenylene are each optionally substituted with one or more groups independently selected from —OH and —O—C_1-4alkyl. Still more preferably, each R^ais independently selected from a single bond and C_1-5alkylene; wherein said alkylene is optionally substituted with one or more groups independently selected from —OH and —O—C_1-4alkyl. Most preferably, each R^ais independently selected from a single bond and C_1-5alkylene.

In the present invention, each R^bis independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. Preferably, each R^bis independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl; wherein said alkyl, said alkenyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. More preferably, each R^bis independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heterocycloalkyl, aryl and heteroaryl; wherein said alkyl, said alkenyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c. Even more preferably, each R^bis independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heterocycloalkyl, aryl and heteroaryl; wherein said alkyl, said alkenyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^cStill more preferably, each R^bis independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, heterocycloalkyl, aryl and heteroaryl; wherein said alkyl, said alkenyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH and —O—C_1-4alkyl. Still more preferably, each R^bis independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl and aryl; wherein said alkyl, said alkenyl and said aryl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH and —O—C_1-4alkyl. Still more preferably, each R^bis independently selected from hydrogen, C_1-5alkyl and aryl; wherein said alkyl and said aryl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH and —O—C_1-4alkyl. Still more preferably, each R^bis independently selected from hydrogen and C_1-5alkyl; wherein said alkyl is optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH and —O—C_1-4alkyl. Most preferably, each R^bis independently selected from hydrogen and C_1-5alkyl; wherein said alkyl is optionally substituted with one or more groups independently selected from halogen.

In the present invention, each R^cis independently selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-S-aryl, —(C_0-3alkylene)-S(C_1-5alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, —(C_0-3alkylene)-CHO, —(C_0-3alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl), and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl, said alkenyl, said alkynyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R^care each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl.

Preferably, each R^cis independently selected from C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, —(C_0-3alkylene)-CHO, —(C_0-3alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl) and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl, said alkenyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R^care each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl.

More preferably, each R^cis independently selected from C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^dand —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl); wherein said alkyl, said alkenyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R^care each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl.

Even more preferably, each R^cis independently selected from C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH and —(C_0-3alkylene)-O—R^d; wherein said alkyl, said alkenyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R^care each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^dand —O—C_1-4alkyl.

Still more preferably, each R^cis independently selected from C_1-5alkyl and C_2-5alkenyl; wherein said alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^dand —O—C_1-4alkyl.

In the present invention, each R^dis independently selected from a monosaccharide, a disaccharide and an oligosaccharide.

R^dmay, e.g., be independently selected from arabinosidyl, galactosidyl, galacturonidyl, mannosidyl, glucosidyl, rhamnosidyl, apiosidyl, allosidyl, glucuronidyl, N-acetyl-glucosamidyl, N-acetyl-mannosidyl, fucosidyl, fucosaminyl, 6-deoxytalosidyl, olivosidyl, rhodinosidyl, and xylosidyl.

Specific examples of R^dinclude disaccharides such as maltoside, isomaltoside, lactoside, melibioside, nigeroside, rutinoside, neohesperidoside glucose(1→3)rhamnoside, glucose(1→4)rhamnoside, and galactose(1→2)rhamnoside.

Specific examples of R^dfurther include oligosaccharides as maltodextrins (maltotrioside, maltotetraoside, maltopentaoside, maltohexaoside, maltoseptaoside, maltooctaoside), galacto-oligosaccharides, and fructo-oligosaccharides.

In some of the compound of the present invention, each R^dis independently selected from arabinosidyl, galactosidyl, galacturonidyl, mannosidyl, glucosidyl, rhamnosidyl, apiosidyl, allosidyl, glucuronidyl, N-acetyl-glucosaminyl, N-acetyl-mannosaminyl, fucosidyl, fucosaminyl, 6-deoxytalosidyl, olivosidyl, rhodinosidyl, and xylosidyl.

The compound of formula (I) may contain at least one OH group in addition to any OH groups in R³, preferably an OH group directly linked to a carbon atom being linked to a neighboring carbon or nitrogen atom via a double bond. Examples of such OH groups include OH groups which are directly attached to aromatic moieties, such as, aryl or heteroaryl groups. One specific example is a phenolic OH group.

Procedures for introducing additional monosaccharides, disaccharides or oligosaccharides at R³, in addition to the rhamnosyl residue, are known in the literature. Examples therefore include the use of cyclodextrin-glucanotranferases (CGTs) and glucansucrases (such as described in EP 1867729 A1) for transfer of glucoside residues at positions C4″-OH and C3″-OH (Shimoda and Hamada 2010, Nutrients 2:171-180, doi:10.3390/nu2020171, Park 2006, Biosci Biotechnol Biochem, 70(4):940-948, Akiyama et al. 2000, Biosci Biotechnol Biochem 64(10): 2246-2249, Kim et al. 2012, Enzyme Microb Technol 50:50-56).

A first preferred example of the compound of formula (I), i.e. a preferred example of a compound to be used as starting material in the methods of the present invention, is a compound of formula (II) or a solvate thereof:

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Many examples of the compound of following formula (II) are disclosed herein, such as, compounds of formulae (IIa), (IIb), (IIc) and (IId). It is to be understood that, if reference is made to the compound of formula (II), this reference also includes any of the compounds of formulae (IIa), (IIb), (IIc), (IId), etc.

In formula (II), R¹, R², R³, R⁴, R⁵and R⁶are as defined with respect to the compound of general formula (I) including the preferred definitions of each of these residues.

In a first proviso concerning the compound of formula (II), the compounds naringenin-5-O-α-L-rhamnopyranoside and eriodictyol-5-O-α-L-rhamnopyranoside are preferably excluded. In a second proviso, R¹in the compound of formula (II) is preferably not methyl if R⁴is hydrogen, R⁵is —OH and custom-character is a double bond.

In preferred compounds of formula (II), R¹is selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^bR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂—NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; and R²is selected from hydrogen, C_1-5alkyl and C_2-5alkenyl. In more preferred compounds of formula (II), R¹is selected from cycloalkyl, heterocycloalkyl, aryl and heteroaryl; wherein said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; and R²is selected from hydrogen and C_1-5alkyl. In even more preferred compounds of formula (II), R¹is selected from aryl and heteroaryl; wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; and R²is selected from hydrogen and C_1-5alkyl. In still more preferred compounds of formula (II), R¹is selected from aryl and heteroaryl; wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; and R²is selected from hydrogen and C_1-5alkyl. Still more preferably, R¹is aryl which is optionally substituted with one or more groups R^c, and R²is —H. In some compounds of formula (II), R¹is aryl which is optionally substituted with one, two or three groups independently selected from —OH, —O—R^dand —O—C_1-4alkyl, and R²is —H. Still more preferably, R¹is phenyl, optionally substituted with one, two or three groups independently selected from —OH, —O—R^dand —O—C_1-4alkyl; and R²is —H.

In alternatively preferred compounds of formula (II), R²is selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —R^a—R^b, —R^a—OR^b, —R^a—OR^d, —R^a—OR^a—OR^b, —R^a—OR^a—OR^d, —R^a—SR^b, —R^a—SR^a—SR^b, —R^a—NR^b, —R^a-halogen, —R^a—(C_1-5haloalkyl), —R^a—CN, —R^a—CO—R^b, —R^a—CO—O—R^b, —R^a—O—CO—R^b, —R^a—CO—NR^bR^b, —R^a—NR^b—CO—R^b, —R^a—SO₂NR^bR^band —R^a—NR^b—SO₂—R^b; wherein said alkyl, said alkenyl, said alkynyl, said heteroalkyl, said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; wherein R²is different from —OH; and R¹is selected from hydrogen, C_1-5alkyl and C_2-5alkenyl. In more preferred compounds of formula (II), R²is selected from cycloalkyl, heterocycloalkyl, aryl and heteroaryl; wherein said cycloalkyl, said heterocycloalkyl, said aryl and said heteroaryl are each optionally substituted with one or more groups R^e; and R¹is selected from hydrogen and C_1-5alkyl. In even more preferred compounds of formula (II), R²is selected from aryl and heteroaryl; wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; and R¹is selected from hydrogen and C_1-5alkyl. In still more preferred compounds of formula (II), R²is selected from aryl and heteroaryl; wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R^c; and R¹is selected from hydrogen and C_1-5alkyl. Still more preferably, R²is aryl which is optionally substituted with one or more groups R^c, and R¹is —H. In some of the compounds of formula (II), R²is aryl which is optionally substituted with one, two or three groups independently selected from —OH, —O—R^dand —O—C_1-4alkyl, and R¹is —H. Still more preferably, R²is phenyl, optionally substituted with one, two or three groups independently selected from —OH, —O—R^dand —O—C_1-4alkyl; and R¹is —H.

each R^ccan preferably independently be selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl, —O-aryl, —S—C_1-4alkyl and —S-aryl.

In preferred compounds of formula (II) each R^dis independently selected from arabinosidyl, galactosidyl, galacturonidyl, mannosidyl, glucosidyl, rhamnosidyl, apiosidyl, allosidyl, glucuronidyl, N-acetyl-glucosamidyl, N-acetyl-mannosidyl, fucosidyl, fucosaminyl, 6-deoxytalosidyl, olivosidyl, rhodinosidyl, and xylosidyl.

The compound of formula (II) may contain at least one OH group in addition to any OH groups in R³, preferably an OH group directly linked to a carbon atom being linked to a neighboring carbon or nitrogen atom via a double bond. Examples of such OH groups include OH groups which are directly attached to aromatic moieties, such as, aryl or heteroaryl groups. One specific example is a phenolic OH group.

R⁴, R⁵and R⁶may each independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^dand —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl).

In some compounds of formula (II), R⁵is —OH, —O—R^dor —O—(C_1-5alkyl). In some compounds of formula (II), R⁴and/or R⁶is/are hydrogen or —OH. Most preferably, R²is H or —(C_2-5alkenyl).

Furthermore, R¹and/or R²may independently be selected from aryl and heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R^c.

A first example of the compound of formula (II) is a compound of the following formula (IIa) or a solvate thereof:

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wherein:

R², R³, R⁴, R⁵and R⁶are as defined with respect to the compound of general formula (I) including the preferred definitions of each of these residues;

each R⁷is independently selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-S-aryl, —(C_0-3alkylene)-S(C_1-5alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, —(C_0-3alkylene)-CHO, —(C_0-3alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl), and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl, said alkenyl, said alkynyl, said aryl and said alkylene and the alkyl or alkylene moieties comprised in any of the aforementioned groups R⁷are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl;

n is an integer of 0 to 5, preferably 1, 2, or 3.

Preferably, each R⁷is independently selected from C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, —(C_0-3alkylene)-CHO, —(C_0-3alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl) and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl, said alkenyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R⁷are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl.

More preferably, each R⁷is independently selected from C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^dand —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl); wherein said alkyl, said alkenyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R⁷are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl.

Even more preferably, each R⁷is independently selected from C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH and —(C_0-3alkylene)-O—R^d; wherein said alkyl, said alkenyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R⁷are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^dand —O—C_1-4alkyl.

The following combination of residues is preferred in compounds of formula (IIa),

R²is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, and —O—C_1-5alkyl; wherein said alkyl, said alkenyl, and the alkyl in said —O—C_1-5alkyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH and —O—R^d;

R⁴is selected from hydrogen, —OH, —O—R^d, C_1-5alkyl, C_2-5alkenyl and —O—C_1-5alkyl; wherein said alkyl, said alkenyl and the alkyl in said —O—C_1-5alkyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH and —O—R^d;

R⁵is selected from hydrogen, —OH, —O—R^d, C_1-5alkyl, C_2-5alkenyl, —O—C_1-5alkyl and —O-aryl; wherein said alkyl, said alkenyl, the alkyl in said —O—C_1-5alkyl and the aryl in said —O-aryl are each optionally substituted with one or more groups R^c;

R⁶is selected from hydrogen, —OH, —O—R^d, C_1-5alkyl and C_2-5alkenyl, wherein said alkyl and said alkenyl are each optionally substituted with one or more groups R^c;

each R^cis independently selected from C_1-5alkyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, —(C_0-3alkylene)-CHO, —(C_0-3alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl), and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl and the alkyl, aryl or alkylene moieties comprised in any of the aforementioned groups R^care each optionally substituted with one or more groups independently selected from halogen, —CF₃, —OH, —O—R^dand —O—C_1-4alkyl; and

n is an integer of 0 to 3.

The following combination of residues is more preferred in compounds of formula (IIa),

R²is selected from hydrogen, C_1-5alkyl and C_2-5alkenyl, wherein said alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d;

R⁴is selected from hydrogen, —OH, —O—R^d, —O—C_1-5alkyl and C_2-5alkenyl wherein the alkyl in said —O—C_1-5alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d;

R⁵is selected from hydrogen, —OH, —O—R^d, —O—C_1-5alkyl and C_2-5alkenyl, wherein the alkyl in said —O—C_1-5alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d;

R⁶is selected from hydrogen, —OH, —O—R^d, —C_1-5alkyl and C_2-5alkenyl, wherein said alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d;

each R⁷is independently selected from C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^dand —(C_0-3alkylene)-O(C_1-5alkyl); wherein the alkyl, alkenyl and alkylene in the group R⁷are each optionally substituted with one or more groups independently selected from halogen, —OH, and —O—R^d; and

n is 0, 1 or 2.

Even more preferably, the compound of formula (IIa), is selected from the following compounds or solvates thereof:

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wherein R³is as defined with respect to the compound of general formula (I).

A second example of the compound of formula (II) is a compound of the following formula (IIb) or a solvate thereof:

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wherein:

R², R³, R⁴, R⁵and R⁶are as defined with respect to the compound of general formula (I) including the preferred definitions of each of these residues;

each R⁷is independently selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-S-aryl, —(C_0-3alkylene)-S(C_1-5alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, —(C_0-3alkylene)-CHO, —(C_0-3alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl), and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl, said alkenyl, said alkynyl, said aryl and said alkylene and the alkyl or alkylene moieties comprised in any of the aforementioned groups R⁷are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl; and

n is an integer of 0 to 5, preferably 1, 2, or 3.

Preferably, each R⁷is independently selected from C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, —(C_0-3alkylene)-CHO, —(C_0-3alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl), and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl, said alkenyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R⁷are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl.

The following combination of residues is preferred in compounds of formula (IIb),

R²is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl and —O—C_1-5alkyl; wherein said alkyl, said alkenyl, and the alkyl in said —O—C_1-5alkyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH and —O—R^d;

R³is as defined with respect to the compound of general formula (I);

R⁴is selected from hydrogen, —OH, —O—R^d, C_1-5alkyl, C_2-5alkenyl and —O—C_1-5alkyl; wherein said alkyl, said alkenyl, and the alkyl in said —O—C_1-5alkyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH and —O—R^d;

R⁶is selected from hydrogen, —OH, —O—R^d, C_1-5alkyl and C_2-5alkenyl; wherein said alkyl and said alkenyl are each optionally substituted with one or more groups R^c;

each R^cis independently selected from C_1-5alkyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^d, alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, —(C_0-3alkylene)-CHO, —(C_0-3alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl), and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl and the alkyl, aryl or alkylene moieties comprised in any of the aforementioned groups R^care each optionally substituted with one or more groups independently selected from halogen, —CF₃, —OH, —O—R^dand —O—C_1-4alkyl; and

n is an integer of 0 to 3.

The following combination of residues is more preferred in compounds of formula (IIb),

R³is as defined with respect to the compound of general formula (I);

R⁴is selected from hydrogen, —OH, —O—R^d, —O—C_1-5alkyl and C_2-5alkenyl, wherein the alkyl in said —O—C_1-5alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d;

R⁵is selected from hydrogen, —OH, —O—R^d, —O—C_1-5alkyl and C_2-5alkenyl, wherein the alkyl in said —O—C_1-5alkyl and said alkylene are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d;

R⁶is selected from hydrogen, —OH, —O—R^d, C_1-5alkyl and C_2-5alkenyl, wherein said alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d;

n is 0, 1 or 2.

Even more preferably, the compound is selected from the following compounds or solvates thereof:

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wherein R³is as defined with respect to the compound of general formula (I).

A third example of the compound of formula (II) is a compound of the following formula (IIc) or a solvate thereof:

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wherein:

R¹, R³, R⁴, R⁵and R⁶are as defined with respect to the compound of general formula (I) including the preferred definitions of each of these residues;

each R⁷is independently selected from C_1-5alkyl, C_2-5alkenyl, C_2-5alkynyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-S-aryl, —(C_0-3alkylene)-S(C_1-5alkylene)-SH, —(C_0-3alkylene)-S(C_1-5alkylene)-S(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, —(C_0-3alkylene)-CHO, —(C_0-3alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl), and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl, said alkenyl, said alkynyl, said aryl and said alkylene and the alkyl or alkylene moieties comprised in any of the aforementioned groups R⁷are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl; and

n is an integer of 0 to 5, preferably 1, 2, or 3.

Preferably, each R⁷is independently selected from C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O-aryl, —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-NH₂, —(C_0-3alkylene)-NH(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-halogen, —(C_0-3alkylene)-(C_1-5haloalkyl), —(C_0-3alkylene)-CN, —(C_0-3alkylene)-CHO, —(C_0-3alkylene)-CO—(C_1-5alkyl), —(C_0-3alkylene)-COOH, —(C_0-3alkylene)-CO—O—(C_1-5alkyl), —(C_0-3alkylene)-O—CO—(C_1-5alkyl), —(C_0-3alkylene)-CO—NH₂, —(C_0-3alkylene)-CO—NH(C_1-5alkyl), —(C_0-3alkylene)-CO—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—CO—(C_1-5alkyl), —(C_0-3alkylene)-N(C_1-5alkyl)-CO—(C_1-5alkyl), —(C_0-3alkylene)-SO₂—NH₂, —(C_0-3alkylene)-SO₂—NH(C_1-5alkyl), —(C_0-3alkylene)-SO₂—N(C_1-5alkyl)(C_1-5alkyl), —(C_0-3alkylene)-NH—SO₂—(C_1-5alkyl), and —(C_0-3alkylene)-N(C_1-5alkyl)-SO₂—(C_1-5alkyl); wherein said alkyl, said alkenyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R⁷are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl and —S—C_1-4alkyl.

Even more preferably, each R⁷is independently selected from C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d; wherein said alkyl, said alkenyl and the alkyl or alkylene moieties comprised in any of the aforementioned groups R⁷are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH, —O—R^dand —O—C_1-4alkyl.

The following combination of residues is preferred in compounds of formula (IIc),

R¹is selected from hydrogen, C_1-5alkyl, C_2-5alkenyl and —O—C_1-5alkyl; wherein said alkyl, said alkenyl, and the alkyl in said —O—C_1-5alkyl are each optionally substituted with one or more groups independently selected from halogen, —CF₃, —CN, —OH and —O—R^d;

R³is as defined with respect to the compound of general formula (I);

R⁶is selected from hydrogen, —OH, —O—R^d, C_1-5alkyl and C_2-5alkenyl, wherein said alkyl and said alkenyl are each optionally substituted with one or more groups R^c;

n is an integer of 0 to 3.

The following combination of residues is more preferred in compounds of formula (IIc),

R¹is selected from hydrogen, C_1-5alkyl and C_2-5alkenyl, wherein said alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d;

R³is as defined with respect to the compound of general formula (I);

each R⁷is independently selected from C_1-5alkyl, C_2-5alkenyl, alkylene)-OH, —(C_0-3alkylene)-O—R^dand —(C_0-3alkylene)-O(C_1-5alkyl); wherein the alkyl, alkenyl and alkylene in the group R⁷are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d; and

n is 0, 1 or 2.

Even more preferred are compounds of formula (IIc), which are is selected from the following compounds or solvates thereof:

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wherein R³is as defined with respect to the compound of general formula (I).

A fourth example of the compound of formula (II) is a compound of the following formula (IId) or a solvate thereof:

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wherein:

R³, R⁴, R⁵, R⁶and R^eare as defined with respect to the compound of general formula (I) including the preferred definitions of each of these residues; and

m is an integer of 0 to 4, preferably 0 to 3, more preferably 1 to 3, even more preferably 1 or 2.

The following combination of residues is preferred in compounds of formula (IId),

R³is as defined with respect to the compound of general formula (I);

R⁶is selected from hydrogen, —OH, —O—R^d, C_1-5alkyl and C_2-5alkenyl, wherein said alkyl and said alkenyl are each optionally substituted with one or more groups R^c;

each R^eis independently selected from —OH, —O—R^d, C_1-5alkyl, C_2-5alkenyl, —O—C_1-5alkyl and —O-aryl; wherein said alkyl, said alkenyl, the alkyl in said —O—C_1-5alkyl and the aryl in said —O-aryl are each optionally substituted with one or more groups R^c; and

m is an integer of 0 to 3.

The following combination of residues is more preferred in compounds of formula (IId),

R³is as defined with respect to the compound of general formula (I);

each R^eis independently selected from —OH, —O—R^d, —O—C_1-5alkyl and C_2-5alkenyl, wherein the alkyl in said —O—C_1-5alkyl and said alkenyl are each optionally substituted with one or more groups independently selected from halogen, —OH and —O—R^d; and

m is 0, 1 or 2.

Even more preferred examples of the compound of formula (IId), are compounds selected from the following compounds or solvates thereof:

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wherein R³is as defined with respect to the compound of general formula (I).

In preferred compounds of formulae (II), (IIa), (IIb), (IIc) and (IId), R³is —O-α-L-rhamnopyranosyl, —O-α-D-rhamnopyranosyl, —O-β-L-rhamnopyranosyl or —O-β-D-rhamnopyranosyl.

A second example of a compound of formula (I) is a compound of formula (III) or a solvate thereof:

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wherein R¹, R², R³, R⁴, R⁵and R⁶are as defined with respect to the compound of general formula (I) including the preferred definitions of each of these residues.

In a preferred example of the compounds of formulae (III), R¹is selected from aryl and heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R^c.

In a preferred example of the compounds of formulae (III), each R^cis independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl, —O-aryl, —S—C_1-4alkyl and —S-aryl.

In a preferred example of the compounds of formulae (III), the compound contains at least one OH group in addition to any OH groups in R³, preferably an OH group directly linked to a carbon atom being linked to a neighboring carbon or nitrogen atom via a double bond.

In a preferred example of the compounds of formulae (III), R⁴, R⁵and R⁶are each independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(CO_—3alkylene)-O(C_1-5alkylene)-O—R^dand —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl).

In a preferred example of the compounds of formulae (III), R⁵is —OH, —O—R^dor —O—(C_1-5alkyl).

In a preferred example of the compounds of formulae (III), R⁴and/or R⁶is/are hydrogen or —OH.

Particular examples of the compound of formula (III) include the following compounds or solvates thereof:

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wherein R³is as defined with respect to the compound of general formula (I).

In a preferred example of the compounds of formula (III), R³is —O-α-L-rhamnopyranosyl, —O-α-D-rhamnopyranosyl, —O-β-L-rhamnopyranosyl or —O-β-D-rhamnopyranosyl.

In a preferred example of the compounds of formula (III), each R^dis independently selected from arabinosidyl, galactosidyl, galacturonidyl, mannosidyl, glucosidyl, rhamnosidyl, apiosidyl, allosidyl, glucuronidyl, N-acetyl-glucosamidyl, N-acetyl-mannosidyl, fucosidyl, fucosaminyl, 6-deoxytalosidyl, olivosidyl, rhodinosidyl, and xylosidyl.

Yet a further example of a compound of formula (I) is a compound of formula (IV) or a solvate thereof:

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wherein R¹, R², R³, R⁴, R⁵, R⁶and R^eare as defined with respect to the compound of general formula (I) including the preferred definitions of each of these residues.

In a preferred example of the compounds of formula (IV), R¹is selected from aryl and heteroaryl, wherein said aryl and said heteroaryl are each optionally substituted with one or more groups R^c.

In a preferred example of the compounds of formula (IV), each R^cis independently selected from halogen, —CF₃, —CN, —OH, —O—R^d, —O—C_1-4alkyl, —O-aryl, —S—C_1-4alkyl and —S-aryl.

In a preferred example of the compounds of formula (IV), the compound contains at least one OH group in addition to any OH groups in R³, preferably an OH group directly linked to a carbon atom being linked to a neighboring carbon or nitrogen atom via a double bond.

In a preferred example of the compounds of formula (IV), R⁴, R⁵and R⁶are each independently selected from hydrogen, C_1-5alkyl, C_2-5alkenyl, —(C_0-3alkylene)-OH, —(C_0-3alkylene)-O—R^d, —(C_0-3alkylene)-O(C_1-5alkyl), —(C_0-3alkylene)-O(C_1-5alkylene)-OH, —(C_0-3alkylene)-O(C_1-5alkylene)-O—R^dand —(C_0-3alkylene)-O(C_1-5alkylene)-O(C_1-5alkyl).

In a preferred example of the compounds of formula (IV), R⁵is —OH, —O—R^dor —O—(C_1-5alkyl).

In a preferred example of the compounds of formula (IV), R⁴and/or R⁶is/are hydrogen or —OH.

Particular examples of the compound of formula (IV) include the following compounds or solvates thereof:

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wherein R³is as defined with respect to the compound of general formula (I).

In a preferred example of the compounds of formula (IV), R³is —O-α-L-rhamnopyranosyl, —O-α-D-rhamnopyranosyl, —O-β-L-rhamnopyranosyl or —O-β-D-rhamnopyranosyl.

In a preferred example of the compounds of formula (IV), each R^dis independently selected from arabinosidyl, galactosidyl, galacturonidyl, mannosidyl, glucosidyl, rhamnosidyl, apiosidyl, allosidyl, glucuronidyl, N-acetyl-glucosamidyl, N-acetyl-mannosidyl, fucosidyl, fucosaminyl, 6-deoxytalosidyl, olivosidyl, rhodinosidyl, and xylosidyl.

The present invention is further described by reference to the following non-limiting figures and examples.

The Figures show:

FIG. 1: Determination of solubility of naringenin-5-O-α-L-rhamnoside (NR1) in water. Defined concentrations of NR1 were 0.22 μm-filtered before injection to HPLC. Soluble concentrations were calculated from peak areas by determined regression curves.

FIG. 2: HPLC-chromatogram of naringenin-5-O-α-L-rhamnoside

FIG. 3: HPLC-chromatogram of naringenin-4′-O-α-L-rhamnoside

FIG. 4: HPLC-chromatogram of prunin (naringenin-7-O-β-D-glucoside)

FIG. 5: HPLC-chromatogram of homoeriodictyol-5-O-α-L-rhamnoside (HEDR1)

FIG. 6: HPLC-chromatogram of HEDR3 (4:1 molar ratio of homoeriodictyol-7-O-α-L-rhamnoside and homoeriodictyol-4′-O-α-L-rhamnoside)

FIG. 7: HPLC-chromatogram of homoeriodictyol-4′-O-β-D-glucoside (HED4′Glc)

FIG. 8: HPLC-chromatogram of hesperetin-5-O-α-L-rhamnoside (HESR1)

FIG. 9: HPLC-chromatogram of hesperetin-3′-O-α-L-rhamnoside (HESR2)

FIG. 10: UV₂₅₄-chromatogram of hesperetin bioconversion 141020, sample injection volume was 1.2 L applied by the pumping system

FIG. 11: ESI-TOF negative mode MS-analysis of fraction 3 from hesperetin bioconversion_141020

FIG. 12: ESI-TOF negative mode MS-analysis of fraction 6 from hesperetin bioconversion_141020

FIG. 13: prepLC UV₂₅₄-chromatogram of PFP-HPLC of fraction 3 bioconversion_141020; the main peak (HESR1) between 3.1 min and 3.5 min was HESR1.

FIG. 14: ESI-TOF negative mode MS-analysis of fraction 3 from 140424_Naringenin-PetC

FIG. 15: ESI-TOF negative mode MS-analysis of fraction 5 from 140424_Naringenin-PetC

FIG. 16: UV-chromatogram of conversion after 24 h in bioreactor unit 1 150603_Naringenin-PetC

FIG. 17: UV₃₃₀chromatogram of an extract from a naringenin biotransformation with PetD

FIG. 18: UV₃₃₀chromatogram of an extract from a naringenin biotransformation with PetC

FIG. 19: UV 210-400 nm absorbance spectra of N5R peaks from figures U1 (middle) and U2 (dark) vs. prunin, the naringenin-7-O-β-D-glucoside (light).

FIG. 20: UV 210-400 nm absorbance spectra of GTF product peak Rf 0.77 (dark) vs. prunin (light).

FIG. 21: UV₃₃₀chromatogram of an extract from a naringenin biotransformation with PetF

FIG. 22: Cytotoxicity of flavonoid-5-O-α-L-rhamnosides on normal human epidermal keratinocytes

FIG. 23: antiinflammatory, protecting, and stimulating activities of flavonoid-5-O-α-L-rhamnosides on normal human epidermal keratinocytes, normal human dermal fibroblasts, and normal human epidermal melanocytes

EXAMPLES

The compounds described in this section are defined by their chemical formulae and their corresponding chemical names. In case of conflict between any chemical formula and the corresponding chemical name indicated herein, the present invention relates to both the compound defined by the chemical formula and the compound defined by the chemical name

Part A: Preparation of 5-O-Rhamnosylated Flavonoids

Example A1—Preparation of Media and Buffers

The methods of the present invention can be used to produce rhamnosylated flavonoids, as will be shown in the appended Examples.

Several growth and biotransformation media were used for the rhmanoslyation of flavonoids. Suitable media thus include: Rich Medium (RM) (Bacto peptone (Difco) 10 g, Yeast extract 5 g, Casamino acids (Difco) 5 g, Meat extract (Difco) 2 g, Malt extract (Difco) 5 g, Glycerol 2 g, MgSO₄×7 H₂O 1 g, Tween 80 0.05 g and H₂O ad 1000 mL at a final pH of about 7.2); Mineral Salt Medium (MSM) (Buffer and mineral salt stock solution were autoclaved. After the solutions had cooled down, 100 mL of each stock solution were joined and 1 mL vitamin and 1 mL trace element stock solution were added. Then sterile water was added to a final volume of 1 L. The stock solutions were: Buffer stock solution (10×) of Na₂HPO₄70 g, KH₂PO₄20 g and H₂O ad 1000 mL; Mineral salt stock solution (10×) of (NH₄)₂SO₄10 g, MgCl₂×6 H₂O 2 g, Ca(NO₃)₂×4 H₂O 1 g and H₂O ad 1000 mL; Trace element stock solution (1000×) of EDTA 500 mg, FeSO₄×7 H₂O 300 mg, CoCl₂×6 H₂O 5 mg, ZnSO₄×7 H₂O 5 mg, MnCl₂×4 H₂O 3 mg, NaMoO₄×2 H₂O 3 mg, NiCl₂×6 H₂O 2 mg, H₃BO₃2 mg, CuCl₂×2 H₂O 1 mg and H₂O ad 200 mL. The solution was sterile filtered. Vitamin stock solution (1000×) of Ca-Pantothenate 10 mg, Cyanocobalamine 10 mg, Nicotinic acid 10 mg, Pyridoxal-HCl 10 mg, Riboflavin 10 mg, Thiamin-HCl 10 mg, Biotin 1 mg, Folic acid 1 mg, p-Amino benzoic acid 1 mg and H₂O ad 100 mL. The solution was sterile filtered); Lysogeny Broth (LB) (Yeast extract 5 g, Peptone 10 g, NaCl 5 g and H₂O ad 1000 mL); Terrific Broth (TB) (casein 12 g, yeast extract 24 g, K₂HPO₄12.5 g, KH₂PO₄2.3 g and H₂O ad 1000 mL at pH 7.2). In some experiments, in particular when the concentration of dissolved oxygen (DO) was above about 50%, nutrients were added to the solution. This was done using a feed solution of Glucose 500 g, MgSO₄10 g, thiamine 1 mg and H₂O ad 1000 mL. In some experiments, in particular when cells expressing glycosyl transferase were harvested prior to starting the production of rhamnosylated flavonoids, cells were resuspended in a buffer solution, in particular phosphate buffer saline (PBS). The solution was prepared using NaCl 150 mM, K₂HPO₄/KH₂PO₄100 mM at a pH of 6.4 to 7.4.

Example A2 Glycosyl Transferases Used for the Production of Rhamnosylated Flavonoids

Several different glycosyl transferases were used in the methods of the present invention to produce rhamnosylated flavonoids. In particular, the glycosyltransferases (GTs) used for flavonoid rhamnoside production were

- 1. GTC, a GT derived metagenomically (AGH18139), preferably having an amino acid sequence as shown in SEQ ID NO:3, encoded by a polynucleotide as shown in SEQ ID NO:4. A codon-optimized sequence for expression in E. coli is shown in SEQ ID NO:27.
- 2. GTD, a GT from Dyadobacter fermentans (WP_015811417), preferably having an amino acid sequence as shown in SEQ ID NO:5, encoded by a polynucleotide as shown in SEQ ID NO:6. A codon-optimized sequence for expression in E. coli is shown in SEQ ID NO:28.
- 3. GTF, a GT from Fibrisoma limi (WP_009280674), preferably having an amino acid sequence as shown in SEQ ID NO:7, encoded by a polynucleotide as shown in SEQ ID NO:8. A codon-optimized sequence for expression in E. coli is shown in SEQ ID NO:29.
- 4. GTS from Segetibacter koreensis (WP_018611930) preferably having an amino acid sequence as shown in SEQ ID NO:9, encoded by a polynucleotide as shown in SEQ ID NO:10. A codon-optimized sequence for expression in E. coli is shown in SEQ ID NO:30.
- 5. Chimera 3 with AAs 1 to 316 of GTD and AAs 324 to 459 of GTC preferably having an amino acid sequence as shown in SEQ ID NO: 58, encoded by a polynucleotide as shown in SEQ ID NO: 59. A codon-optimized sequence for expression in E. coli is shown in SEQ ID NO: 60.
- 6. Chimera 4 with AAs 1 to 268 of GTD and AAs 276 to 459 of GTC preferably having an amino acid sequence as shown in SEQ ID NO: 61, encoded by a polynucleotide as shown in SEQ ID NO: 62. A codon-optimized sequence for expression in E. coli is shown in SEQ ID NO: 63.
- 7. Chimera 1 frameshift with AAs 1 to 234 of GTD and AAs 242 to 443 of GTC preferably having an amino acid sequence as shown in SEQ ID NO: 56, encoded by a polynucleotide as shown in SEQ ID NO: 57.

The GT genes were amplified by PCR using respective primers given in Table A1. Purified PCR products were ligated into TA-cloning vector pDrive (Qiagen, Germany) Chemically competent E. coli DH5α were transformed with ligation reactions by heat shock and positive clones verified by blue/white screening after incubation. GT from Segetibacter koreensis was directly used as codon-optimized nucleotide sequence.

Chimera 3 and chimera 4 were created from the codon-optimized nucleotide sequences from GTD and GTC, while chimera 1 was constructed from the SEQ ID NO:4 and SEQ ID NO:6. Chimera 1 was created according to the ligase cycling reaction method described by Kok (2014) ACS Synth Biol 3(2):97-106. Thus, the two nucleotide sequences of each chimeric fragment were amplified via PCR and were assembled using a single-stranded bridging oligo which is complementary to the ends of neighboring nucleotide parts of both fragments. A thermostable ligase was used to join the nucleotides to generate the full-length sequence of the chimeric enzyme.

Chimera 3 and chimera 4 were constructed according to the AQUA cloning method described by Beyer (2015) PLoS ONE 10(9):e0137652. Therefore, the nucleotide fragments were amplified with complementary regions of 20 to 25 nucleotides, agarose-gel purified, mixed in water, incubated for 1 hour at room temperature and transformed into chemically competent E. coli DH5α. The primers used for the chimera construction are listed in Table A2.

TABLE A1

Primers used for the amplification of the GT genes by PCR

Enzyme

Primer name

Sequence (5′→3′)

GTC

GTC-NdeI-for

CATATGAGTAATTTATTTTCTTCACAAAC

GTC-BamHI-rev

GGATCCTTAGTATATCTTTTCTTCTTC

GTD

GTF_XhoI_for

CTCGAGATGACGAAATACAAAAATGAAT

GTF_BamHI_rev

GGATCCTTAACCGCAAACAACCCGC

GTF

GTL_XhoI_for

CTCGAGATGACAACTAAAAAAATCCTGTT

GTL_BamHI_rev

GGATCCTTAGATTGCTTCTACGGCTT

GTS

GTSopt_pET_fw

GGGAATTCCATATGATGAAATATATCAGCTCCATTCAG

GTSopt_pET_rv

CGGGATCCTTAAACCAGAACTTCGGCCTGATAG

TABLE A2

Primers used for the construction of chimeric enzymes

Enzyme

Primer name

Sequence (5′→3′)

Chimera 1

Bridge_P1_pETGTD

GCGGCCATATCGACGACGACGACAAGCATATGACGAAATAC

AAAAATGAATTAACAGGT

Bridge_P1_GTCpET

GGAAGAAGAAAAGATATACTAAGGATCCGGCTGCT

AACAAAGCCCGAAAGG

Chim_P1_D_Nde_for

CATATGACGAAATACAAAAATGAATT

Chim_P1_D_rev

GCGGTCATACTCAAATGATT

Chim_P1_C_for

AGTGATCTGGGAAAAAATATC

Chim_P1_C_Bam_rev

GGATCCTTAGTATATCTTTTCTTCTTCCT

Chimera 3

GTDopt_pEt_fw

GGGAATTCCATATGATGACCAAATACAAAAATG

Chim3_pET_rv

CGGGATCCTTAGTAAATCTTTTCTTCTTCCTTC

1r-Chim3-opt-o(Chim3-

TGCCCTGAGGAAAGCGCGCACGTAATTC

opt)

2f-Chim3-opt-o(Chim3-

TGCGCGCTTTCCTCAGGGCAACTTAATC

opt)

1f-Assembly-o(Vec)

TGACGATAAGGATCGATGGGGATCCATGACCAAATACAAA

1r-Assembly-o(Vec)

TATGGTACCAGCTGCAGATCTCGAGTTAGTAAATCTTTTCTTC

Chimera 4

GTDopt_pEt_fw

GGGAATTCCATATGATGACCAAATACAAAAATG

Chim3_pET_rv

CGGGATCCTTAGTAAATCTTTTCTTCTTCCTTC

1r-Chim4_GTD-

CGATTTTGCGCCCATATTGTAACAACTTTTGA

o(Chim4_GTC)

2f-Chim4_GTC-

ACAATATGGGCGCAAAATCGTCGTAGTC

o(Chim4_GTD)

1f-Assembly-o(Vec)

TGACGATAAGGATCGATGGGGATCCATGACCAAATACAAA

1r-Assembly-o(Vec)

TATGGTACCAGCTGCAGATCTCGAGTTAGTAAATCTTTTCTTC

To establish expression hosts purified pDrive::GT vectors were incubated with respective endonucleases (Table A1) and the fragments of interest were purified from Agarose after gel electrophoresis. Alternatively, the amplified and purified PCR product was directly incubated with respective endonucleases and purified from agarose gel after electrophoresis. The fragments were ligated into prepared pET19b or pTrcHisA plasmids and competent E. coli Rosetta gami 2 (DE3) were transformed by heat shock. Positive clones were verified after overnight growth by direct colony PCR using T7 promoter primers and the GT gene reverse primers, respectively.

Altogether, seven production strains were established:

PetC

E. coli Rosetta gami 2 (DE3) pET19b::GTC

PetD

E. coli Rosetta gami 2 (DE3) pET19b::GTD

PetF

E. coli Rosetta gami 2 (DE3) pET19b::GTF

PetS

E. coli Rosetta gami 2 (DE3) pET19b::GTS

PetChim1fs

E. coli Rosetta gami 2 (DE3) pET19b::Chimera 1

frameshift

PetChim3

E. coli Rosetta gami 2 (DE3) pET19b::Chimera 3

PetChim4

E. coli Rosetta gami 2 (DE3) pET19b::Chimera 4

Example A3—Production of Rhamnosylated Flavonoids in Biotransformations

Three kinds of whole cell bioconversion (biotransformation) were performed. All cultures were inoculated 1/100 with overnight pre-cultures of the respective strain. Pre-cultures were grown at 37° C. in adequate media and volumes from 5 to 100 mL supplemented with appropriate antibiotics.

1. Analytical Small Scale and Quantitative Shake Flask Cultures

For analytical activity evaluations, 20 mL biotransformations were performed in 100 mL Erlenmeyer flasks while quantitative biotransformations were performed in 500 mL cultures in 3 L Erlenmeyer flasks. Bacterial growth was accomplished in complex media, e.g. LB, TB, and RM, or in M9 supplemented with appropriate antibiotics at 28° C. until an OD₆₀₀of 0.8. Supplementation of 50 or 100 μM Isopropyl-β-D-thiogalactopyranoside (IPTG) induced gene expression overnight (16 h) at 17° C. and 175 rpm shaking. Subsequently, a polyphenolic substrate, e.g. Naringenin, Hesperetin or else, in concentrations of 200-800 μM was added to the culture. Alternatively, the polyphenolic substrate was supplemented directly with the IPTG. A third alternative was to harvest the expression cultures by mild centrifugation (5.000 g, 18° C., 10 min) and suspend in the same volume of PBS, supplied with 1% (w/v) glucose, optionally biotin and/or thiamin, each at 1 mg/L, the appropriate antibiotic and the substrate in above mentioned concentrations. All biotransformation reactions in 3 L shake flasks were incubated at 28° C. up to 48 h at 175 rpm.

2. Quantitative bioreactor (fermenter) cultures

In order of a monitorable process bioconversions were performed in volumes of 0.5 L in a Dasgip fermenter system (Eppendorf, Germany) The whole process was run at 26 to 28° C. and kept at pH 7.0. The dissolved oxygen (DO) was kept at 30% minimum. During growth the DO rises due to carbohydrate consumption. At DO of 50% an additional feed with glucose was started with 1 mL/h following the equation

y=e^0.1x

whereby y represents the added volume (mL) and x the time (h).

For cell growth the bacterial strains were grown in LB, TB, RM or M9 overnight. At OD₆₀₀of 10 to 50 50 μM of IPTG and the polyphenolic substrate (400-1500 μM) were added to the culture. The reaction was run for 24 to 48 h.

All bioconversion reactions were either stopped by cell harvest through centrifugation (13,000 g, 4° C., 20 min) followed by sterile filtration with a 0.22 μM PES membrane (Steritop™, Carl Roth, Germany) Alternatively, cultures were harvested by hollow fibre membrane filtration techniques, e.g. TFF Centramed system (Pall, USA). Supernatants were purified directly or stored short-term at 4° C. (without light).

Qualitative Analyses of Biotransformation Reactions and Products

Biotransformation products were determined by thin layer chromatography (TLC) or by HPLC.

For qualitative TLC analysis, 1 mL culture supernatant was extracted with an equal amount ethyl acetate (EtOAc). After centrifugation (5 min, 3,000 g) the organic phase was transferred into HPLC flat bottom vials and was used for TLC analysis. Samples of 20 μL were applied on 20×10 cm²(HP)TLC silica 60 F₂₅₄plates (Merck KGaA, Darmstadt, Germany) versus 200 pmol of reference flavonoids by the ATS 4 (CAMAG, Switzerland). To avoid carryover of substances, i.e. prevent false positives, samples were spotted with double syringe rinsing in between. The sampled TLC plates were developed in EtOAc/acetic acid/formic acid/water (EtOAc/HAc/HFo/H₂O) 100:11:11:27. After separation the TLC plates were dried in hot air for 1 minute. The chromatograms were read and absorbances of the separated bands were determined densitometrically depending on the absorbance maximum of the educts at 285 to 370 nm (D2) by a TLC Scanner 3 (CAMAG, Switzerland).

Analytical HPLC Conditions

HPLC analytics were performed on a VWR Hitachi LaChrom Elite device equipped with diode array detection.

Column: Agilent Zorbax SB-C18 250×4.6 mm, 5 μM

Flowrate: 1 mL/min

Mobile phases: A: H₂O+0.1% Trifluoro acetic acid (TFA), B: ACN+0.1% TFA 0-5′:5% B, 5-15′: 15% B, 15-25′: 25% B, 25-25′: 35% B, 35-45′: 40%, 45-55′ 100% B, 55-63′: 5% B

Sample injection volume 100-500 μL

MS and MS/MS analyses were obtained on a microOTOF-Q with electrospray ionization (ESI) from Bruker (Bremen, Germany) The ESI source was operated at 4000 V in negative ion mode. Samples were injected by a syringe pump and a flow rate of 200 μL/min.

In order to purify the polyphenolic glycosides two different purification procedures were applied successfully.

1. Extraction and subsequent preparative HPLC

- 1.1 In liquid-liquid extractions bioconversion culture supernatants were extracted twice with half a volume of iso-butanol or EtOAc.
- 1.2 In solid phase extractions (SPE) supernatants were first bound on suitable polymeric matrices, e.g. Amberlite XAD resins or silica based functionalized phases, e.g. C-18, and subsequently eluted with organic solvents, e.g. ACN, methanol (MeOH), EtOAc, dimethyl sulfoxide (DMSO) et al. or with suitable aqueous solutions thereof, respectively.
- Organic solvents were evaporated and the residuum completely dissolved in water-acetonitrile (H₂O-ACN) 80:20. This concentrate was further processed by HPLC as described below.
  
  2. Direct fractionation by preparative HPLC
- Sterile filtered (0.2 μm) biotransformation culture supernatants or pre-concentrated extracts were loaded on adequate RP18 columns (5 μm, 250 mm) and fractionated in a H₂O-ACN gradient under following general conditions:
- System: Agilent 1260 Infinity HPLC system.
- Column: ZORBAX SB-C18 prepHT 250×21.2 mm, 7 μm.
- Flowrate: 20 mL/min
- Mobile Phase: A: Water+0.1 formic acid
  - B: ACN+0.1 formic acid

Gradient:

0-5 min

5-30% B

5-10 min

30% B

10-15 min

35% B

15-20 min

40% B

20-25 min

100% B

- Fractions containing the polyphenolic glycosides were evaporated and/or freeze dried. Second polishing steps were performed with a pentafluor-phenyl (PFP) phase by HPLC to separate double peaks or impurities.

The rhamnose transferring activity was shown with enzymes GTC, GTD, GTF and GTS and the three chimeric enzymes chimera 1 frameshift, chimera 3 and chimera 4 in preparative and analytical biotransformation reactions. The enzymes were functional when expressed in different vector systems. GT-activity could be already determined in cloning systems, e.g. E. coli DH5α transformed with pDrive vector (Qiagen, Germany) carrying GT-genes. E. coli carrying pBluescript II SK+ with inserted GT-genes also was actively glycosylating flavonoids. For preparative scales the production strains PetC, PetD, PetF, PetS, PetChim1fs, PetChim3 and PetChim4 were successfully employed. Products were determined by HPLC, TLC, LC-MS and NMR analyses.

Biotransformation of the Flavanone Hesperetin Using E. coli Rosetta Gami 2 (DE3) pET19b::GTC (PetC)

In a preparative scale reaction hesperetin (3′,5,7-Trihydroxy-4′-methoxyflavanone, 2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, CAS No. 520-33-2) was converted. The biotransformation was performed following general preparative shake flask growth and bioconversion conditions.

The bioconversion of hesperetin (>98%, Cayman, USA) was monitored by HPLC analyses of 500 μL samples taken at start (T=0), 3 h and 24 h reaction at 28° C. The culture supernatant was loaded directly via pump flow to a preparative RP18 column (Agilent, USA). Stepwise elution was performed and seven fractions were collected according to FIG. 10 and table A2.

All seven fractions subsequently were analyzed by HPLC and ESI-Q-TOF MS analyses. MS analyses in negative ion mode revealed fraction 3 and fraction 6 to contain a compound each with the molecular weight of 448 Da corresponding to hesperetin-O-rhamnoside (C₂₂H₂₄O₁₀) (FIGS. 11 and 12 table A2). To further purify the two compounds fractions 3 and 6 were lyophilized and dissolved in 30% ACN.

Final purification was performed by HPLC using a PFP column The second purification occurred on a Hypersil Gold PFP, 250×10 mm, 5 μm purchased from Thermo Fischer Scientific (Langerwehe, Germany) and operated at a flow rate of 6 mL/min (Mobile Phase: A: Water, B: ACN, linear gradient elution (0′-8′:95%-40% A, 8′-13′:100% B)(FIG. 13). Subsequently, ESI-TOF MS analyses of the PFP fractions identified the target compounds designated HESR1 and HESR2 in respective fractions (table A3).

After lyophilization NMR analyses elucidated the molecular structure of HESR1 and HESR2, respectively (Example B-2). HESR1 turned out to be the hesperetin-5-O-α-L-rhamnoside and had a RT of 28.91 min in analytical HPLC conditions. To this point, this compound has ever been isolated nor synthesized before.

TABLE A2

Fractionation of hesperetin bioconversion by prepLC separation

Frac

Volume

BeginTime

EndTime

# #

Well

Location

[μl]

[min]

Description

ESI-MS

1 1

Vial

201

20004.17

3.4999

4.5001 Time

2 1

Vial

202

58004.17

4.9999

7.9001 Time

3 1

Vial

203

17804.17

7.9999

8.8901 Time

HESR1

448

4 1

Vial

204

20791.67

8.9505

9.9901 Time

5 1

Vial

205

39012.50

10.0495

12.0001 Time

6 1

Vial

206

38004.17

12.0999

14.0001 Time

HESR2

448

7 1

Vial

207

40004.17

17.9999

20.0001 Time

TABLE A3

Peak table of PFP-HPLC of fraction 3 hesperetin bioconversion

Width

RT [min]

Type

[min]

Area

Height

Area %

Name

2.030

0.1794

866.4182

75.7586

3.9105

2.507

0.1642

493.0764

43.5284

2.2254

2.686

0.0289

20.4545

9.5811

0.0923

2.772

0.0861

85.4639

15.0938

0.3857

2.939

0.0806

119.9032

23.8914

0.5412

3.264

0.1016

16549.5371

2365.6169

74.6942

HESR1

3.488

0.0977

957.1826

140.0522

4.3201

3.742

0.0932

2007.7089

320.0400

9.0615

4.047

0.0816

74.1437

14.5014

0.3346

4.467

0.1241

190.8758

23.6774

0.8615

5.238

0.1326

121.1730

13.5104

0.5469

5.501

0.1617

315.1474

27.9130

1.4224

6.192

0.1654

43.3605

3.8503

0.1957

10.368

0.4019

296.8163

9.8411

1.3396

12.464

0.1204

15.1287

1.7240

0.0683

Biotransformation of the Flavanone Naringenin Using PetC in a Preparative Shake Flask Culture

Naringenin (4′,5,7-Trihydroxyflavanone, 2,3-dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, CAS No. 67604-48-2) was converted in a preparative scale reaction. The biotransformation was performed following general preparative shake flask growth and bioconversion conditions.

The bioconversion of naringenin (98%, Sigma-Aldrich, Switzerland) was controlled by HPLC analyses of a 500 μL sample after 24 h reaction. The culture supernatant was directly loaded via pump flow to a preparative RP18 column. Stepwise elution was performed and seven fractions were collected according to table A4.

All seven fractions subsequently were analyzed by HPLC and ESI-TOF MS analyses. MS analyses in negative ion mode revealed fraction 3 and fraction 5 to contain a compound each with the molecular weight of 418 Da which is the molecular weight of naringenin-O-rhamnoside (C₂₁H₂₂O₉)(table A4). The two compounds designated NR1 and NR2 were lyophilized. HPLC analysis in analytical conditions revealed RTs of approx. 27.2 min for NR1 and 35.7 min for NR2, respectively. NMR analyses elucidated the molecular structure of NR1 (Example B-3). NR1 was identified to be an enantiomeric 1:1 mixture of S- and R-naringenin-5-O-α-L-rhamnoside (N5R). Since the used precursor also was composed of both enantiomers the structure analysis proved that both isomers were converted by GTC. To our knowledge this is the first report that naringenin-5-O-α-L-rhamnoside has ever been biosynthesized. The compound was isolated from plant material (Shrivastava (1982) Ind J Chem Sect B 21(6):406-407). However, the rare natural occurrence of this scarce flavonoid glycoside has impeded any attempt of an industrial application.

In contrast, the first time bioconversion of naringenin-5-O-α-L-rhamnoside opens the way of a biotechnological production process for this compound. Until now the biotechnological production was only shown for e.g. naringenin-7-O-α-L-xyloside and naringenin-4′-O-β-D-glucoside (Simkhada (2009) Mol. Cells 28:397-401, Werner (2010) Bioprocess Biosyst Eng 33:863-871).

TABLE A4

Fractionation of naringenin bioconversion by prepLC separation

Frac

Volume

BeginTime

EndTime

# #

Well

Location

[μl]

[min]

Description

ESI-MS

1 1

Vial

201

31518.75

4.6963

6.4407 Time

2 1

Vial

202

17328.75

6.5074

7.4634 Time

3 1

Vial

203

34638.75

7.5301

9.4478 Time

NR1

418

4 1

Vial

204

43905.00

9.5130

11.9455 Time

5 1

Vial

205

115995.00

12.0109

18.4484 Time

NR2

418

6 1

Vial

206

71111.25

18.5151

22.4590 Time

7 1

Vial

207

80047.50

22.5242

26.9647 Time

Biotransformation of Naringenin Using E. coli Rosetta Gami 2 (DE3)pET19b::GTC (PetC) in a Monitored Bioreactor System

Next to production of naringenin rhamnosides in shake flask cultures a bioreactor process was successfully established to demonstrate applicability of scale-up under monitored culture parameters.

In a Dasgip fermenter system (Eppendorf, Germany) naringenin was converted in four fermenter units in parallel under conditions stated above.

At an OD₆₀₀of 50 expression in PetC was induced by IPTG while simultaneously supplementation of 0.4 g of naringenin (98% CAS No. 67604-48-2, Sigma-Aldrich, Switzerland) per unit was performed. Thus, the final concentration was 2.94 mM of substrate.

After bioconversion for 24 h the biotransformation was finished and centrifuged. Subsequently, the cell free supernatant was extracted once with an equal volume of iso-butanol by shaking intensively for one minute. Preliminary extraction experiments with defined concentrations of naringenin rhamnosides revealed an average efficiency of 78.67% (table A5).

HPLC analyses of the bioreactor reactions indicated that both products, NR1 (RT 27,28′) and NR2 (RT 35.7′), were built successfully (FIG. 16). ESI-MS analyses verified the molecular mass of 418 Da for both products. Quantitative analysis of the bioconversion products elucidated the reaction yields. Concentration calculations were done from peak areas after determination regression curves of NR1 and NR2 (table A6). NR1 yielded an average product concentration of 393 mg/L, NR2 as the byproduct yielded an average 105 mg/L.

TABLE A5

Extraction of naringenin biotransformation

products from supernatant with iso-butanol

Extraction mit iso-butanol

1 ml/1 mL 1′ shaking

Mean

Loss %

Std Dev.

75.75160033

78.6707143

21.32928571

2.73747541

82.49563254

76.42705533

80.00856895

TABLE A6

HPLC chromatogram peak area and resulting

product concentrations of NR1 and NR2

NR1

NR2

Concentration

Peak area

mg/mL

Peak area

mg/mL

Unit 1 26° C. 24 h

232620332

0.33231476

64179398

0.091684854

Unit 2 28° C. 24 h

192866408

0.27552344

57060698

0.081515283

Unit 3 26° C. 24 h

235176813

0.335966876

61065093

0.087235847

Unit 4 28° C. 24 h

204937318

0.292767597

49803529

0.071147899

Unit 1 26° C. 24 h

232620332

0.422412283

64179398

0.116542547

Unit 2 28° C. 24 h

192866408

0.350223641

57060698

0.103615791

Unit 3 26° C. 24 h

235176813

0.427054564

61065093

0.110887321

Unit 4 28° C. 24 h

204937318

0.372143052

49803529

0.090437591

Average

0.392958385

0.105370812

Biotransformation of Narengenin Using E. coli Rosetta Gami 2 (DE3)pET19b::GTC (PetC), E. coli Rosetta Gami 2 (DE3) pET19b::GTD (PetD), E. coli Rosetta Gami 2 (DE3) pET19b::GTF (PetF), E. coli Rosetta Gami 2 (DE3) pET19b::GTS (PetS), E. coli Rosetta Gami 2 (DE3) pET19b::Chimera 1 Frameshift (PetChim1fs), E. coli Rosetta Gami 2 (DE3)pET19b::Chimera 3 (PetChim3) and E. coli Rosetta Gami 2 (DE3)pET19b::Chimera 4 (PetChim4), Respectively

To determine the regio specificities of GTC, GTD, GTF and GTS as well as the three chimeric enzymes chimera 1 frameshift, chimera 3 and chimera 4 biotransformations were performed in 20 mL cultures analogously to preparative flask culture bioconversions using naringenin as a substrate among others. To purify the formed flavonoid rhamnosides, the supernatant of the biotransformation was loaded on a C₆H₅solid phase extraction (SPE) column. The matrix was washed once with 20% acetonitrile. The flavonoid rhamnosides were eluted with 100% acetonitrile. Analyses of the biotransformations were performed using analytical HPLC and LC-MS. For naringenin biotransformations analyses results of the formed products NR1 and NR2 of each production strain are listed in Table A7 and A8, respectively.

TABLE A7

Formed NR1 products in bioconversions of naringenin

with different production strains

strain

NR1 retention time [min] HPLC

ESI-MS

ESI-MSMS

PetC

27.32

418

272

PetD

27.027

418

272

PetF

26.627

418

272

PetS

26.833

418

272

PetChim1fs

26.673

418

272

PetChim3

26.72

418

272

PetChim4

26.727

418

272

TABLE A8

Formed NR2 products in bioconversions of naringenin

with different production strains

strain

NR2 retention time [min] HPLC

ESI-MS

ESI-MSMS

PetC

35.48

418

272

PetD

35.547

418

272

PetF

35.26

418

272

PetS

35.28

418

272

PetChim1fs

35.080

418

272

PetChim3

35.267

418

272

PetChim4

35.267

418

272

Biotransformation of the Flavanone Homoeriodictyol (HED) Using PetC

In preparative scale HED (5,7-Dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4-chromanone, CAS No. 446-71-9) was glycosylated by PetC. The biotransformation was performed following general preparative shake flask growth and bioconversion conditions.

The bioconversion of HED was monitored by HPLC analyses. The culture supernatant was loaded directly via pump flow to a preparative RP18 column (Agilent, USA). Stepwise elution was performed and nine fractions were collected according to table A5.

All nine fractions subsequently were analyzed by HPLC and ESI-TOF MS analyses. MS analyses of fractions 5 and 8 in negative ion mode showed that both contained a compound with the molecular weight of 448 Da which corresponded to the size of a HED-O-rhamnoside and were designated HEDR1 and HEDR3. MS analysis of fraction 7 (HEDR2) gave a molecular weight of 434 Da. However, ESI MS/MS analyses of all three fractions identified a leaving group of 146 Da suggesting a rhamnosidic residue also in fraction 7.

After HPLC polishing by a (PFP) phase and subsequent lyophilization the molecular structure of HEDR1 was solved by NMR analysis (Example B-1). HEDR1 (RT 28.26 min in analytical HPLC) was identified as the pure compound HED-5-O-α-L-rhamnoside.

TABLE A9

Fractionation of HED bioconversion by prepLC separation

Frac

Volume

BeginTime

EndTime

Description

# #

Well

Location

[μl]

[min]

[compound]

ESI-MS

1 1

Vial

201

22503.75

5.0999

6.3501 Time

2 1

Vial

202

28593.75

6.4115

8.0001 Time

3 1

Vial

203

34927.50

8.0597

10.0001 Time

4 1

Vial

204

20141.25

10.0611

11.1801 Time

5 1

Vial

205

13695.00

11.2392

12.0001 Time

HEDR1

448

6 1

Vial

206

34931.25

12.0594

14.0001 Time

7 1

Vial

207

25203.75

15.5999

17.0001 Time

HEDR2

434

8 1

Vial

208

38246.25

17.0753

19.2001 Time

HEDR3

448

9 1

Vial

209

66603.75

19.2999

23.0001 Time

HED

302

Biotransformation Reactions Using PetC of the Isoflavone Genistein Using PetC

In preparative scale genistein (4′,5,7-Trihydroxyisoflavone, 5,7-dihydroxy-3-(4-hydroxyphenyl)chromen-4-one, CAS No. 446-72-0) was glycosylated in bioconversion reactions using PetC. The biotransformation was performed in PBS following general preparative shake flask growth and bioconversion conditions.

The bioconversion of genistein was monitored by HPLC analyses. The genistein aglycon showed a RT of approx. 41 min. With reaction progress four peaks of reaction products (GR1-4) with RTs of approx. 26 min, 30 min, 34.7 min, and 35.6 min accumulated in the bioconversion (table A10). The reaction was stopped by cell harvest after 40 h and in preparative RP18 HPLC stepwise elution was performed. All fractions were analyzed by HPLC and ESI-Q-TOF MS analyses.

Fractions 3, 4, and 5, respectively, showed the molecular masses of genistein rhamnosides in MS analyses. Fraction 3 consisted of two separated major peaks (RT 26 min and 30 min) Fraction 4 showed a double peak of 34.7 min and 35.6 min, fraction 5 only the latter product peak at RT 35.6 min. Separate MS analyses of the peaks in negative ion mode revealed that all peaks contained compounds with the identical molecular masses of 416 which corresponded to the size of genistein-O-rhamnosides. NMR analysis of GR1 identified genistein-5,7-di-O-α-L-rhamnoside (Example B-9).

Biotransformation of the Isoflavone Biochanin A Using PetC

In preparative scale biochanin A (5,7-dihydroxy-3-(4-methoxyphenyl)chromen-4-one, CAS No. 491-80-5) was glycosylated in bioconversion reactions using PetC. The biotransformation was performed following general preparative shake flask growth and bioconversion conditions. The bioconversion of biochanin A was monitored by HPLC. The biochanin A aglycon showed a RT of approx. 53.7 min With reaction progress three product peaks at approx. 32.5′, 36.6′, and 45.6′ accumulated in the bioconversion (table A10). These were termed BR1, BR2, and BR3, respectively. The reaction was stopped by cell harvest after 24 h through centrifugation (13,000 g, 4° C.). The filtered supernatant was loaded to a preparative RP18 column and fractionated by stepwise elution. All fractions were analyzed by HPLC and ESI-Q-TOF MS analyses.

The PetC product BR1 with a RT of 32.5 min was identified by NMR as the 5,7-di-O-α-L-rhamnoside of biochanin A (Example B-4). NMR analysis of BR2 (RT 36.6′) gave the 5-O-α-L-rhamnoside (example B-5). In accordance to 5-O-α-L-rhamnosides of other flavonoids, e.g. HED-5-O-α-L-rhamnoside, BR2 was the most hydrophilic mono-rhamnoside with a slight retardation compared to HEDR1. Taking into account the higher hydrophobicity of the precursor biochanin A (RT 53.5′) due to less hydroxy groups and its C4′-methoxy function in comparison to a C4′-OH of genistein (RT 41′) the retard of BR2 compared to GR2 could be explained.

Biotransformation of the Flavone Chrysin Using PetC

In preparative scale chrysin (5,7-Dihydroxyflavone, 5,7-Dihydroxy-2-phenyl-4-chromen-4-one, CAS No. 480-40-0) was glycosylated in bioconversion reactions using PetC. The biotransformation was performed following stated preparative shake flask conditions in PBS.

The bioconversion of chrysin was monitored by HPLC analyses. The chrysin aglycon showed a RT of 53.5 min. In PetC bioconversions three reaction product peaks accumulated in the reaction, CR1 at RT 30.6 min, CR2 at RT36.4 min, and CR3 at RT43.4, respectively (table A10). All products were analyzed by HPLC and ESI-Q-TOF MS analyses.

CR1 was further identified by NMR as the 5,7-di-O-α-L-rhamnoside of chrysin (Example B-6) and in NMR analysis CR2 turned out to be the 5-O-α-L-rhamnoside (Example B-7). Like BR2, CR2 was also less hydrophilic than the 5-O-rhamnosides of flavonoids with free OH-groups at ring C, e.g. hesperetin and naringenin, although CR2 was the most hydrophilic mono-rhamnoside of chrysin.

Biotransformation of the Flavone Diosmetin Using PetC

Diosmetin (5,7-Trihydroxy-4′-methoxyflavone, 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl) chromen-4-one, CAS No. 520-34-3) was glycosylated in bioconversion reactions using PetC. The biotransformation was performed as stated before.

The bioconversion of diosmetin was monitored by HPLC. The diosmetin aglycon showed a RT of 41.5 min using the given method. With reaction progress three peaks of putative reaction products at 26.5′ (DR1), 29.1′ (DR2), and 36′ (DR3) accumulated (table A10).

The product DR2 with a RT of 29.1 min was further identified as the 5-O-α-L-rhamnoside of diosmetin (D5R) (Example B-10). DR1 was shown by ESI-MS analysis to be a di-rhamnoside of diosmetin. In accordance with the 5-O-α-L-rhamnosides of other flavonoids, e.g. hesperetin, DR2 had a similar retention in analytical RP18 HPLC-conditions.

Table A10 summarizes all reaction products of PetC biotransformations with the variety of flavonoid precursors tested.

TABLE A10

Compilation of applied precursors and corresponding rhamnosylated products

NMR

Elucidated

Precursor

Products

RT [min]

ESI-MS

(Part B)

Structure

Homoeriodictyol

42.4

302.27

HEDR1

28.1

448.11

B-1

5-O-α-L-rhamnoside

HEDR2

34.6

434.13

HEDR3

Double

448.11

Peak

35.8/36.4

Hesperetin

41.1

302.27

HESdiR

26.3

594.12

—

3′,5-di-O-α-L-rhamnoside

HESR1

28.2

448.15

B-2

5-O-α-L-rhamnoside

HESR

448.15

Naringenin

40.8

272.26

NR1

27.2

418.1

B-3

5-O-α-L-rhamnoside

NR2

25.7

418.1

Biochanin A

53.7

284.26

BR1

32.5

—

B-4

5,7-di-O-α-L-rhamnoside

BR2

36.6

430.15

B-5

5-O-α-L-rhamnoside

BR3

45.6

430.15

—

Chrysin

53.0

254.24

CR1

30.6

—

B-6

5,7-di-O-α-L-rhamnoside

CR2

36.4

400.14

B-7

5-O-α-L-rhamnoside

CR3

43.4

400.14

—

Silibinin

39.8

482.44

SR1

32.5

628.15

B-8

5-O-α-L-rhamnoside

Genistein

40.8

270.24

GR1

25.9

—

B-9

5,7-di-O-α-L-rhamnoside

GR2

30.0

416.15

GR3

34.7

416.15

GR4

35.6

416.15

Diosmetin

41.5

300.26

DR1

26.5

—

Di-O-α-L-rhamnoside

DR2

29.1

446.15

B-10

5-O-α-L-rhamnoside

DR3

36.0

446.15

Part B: NMRanalyses of the Rhamnosylated Flavonoids

The following Examples were prepared according to the procedure described above in Part A.

Example B-1: HED-5-O-α-L-rhamnoside

embedded image

¹H NMR ((600 MHz Methanol-d₄): δ=7.06 (d, J=2.0 Hz, 1H), 7.05 (d, J=2.1 Hz, 1H), 6.91 (dt, J=8.2, 2.1, 0.4 Hz, 1H), 6.90 (ddd, J=8.1, 2.0, 0.6 Hz, 1H), 6.81 (d, J=8.1 Hz, 1H), 6.80 (d, J 8.1 Hz, 1H), 6.32 (d, J=2.3 Hz, 1H), 6.29 (d, J=2.3 Hz, 1H), 6.09 (t, J=2.3 Hz, 2H), 5.44 (d, J=1.9 Hz, 1H), 5.40 (d, J=1.9 Hz, 1H), 5.33 (dd, J=7.7, 2.9 Hz, 1H), 5.31 (dd, J=8.1, 3.0 Hz, 1H), 4.12 (ddd, J 11.2, 3.5, 1.9 Hz, 2H), 4.08 (dd, J=9.5, 3.5 Hz, 1H), 4.05 (dd, J=9.5, 3.5 Hz, 1H), 3.87 (s, 3H), 3.87 (s, 3H), 3.69-3.60 (m, 2H), 3.46 (td, J=9.5, 5.8 Hz, 2H), 3.06-3.02 (m, 1H), 3.02-2.98 (m, 1H), 2.64 (ddd, J=16.6, 15.5, 3.0 Hz, 2H), 1.25 (d, J=6.2 Hz, 3H), 1.23 (d, J=6.3 Hz, 3H).

Example B-2: Hesperetin-5-O-α-L-rhamnoside

embedded image

¹H-NMR (400 MHz, DMSO-d₆): δ=1.10 (3H, d, J=6.26 Hz, CH₃), 2.45 (m, H-3(a), superimposed by DMSO), 2.97 (1H, dd, J=12.5, 16.5 Hz, H3(b)), 3.27 (1H, t, 9.49 Hz, H(b)), 3.48 (m, H(a), superimposed by HDO), 3.76 (3H, s, OCH3), 3.9-3.8 (2H, m, H(c), Hd), 5.31 (1H, d, 1.76 Hz, He), 5.33 (1H, dd, 12.5, 2.83 Hz, H2), 6.03 (1H, d, 2.19 Hz, H6/H8), 6.20 (1H, d, 2.19 Hz, H6/H8), 6.86 (1H, dd, 8.2, 2.0 Hz, H6′), 6.90 (1H, d, 2.0 Hz, H2′), 6.93 (1H, d, 8.2 Hz, H5′)

Example B-3: Naringenin-5-O-α-L-rhamnoside

embedded image

¹H NMR (600 MHz, DMSO-d₆): δ=7.30 (d, J=6.9 Hz, 2H), 7.29 (d, J=6.9 Hz, 2H), 6.79 (d, J=8.6 Hz, 2H), 6.78 (d, J=8.6 Hz, 2H), 6.22 (d, J=2.3 Hz, 1H), 6.20 (d, J=2.2 Hz, 1H), 6.02 (d, J=2.2 Hz, 1H), 6.01 (d, J=2.2 Hz, 1H), 5.38 (dd, J=12.7, 3.1 Hz, 1H), 5.35 (dd, J=13.0, 2.5 Hz, 1H), 5.31 (d, J=1.8 Hz, 1H), 5.27 (d, J=1.9 Hz, 1H), 3.90-3.88 (m, 1H), 3.88-3.85 (m, 1H), 3.85-3.80 (m, 2H), 3.50 (dq, J=9.2, 6.2 Hz, 1H), 3.48 (dq, J=9.1, 6.2 Hz, 1H), 3.29 (t, J=9.8 Hz, 2H), 3.07-2.98 (m, 2H), 2.55-2.48 (m, 2H), 1.12 (d, J=6.2 Hz, 3H), 1.10 (d, J=6.2 Hz, 3H).

¹³C NMR (151 MHz, DMSO-d₆): δ=187.75, 187.71, 164.04, 163.92, 163.80, 158.33, 158.23, 157.48, 157.44, 129.26, 129.24, 129.18, 129.15, 128.07, 128.00, 115.00, 105.19, 105.06, 98.58, 98.44, 97.25, 96.85, 96.77, 96.64, 78.03, 77.97, 71.67, 71.65, 69.98, 69.95, 69.66, 69.64, 44.78, 44.74, 17.80, 17.75.

Example B-4: Biochanin A-5,7-di-O-α-L-rhamnoside

embedded image

¹H NMR (400 MHz DMSO-d₆): δ=8.21 (s, 1H), 7.43 (d, J=8.5 Hz, 2H), 6.97 (d, J=8.6 Hz, 2H), 6.86 (d, J=1.8 Hz, 1H), 6.74 (d, J=1.8 Hz, 1H), 5.53 (d, J=1.6 Hz, 1H), 5.41 (d, J=1.6 Hz, 1H), 5.15 (s, 1H), 5.00 (s, 1H), 4.93 (s, 1H), 4.83 (s, 1H), 4.70 (s, 1H), 3.93 (br, 1H), 3.87 (br, 1H), 3.85 (br, 1H), 3.77 (s, 3H), 3.64 (dd, J=9.3, 3.0 Hz, 1H), 3.54 (dq, J=9.4, 6.4 Hz, 1H), 3.44 (dq, J=9.4, 6.4 Hz, 1H), 3.34 (br, 1H), 1.13 (d, J=6.1 Hz, 3H), 1.09 (d, J=6.1 Hz, 3H)

Example B-5: Biochanin A 5-O-α-L-rhamnoside

embedded image

¹H NMR (400 MHz DMSO-d₆): δ=8.21 (s, 1H), 7.42 (d, J=8.7 Hz, 2H), 6.96 (d, J=8.7 Hz 2H), 6.55 (d, J=1.9 Hz, 1H), 6.48 (d, J=1.9 Hz, 1H), 5.33 (d, J=1.7 Hz, 1H), 5.1-4.1 (br, nH), 3.91 (br, 1H), 3.86 (d, J=9.7, 1H), 3.77 (s, 3H), 3.48 (br, superimposed by impurity, 1H), 3.44 (impurity), 3.3 (superimposed by HDO), 1.10 (d, J=6.2 Hz, 3H)

Example B-6: Chrysin-di-5,7-O-α-L-rhamnoside

embedded image

¹H NMR (400 MHz DMSO-d₆): δ=8.05 (m, 2H), 7.57 (m, 3H), 7.08 (s, 1H), 6.76 (d, J=2.3 Hz, 1H), 6.75 (s, 1H), 5.56 (d, J=1.6 Hz, 1H), 5.42 (d, J=1.6 Hz, 1H), 5.17 (s, 1H), 5.02 (s, 1H), 4.94 (s, 1H), 4.86 (s, 1H), 4.71 (s, 1H), 3.97 (br, 1H), 3.88 (dd, J=9.5, 3.1 Hz, 1H), 3.87 (br, 1H), 3.66 (dd, J=9.3, 3.4 Hz, 1H), 3.56 (dq, J=9.4, 6.2 Hz, 1H), 3.47 (dq, J=9.4, 6.2 Hz, 1H), 3.32 (superimposed by HDO, 2H), 1.14 (d, J=6.2 Hz, 3H), 1.11 (d, J=6.2 Hz, 3H)

Example B-7: Chrysin-5-O-α-L-rhamnoside

embedded image

¹H NMR (400 MHz DMSO-d₆): δ=8.01 (m, 2H), 7.56 (m, 3H), 6.66 (s, 1H), 6.64 (d, J=2.1 Hz, 1H), 6.55 (d, J=2.1 Hz, 1H), 5.33 (d, J=1.5 Hz, 1H), 5.01 (s, 1H), 4.85 (d, J=4.7 Hz, 1H), 4.69 (s, 1H), 3.96 (br, 1H), 3.87 (md, J=8.2 Hz, 1H), 3.54 (dq, J=9.4, 6.2 Hz, 1H), 3.3 (superimposed by HDO), 1.11 (d, J=6.1 Hz, 3H)

Example B-8: Silibinin-5-O-α-L-rhamnoside

embedded image

¹H NMR (400 MHz DMSO-d₆): δ=7.05 (dd, J=5.3, 1.9 Hz, 1H), 7.01 (br, 1H), 6.99 (ddd, J=8.5, 4.4, 1.8 Hz, 1H), 6.96 (dd, J=8.3, 2.3 Hz, 1H), 6.86 (dd, J=8.0, 1.8 Hz, 1H), 6.80 (d, J=8.0 Hz, 1H), 6.25 (d, J=1.9 Hz, 1H), 5.97 (dd, J=2.1, 3.7 Hz, 1H), 5.32 (d, J=1.6 Hz, 1H), 5.01 (d, J=11.2 Hz, 1H), 4.90 (d, J=7.3 Hz, 1H), 4.36 (ddd, J=11.2, 6.5, 4.6 Hz, 1H), 4.16 (ddd, J=7.6, 3.0, 4.6 Hz, 1H), 3.89 (m, 1H), 3.83 (br, 1H), 3.77 (d, J=1.8 Hz, 1H), 3.53 (m, 3H), 3.30 (superimposed by HDO, 3H), 1.13 (d, J=6.2 Hz, 3H)

Example B-9: Genistein-5,7-di-O-α-L-rhamnoside

embedded image

¹H NMR (400 MHz DMSO-d₆): δ=8.16 (s, 1H), 7.31 (d, J=8.4 Hz, 2H), 6.85 (d, J=2.1 Hz, 1H), 6.79 (d, J=8.4 Hz, 2H), 6.73 (d, J=2.1 Hz, 1H), 5.52 (d, J=1.8 Hz, 1H), 5.40 (d, J=1.8 Hz, 1H), 5.14 (d, J=3.8 Hz, 1H), 4.99 (d, J=3.8 Hz, 1H), 4.92 (d, J=5.2 Hz, 1H), 5.83 (d, J=5.2 Hz, 1H), 5.79 (d, J=5.5 Hz, 1H), 4.69 (d, J=5.5 Hz, 1H), 3.93 (br, 1H), 3.87 (br, 1H), 3.85 (br, 1H), 3.64 (br, 1H), 3.44 (m, 2H), 3.2 (superimposed by HDO, 2H), 1.12 (d, J=6.2 Hz, 3H), 1.09 (d, J=6.2 Hz, 3H)

Example B-10: Diosmetin-5-O-α-L-rhamnoside

embedded image

¹H NMR (600 MHz DMSO-d₆): δ=7.45 (dd, J=8.5, 2.3 Hz, 1H), 7.36 (d, J=2.3 Hz, 1H), 7.06 (d, J=8.6 Hz, 1H), 6.61 (d, J=2.3 Hz, 1H), 6.54 (d, J=2.3 Hz, 1H), 6.45 (s, 1H), 5.32 (d, J=1.7 Hz, 1H), 3.96 (dd, J=3.5, 2.0 Hz, 1H), 3.86 (m, 1H), 3.85 (s, 3H), 3.54 (dq, J=9.4, 6.3 Hz, 1H), 3.30 (superimposed by HDO, 1H), 1.11 (d, J=6.2, 3H)

Part C: Solubility

FIG. 1 illustrates the amounts of Naringenin-5-rhamnoside recaptured from a RP18 HPLC-column after loading of a 0.2 μm filtered solution containing defined amounts up to 25 mM of the same. Amounts were calculated from a regression curve. The maximum water solubility of Naringenin-5-rhamnoside approximately is 10 mmol/L, which is equivalent to 4.2 g/L.

The hydrophilicity of molecules is also reflected in the retention times in a reverse phase (RP) chromatography. Hydrophobic molecules have later retention times, which can be used as qualitative determination of their water solubility.

HPLC-chromatography was performed using a VWR Hitachi LaChrom Elite device equipped with diode array detection under the following conditions:

Column: Agilent Zorbax SB-C18 250×4.6 mm, 5 μM, Flow 1 mL/min

Mobile phases: A: H₂O+0.1% Trifluoro acetic acid (TFA);

B: ACN+0.1% TFA

Sample injection volume: 500 μL;

Gradient: 0-5 min: 5% B, 5-15 min: 15% B, 15-25 min: 25% B, 25-25 min: 35% B, 35-45 min: 40%, 45-55 min: 100% B, 55-63 min: 5% B

TABLE B1

contains a summary of the retention times

according to FIGS. 2-9 and Example A-2.

N-5-O-α-L-

N-7-O-β-D-

N-4′-O-α-L-

Order of elution

rhamnoside

glucoside

rhamnoside

Retention time [min]

27.3

30.9

Order of elution

HED-5-O-α-L-

HED-4′-O-β-D-

HEDR3

rhamnoside

glucoside

Retention time [min]

28.3

30.1

35.8

Order of elution

HES-5-O-α-L-

HESR2

HES-7-O-β-D-

rhamnoside

glucoside

Retention time [min]

28.9

Generally, it is well known that glucosides of lipophilic small molecules in comparison to their corresponding rhamnosides are better water soluble, e.g. isoquercitrin (quercetin-3-glucoside) vs. quercitrin (quercetin-3-rhamnosides). Table B1 comprehensively shows the 5-O-α-L-rhamnosides are more soluble than α-L-rhamnosides and β-D-glucosides at other positions of the flavonoid backbone. All the 5-O-α-L-rhamnosides eluted below 30 min in RP18 reverse phase HPLC. In contrast, flavanone glucosides entirely were retained at RTs above 30 min independent of the position at the backbone. In case of HED it was shown that among other positions, here C4′ and C7, the differences concerning the retention times of the α-L-rhamnosides were marginal, whereas the C5 position had a strong effect on it. This was an absolutely unexpected finding.

The apparent differences of the solubility are clearly induced by the attachment site of the sugar at the polyphenolic scaffold. In 4-on-5-hydroxy benzopyrans the OH-group and the keto-function can form a hydrogen bond. This binding is impaired by the substitution of an α-L-rhamnoside at C5 resulting in an optimized solvation shell surrounding the molecule. Further, in aqueous environments the hydrophilic rhamnose residue at the C5 position might shield a larger area of the hydrophobic polyphenol with the effect of a reduced contact to the surrounding water molecules. Another explanation would be that the occupation of the C5 position more effectively forms a molecule with a spatial separation a hydrophilic saccharide part and a hydrophobic polyphenolic part. This would result in emulsifying properties and the formation of micelles. An emulsion therefore enhances the solubility of the involved compound.

Part D: Activity of Rhamnosylated Flavonoids

Example D-1: Cytotoxicity of flavonoid-5-O-α-L-rhamnosides

To determine the cytotoxicity of flavonoid-5-O-α-L-rhamnosides tests were performed versus their aglycon derivatives in cell monolayer cultures. For this purpose concentrations ranging from 1 μM to 250 μM were chosen. The viability of normal human epidermal keratinocytes (NHEK) was twice evaluated by a MTT reduction assay and morphological observation with a microscope. NHEK were grown at 37° C. and 5% CO₂aeration in Keratinocyte-SFM medium supplemented with epidermal growth factor (EGF) at 0.25 ng/mL, pituitary extract (PE) at 25 μg/mL and gentamycin (25 μg/mL) for 24 h and were used at the 3rd passage. For cytotoxicity testing, pre-incubated NHEK were given fresh culture medium containing a specific concentration of test compound and incubated for 24 h. After a medium change at same test concentration cells were incubated a further 24 h until viability was determined. Test results are given in Table B2 and illustrated in FIG. 10.

TABLE B2

Cytotoxicity of flavonoid-5-O-α-L-rhamnosides on normal human epidermal

keratinocytes

[μM] from stock solution at 100 mM in DMSO

Compound

Control

2.5

100

250

Hesperetin

Viability (%)

103

107

101

106

102

106

109

106

105

109

106

100

Mean

100

105

103

106

103

108

106

Morph. obs.

+/−

Hes-5-Rha

Viability (%)

118

103

108

113

103

112

108

102

Mean

100

101

Morph. obs.

Naringenin

Viability (%)

104

105

Mean

100

Morph. obs.

+/−,*

Nar-5-Rha

Viability (%)

101

104

111

100

Mean

100

101

Morph. obs.

+/−

Cytotoxicity measurements on monolayer cultures of NHEK demonstrated a better compatibility of the 5-O-α-L-rhamnosides versus their flavonoid aglycons at elevated concentration. Up to 100 μM no consistent differences were observed (FIG. 10). However, at 250 μM concentration of the aglycons hesperetin and naringenin the viability of NHEK was decreased to about 50% while the mitochondrial activity of NHEK treated with the corresponding 5-O-α-L-rhamnosides was still unaffected compared to lower concentrations. In particular these results were unexpected as the solubility of flavonoid aglycons generally is below 100 μM in aqueous phases while that of glycosidic derivatives is above 250 μM. These data clearly demonstrated that the 5-O-α-L-rhamnosides were less toxic to the normal human keratinocytes.

Example D-2: Anti-Inflammatory Properties

Anti-Inflammatory Potential

NHEK were pre-incubated for 24 h with the test compounds. The medium was replaced with the NHEK culture medium containing the inflammatory inducers (PMA or Poly I:C) and incubated for another 24 hours. Positive and negative controls ran in parallel. At the endpoint the culture supernatants were quantified of secreted IL-8, PGE2 and TNF-α, by means of ELISA.

Anti-Inflammatory Effects of 5-O-Rhamnosides in NHEK Cell Cultures

TABLE B3

Inhibition of 5-O-rhamnosides on Cytokine release in human keratinocytes (NHEK)

% stim.

Compound

Cytokine [pg/mL]

control

Inhibition

Conc.

Stimulation

Type

Mean

p⁽¹⁾

Non-

Control

126

***

100

***

stimulat

157

127

Stimulated

Control

1846

1569

141

100

—

conditions:

1480

PMA - 1 μg/ml

1381

Indomethacin

***

106

***

10⁻⁶M

Dexamethasone

1318

1437

168

—

10⁻⁶M

1556

HESR1

PMA

PGE₂

582

507

107

—

(HES-5-

431

Rha)

IL-8

3242

2843

564

—

100 μM

2445

poly(I:C)

IL-8

2617

2793

250

2970

TNFα

416

423

429

NR1

PMA

PGE₂

851

1271

594

—

(N-5-

1691

Rha)

IL-8

2572

2564

—

100 μM

2555

poly(I:C)

IL-8

3055

3154

140

3253

TNFα

516

Compared to control experiments the 5-O-rhamnosides showed anti-inflammatory activities on human keratinocytes concerning three different inflammation markers IL-8, TNFα, and PGE2 under inflammatory stimuli (PMA, poly(I:C)). Especially, the activity of HESR1 on PGE2 was remarkable with a 74% inhibition. An anti-inflammatory activity is well documented for flavonoid derivatives. And several authors reported their action via COX, NFκB, and MAPK pathways (Biesalski (2007) Curr Opin Clin Nutr Metab Care 10(6):724-728, Santangelo (2007) Ann Ist Super Sanita 43(4): 394-405). However, the exceptional water solubility of flavonoid-5-O-rhamnosides disclosed here allows much higher intracellular concentrations of these compounds than achievable with their rarely soluble aglycon counterparts. The aglycon solubilities are mostly below their effective concentration. Thus, the invention enables higher efficacy for anti-inflammatory purposes.

Among many other regulatory activities TNFα also is a potent inhibitor of hair follicle growth (Lim (2003) Korean J Dermatology 41: 445-450). Thus, TNFα inhibiting compounds contribute to maintain normal healthy hair growth or even stimulate it.

Example D-3: Antioxidative Properties

Antioxidative Effects of 5-O-Rhamnosides in NHEK Cell Cultures

Pre-incubated NHEK were incubated with the test compound for 24 h. Then the specific fluorescence probe for the measurement of hydrogen peroxide (DHR) or lipid peroxides (C₁₁-fluor) was added and incubated for 45 min. Irradiation occurred with in H₂O₂determination UVB at 180 mJ/cm²(+UVA at 2839 mJ/cm²) or UVB at 240 mJ/cm²(+UVA at 3538 mJ/cm²) in lipid peroxide, respectively, using a SOL500 Sun Simulator lamp. After irradiation the cells were post-incubated for 30 min before flow-cytometry analysis.

TABLE B4

Protection of 5-O-rhamnosides against UV-induced H₂O₂stress in NHEK cells

% irradiated

Test

H₂O₂(AU)

control

Protection

compound

Concentration

(DHR GMFI)

Mean

p⁽¹⁾

Non-Irradiated

No DHR

—

8.77

—

condition

probe

Control

311

316.33

100

319

Irradiated

Control

1770

1846.83

209

100

—

conditions:

1307

180 mJ/cm²UVB

2388

(2839 mJ/cm²UVA)

1182

2169

2265

BHA

100 μM

740

776

834

754

Vit. E

50 μM

628

655

650

687

HESR1

100 μM

1046

1152

150

—

1258

NR1

100 μM

2531

2516.5

136

—

−44

2502

TABLE B5

Protection of 5-O-rhamnosides against UV-induced lipid peroxide in NHEK cells

% Irradiated

Test

C11-fluor (AU)

control

Protection

compound

Concentration

GMFI

1/GMFI

Mean

p⁽¹⁾

Non-

No C11-

—

3.1E−01

1.1E−02

—

Irradiated

fluor

3.0E−01

condition

probe

3.3E−01

Control

—

9049

1.1E−04

7.6E−06

***

100

***

10874

9.2E−05

8504

1.2E−04

Irradiated

Control

2273

4.4E−04

4.6E−04

1.2E−05

100

—

conditions:

2072

4.8E−04

240 mJ/cm²

2166

4.6E−04

UVB

BHT

50 μM

3139

3.2E−04

3.3E−04

8.5E−06

***

(3538 mJ/cm²UVA)

3047

3.3E−04

2877

3.5E−04

HESR1

100 μM

1671

6.0E−04

6.4E−04

6.3E−05

—

1455

6.9E−04

NR1

100 μM

2414

4.1E−04

4.3E−04

2.1E−05

—

2255

4.4E−04

An anti-oxidative function of the tested flavonoid-5-O-rhamnosides could be observed for HESR1 on mitochondrially produced hydrogen peroxides species and for NR1 on lipid peroxides, respectively. However, it is argued that these parameters are influenced also by different intracellular metabolites and factors, e.g. gluthation. Hence, interpretation of anti-oxidative response often is difficult to address to a single determinant.

Example D-4: Stimulating Properties of 5-O-rhamnosides

Tests were performed with normal human dermal fibroblast cultures at the 8^thpassage. Cells were grown in DMEM supplemented with glutamine at 2 mM, penicillin at 50 U/mL and streptomycin (50 μg/mL) and 10% of fetal calf serum (FCS) at 37° C. in a 5% CO₂atmosphere.

Stimulation of Flavonoid-5-O-rhamnosides on Syntheses of Procollagen I, Release of VEGF, and Fibronectin Production in NHDF Cells

Fibroblasts were cultured for 24 hours before the cells were incubated with the test compounds for further 72 hours. After the incubation the culture supernatants were collected in order to measure the released quantities of procollagen I, VEGF, and fibronectin by means of ELISA. Reference test compounds were vitamin C (procollagen I), PMA (VEGF), and TGF-β (fibronectin).

TABLE B6

Stimulation of 5-O-rhamnosides on procollagen I synthesis in NHDF cells

Basic data

Normalized data

Treatment

Procollagen I

Compound

Conc.

(ng/ml)

Mean

Control

p⁽¹⁾

Stimulation

p⁽¹⁾

Control

—

1893

1667

122

100

—

1473

1637

Vitamin C

20 μg/ml

4739

5272

323

316

***

216

***

5854

5225

NR1

100 μM

1334

1097

335

—

−34

860

HESR1

100 μM

1929

1968

118

—

2007

TABLE B7

Stimulation of 5-O-rhamnosides on VEGF release in NHDF cells

Basic data

Normalized data

Treatment

VEGF

Mean VEGF

Compound

Conc.

(pg/ml)

Control

(%)

p⁽¹⁾

Stimulation

(%)

p⁽¹⁾

Control

—

100

—

PMA

1 μg/ml

150

148

205

***

105

***

150

143

NR1

100 μM

128

—

HESR1

100 μM

101

—

TABLE B8

Stimulation of 5-O-rhamnosides on fibronectin synthesis in NHDF cells

Basic data

Normalized data

Treatment

Fibronectin

Mean

Compound

Conc.

(ng/ml)

Control

(%)

p⁽¹⁾

Stimulation

(%)

p⁽¹⁾

Control

—

6017

6108

100

—

6281

6027

TGF-β

10 ng/ml

10870

####

181

***

11178

11128

NR1

100 μM

6833

7326

698

120

—

7820

HESR1

100 μM

5843

5853

—

−4

5864

Results demonstrated that flavonoid-5-O-rhamnosides can positively affect extracellular matrix components. HESR1 stimulated procollagen I synthesis in NHDF by about 20% at 100 μM. NR1 at 100 μM had a stimulating effect on fibronectin synthesis with an increase of 20% in NHDF. Both polymers are well known to be important extracellular tissue stabilization factors in human skin. Hence substances promoting collagen synthesis or fibronectin synthesis support a firm skin, reduce wrinkles and diminish skin aging. VEGF release was also stimulated approx. 30% by NR1 indicating angiogenic properties of flavonoid-5-O-rhamnosides. Moderate elevation levels of VEGF are known to positively influence hair and skin nourishment through vascularization and thus promote e.g. hair growth (Yano (2001) J Clin Invest 107:409-417, KR101629503B1). Also, Fibronectin was described to be a promoting factor on human hair growth as stated in US 2011/0123481 A1. Hence, NR1 stimulates hair growth by stimulating the release of VEGF as well as the synthesis of fibronectin in normal human fibroblasts.

Stimulation of Flavonoid-5-O-rhamnosides on MMP-1 Release in UVA-Irradiated NHDF

Human fibroblasts were cultured for 24 hours before the cells were pre-incubated with the test or reference compounds (dexamethasone) for another 24 hours. The medium was replaced by the irradiation medium (EBSS, CaCl₂) 0.264 g/L, MgClSO₄0.2 g/L) containing the test compounds) and cells were irradiated with UVA (15 J/cm²). The irradiation medium was replaced by culture medium including again the test compounds incubated for 48 hours. After incubation the quantity of matrix metallopeptidase 1 (MMP-1) in the culture supernatant was measured using an ELISA kit.

TABLE B10

Stimulation of 5-O-rhamnosides on UV-induced MMP-1 release in NHDF cells

Basic data

Mean

Normalized data

Treatment

MMP-1

Irradiated

Test compound

Conc.

(ng/ml)

control

(%)

p⁽¹⁾

Protection

(%)

p⁽¹⁾

Non-

Control

—

28.1

25.5

1.6

100

Irradiated

26.1

22.5

Irradiated conditions:

Control

—

83.7

71.0

7.1

100

—

15 J/cm²UVA

59.1

70.3

Dexamethasone

10⁻⁷M

2.5

2.9

0.2

***

150

***

3.1

3.2

NR1

100 μM

211.7

240.3

40.3

338

—

−372

268.8

HESR1

100 μM

87.0

82.2

6.8

116

—

−25

77.4

Flavonoid-5-O-rhamnosides showed high activities on MMP-1 levels in NHDF. NR1 caused a dramatic upregulation of MMP-1 biosynthesis nearly 4-fold in UV-irradiated conditions.

MMP-1 also known as interstitial collagenase is responsible for collagen degradation in human tissues. Here, MMP-1 plays important roles in pathogenic arthritic diseases but was also correlated with cancer via metastasis and tumorigenesis (Vincenti (2002) Arthritis Res 4:157-164, Henckels (2013) F₁₀₀₀Research 2:229). Additionally, MMP-1 activity is important in early stages of wound healing (Caley (2015) Adv Wound Care 4: 225-234). Thus, MMP-1 regulating compounds can be useful in novel wound care therapies, especially if they possess anti-inflammatory and VEGF activities as stated above.

NR1 even enables novel therapies against arthritic diseases via novel biological regulatory targets. For example, MMP-1 expression is regulated via global MAPK or NFκB pathways (Vincenti and Brinckerhoff 2002, Arthritis Research 4(3):157-164). Since flavonoid-5-O-rhamnosides are disclosed here to possess anti-inflammatory activities and reduce IL-8, TNFα, and PGE-2 release, pathways that are also regulated by MAPK and NFκB. Thus, one could speculate that MMP-1 stimulation by flavonoid-5-O-rhamnosides is due to another, unknown pathway that might be addressed by novel pharmaceuticals to fight arthritic disease.

Current dermocosmetic concepts to reduce skin wrinkles address the activity of collagenase. Next to collagenase inhibition one contrary concept is to support its activity. In this concept misfolded collagene fibres that solidify wrinkles within the tissue are degraded by the action of collagenases. Simultaneously, new collagene has to be synthesized by the tissue to rebuild skin firmness. In this concept, the disclosed flavonoid-5-O-rhamnosides combine ideal activities as they show stimulating activity of procollagen and MMP-1.

Finally, MMP-1 upregulating flavonoid-5-O-rhamnosides serve as drugs in local therapeutics to fight abnormal collagene syndroms like Dupuytren's contracture.

Example D-5: Modulation of Transcriptional Regulators by Flavonoid-5-O-Rhamnosides

NF-κB Activity in Fibroblasts

NIH3T3-KBF-Luc cells were stably transfected with the KBF-Luc plasmid (Sancho (2003) Mol Pharmacol 63:429-438), which contains three copies of NF-κB binding site (from major histocompatibility complex promoter), fused to a minimal simian virus 40 promoter driving the luciferase gene. Cells (1×10⁴for NIH3T3-KBF-Luc) were seeded the day before the assay on 96-well plate. Then the cells were treated with the test substances for 15 min and then stimulated with 30 ng/ml of TNFα. After 6 h, the cells were washed twice with PBS and lysed in 50 μl lysis buffer containing 25 mM Tris-phosphate (pH 7.8), 8 mM MgCl₂, 1 mM DTT, 1% Triton X-100, and 7% glycerol during 15 min at RT in a horizontal shaker. Luciferase activity was measured using a GloMax 96 microplate luminometer (Promega) following the instructions of the luciferase assay kit (Promega, Madison, Wis., USA). The RLU was calculated and the results expressed as percentage of inhibition of NF-κB activity induced by TNFα (100% activation) (tables B10.1-B10.3). The experiments for each concentration of the test items were done in triplicate wells.

TABLE B10.1

Influence of 5-O-rhamnosides on NF-κB activity in NIH3T3 cells

RLU

RLU 1

RLU 2

RLU 3

MEAN

specific

Activation

Control

38240

38870

34680

37263

TNFα 30 ng/ml

115870

120220

121040

119043

81780

100.0

+30 ng/ml TNFα

HESR1 10 μM

186120

181040

182280

183147

145883

178.4

HESR1 25 μM

218940

216580

213320

216280

179017

218.9

NR1 10 μM

134540

126580

130240

130453

93190

114.0

NR1 25 μM

151080

151840

143870

148930

111667

136.5

Chrysin 10 μM

301630

274240

303950

293273

256010

313.0

Chrysin 25 μM

273410

272580

285980

277323

240060

293.5

TABLE B10.2

Influence of 5-O-rhamnosides on NF-κB activity in NIH3T3 cells

RLU

RLU 1

RLU 2

RLU 3

MEAN

specific

Activation

Control

23060

23330

23700

23363

TNFα 30 ng/ml

144940

156140

160200

153760

130397

100.0

+30 ng/ml TNFα

CR1 10 μM

157870

169000

173010

166627

143263

109.9

CR1 25 μM

175140

183630

183960

180910

157547

120.8

CR2 10 μM

156600

160140

151070

155937

132573

101.7

CR2 25 μM

170390

179220

163490

171033

147670

113.2

Diosmetin 10 μM

398660

411390

412940

407663

384300

294.7

Diosmetin 25 μM

448530

452660

451610

450933

427570

327.9

DR2 10 μM

211150

215320

213260

213243

189880

145.6

DR2 25 μM

245900

241550

234880

240777

217413

166.7

Biochanin A 10 μM

588070

586440

579220

584577

561213

430.4

Biochanin A 25 μM

570360

573190

594510

579353

555990

426.4

BR1 10 μM

259120

247590

229500

245403

222040

170.3

BR1 25 μM

211660

208010

203720

207797

184433

141.4

BR2 10 μM

205410

202640

202940

203663

180300

138.3

BR2 25 μM

237390

235850

235350

236197

212833

163.2

TABLE B10.3

Influence of 5-O-rhamnosides on NF-κB activity in NIH3T3 cells

RLU

RLU 1

RLU 2

RLU 3

MEAN

specific

Activation

Control

32200

33240

33100

32847

TNFα 30 ng/ml

179150

179270

184270

180897

148050

100.0

+30 ng/ml

Silibinin 10 μM

249050

238550

231180

239593

206747

139.6

TNFα

Silibinin 25 μM

212420

210050

200660

207710

174863

118.1

SR1 10 μM

269710

262180

254090

261993

229147

154.8

SR1 25 μM

174940

171280

171730

172650

139803

94.4

It was reported that NF-κB activity is reduced by many flavonoids (Prasad (2010) Planta Med 76: 1044-1063). Chrysin was reported to inhibit NF-κB activity through the inhibition of IκBα phosphorylation (Romier(2008) Brit J Nutr 100: 542-551). However, when NIH3T3-KBF-Luc cells were stimulated with TNFα the activity of NF-κB was generally co-stimulated by flavonoids and their 5-O-rhamnosides at 10 μM and 25 μM, respectively.

STAT3 Activity

HeLa-STAT3-luc cells were stably transfected with the plasmid 4×M67 pTATA TK-Luc. Cells (20×10³cells/ml) were seeded 96-well plate the day before the assay. Then the cells were treated with the test substances for 15 mM and then stimulated with IFN-γ 25 IU/ml. After 6 h, the cells were washed twice with PBS and lysed in 50₁11 lysis buffer containing 25 mM Tris-phosphate (pH 7.8), 8 mM MgCl₂, 1 mM DTT, 1% Triton X-100, and 7% glycerol during 15 mM at RT in a horizontal shaker. Luciferase activity was measured using GloMax 96 microplate luminometer (Promega) following the instructions of the luciferase assay kit (Promega, Madison, Wis., USA). The RLU was calculated and the results were expressed as percentage of inhibition of STAT3 activity induced by IFN-γ (100% activation) (tables B11.1-B11.3). The experiments for each concentration of the test items were done in triplicate wells.

TABLE B11.1

STAT3 activation by flavonoids and their 5-O-rhamnosides in HeLa cells

RLU

RLU 1

RLU 2

RLU 3

MEAN

specific

% Activation

Control

2060

2067

1895

2007

IFNγ 25 U/ml

12482

15099

15993

14525

12517

100

+IFNγ 25 U/ml

HESR1 25 μM

13396

12243

12859

12833

10825

86.48

HESR1 50 μM

14303

13124

11985

13137

11130

88.92

NR1 25 μM

10925

8301

8752

9326

7319

58.47

NR1 50 μM

18272

6426

7599

10766

8758

69.97

Chrysin 25 μM

28031

22367

17504

22634

20627

164.78

Chrysin 50 μM

27912

3531

16304

15916

13908

111.11

C57dR 25 μM

11316

1954

8493

7254

5247

41.92

C57dR 50 μM

9196

2358

6307

5954

3946

31.53

C5R 25 μM

7897

2398

5326

5207

3200

25.56

C5R 50 μM

6897

7665

10507

8356

6349

50.72

Diosmetin 25 μM

16337

14303

17066

15902

13895

111.00

Diosmetin 50 μM

9189

7751

7857

8266

6258

50.00

D5R 25 μM

12137

10269

9275

10560

8553

68.33

D5R 50 μM

13005

10547

10143

11232

9224

73.69

TABLE B11.2

STAT3 activation by flavonoids and their 5-O-rhamnosides in HeLa cells

RLU

RLU 1

RLU 2

RLU 3

MEAN

specific

% Activation

Control

1875

1815

1835

IFNγ 25 U/ml

9659

9851

10116

9875

8040

100

+IFNγ 25 U/ml

Biochanin A 25 μM

9732

9023

8911

9222

7387

91.87

Biochanin A 50 μM

6804

12097

11786

10229

8394

104.40

BR1 25 μM

8162

12819

11157

10713

8878

110.41

BR1 50 μM

12336

11620

12104

12020

10185

126.67

BR2 25 μM

11157

10163

10660

8825

109.76

BR2 50 μM

7983

9023

11110

9372

7537

93.74

Silibinin 25 μMI

12389

11170

11210

11590

9755

121.32

Silibinin 50 μM

12157

11885

10540

11527

9692

120.55

TABLE B11.3

STAT3 activation by flavonoids and their 5-O-rhamnosides in HeLa cells

RLU

% Acti-

RLU 1

RLU 2

RLU 3

MEAN

specific

vation

Control

2312

2233

2173

2239

IFNγ 25 U/ml

11375

10852

11269

11165

9158

100

SR1 25 μM +

9507

11653

10203

10454

8447

92.24

IFNγ 25 U/ml

SR1 50 μM +

10090

11355

10938

10794

8787

95.95

IFNγ 25 U/ml

STAT3 is a transcriptional factor of many genes related to epidermal homeostasis. Its activity has effects on tissue repair and injury healing but also is inhibiting on hair follicle regeneration (Liang (2012) J Neurosci32:10662-10673). STAT3 activity may even promote melanoma and increases expression of genes linked to cancer and metastasis (Cao(2016) Sci. Rep. 6, 21731).

Example D-6: Alteration of Glucose Uptake into Cells by Flavonoid 5-O-Rhamnosides

Determination of Glucose Uptake in Keratinocytes

HaCaT cells (5×10⁴) were seeded in 96-well black plates and incubated for 24 h. Then, medium was removed and the cells cultivated in OptiMEM, labeled with 50 μM 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose and treated with the test substances or the positive control, Rosiglitazone, for 24 h. Medium was removed and the wells were carefully washed with PBS and incubated in PBS (100 μl/well). Finally the fluorescence was measured using the Incucyte FLR software, the data were analyzed by the total green object integrated intensity (GCU×μm2×Well) of the imaging system IncuCyte HD (Essen BioScience). The fluorescence of Rosiglitazone is taken as 100% of glucose uptake, and the glucose uptake was calculated as (% Glucose uptake)=100(T−B)/(R−B), where T (treated) is the fluorescence of test substance-treated cells, B (Basal) is the fluorescence of 2-NBDG cells and P (Positive control) is the fluorescence of cells treated with Rosiglitazone. Results of triplicate measurements are given in tables B12.1 and B12.2.

TABLE B12.1

Influence of flavonoid 5-O-rhamnosides on Glucose uptake in HaCaT cells

RFU

Glucose

Measure 1

Measure 2

Measure 3

Mean

specific

uptake

Control

8945

6910

3086

6314

0.0

2NBDG 50 μM

176818

359765

312467

283017

276703

0.0

+2NBDG 50 μM

Rosiglitazone

776381

707003

1141924

875103

868789

100.0

80 μM

HESR1 25 μM

756943

549324

384251

563506

557192

64.1

HESR1 50 μM

501977

642949

529620

558182

551868

63.5

NR1 25 μM

493970

1160754

649291

768005

761691

87.7

NR1 50 μM

278134

256310

257198

263881

257567

29.6

CR1 25 μM

291406

358114

628963

426161

419847

48.3

CR1 50 μM

619992

595330

174412

463245

456931

52.6

CR2 25 μM

427937

431593

390512

416681

410367

47.2

CR2 50 μM

771478

1100390

923151

931673

925359

106.5

DR2 25 μM

632398

940240

197738

590125

583811

67.2

DR2 50 μM

2958363

1297231

2493030

2249541

2243227

258.2

TABLE B12.2

Influence of flavonoid 5-O-rhamnosides on Glucose uptake in HaCaT cells

RFU

Glucose

Measure 1

Measure 2

Measure 3

Mean

specific

uptake

Control

44637

49871

4750

33086

0.0

2NBDG 50 μM

492141

470496

873235

611957

578871

0.0

+2NBDG 50 μM

Rosiglitazone

923011

1440455

1584421

1315962

1282877

100.0

80 μM

BR1 25 μM

730362

661244

400131

597246

564160

44.0

BR1 50 μM

899548

626443

743535

756509

723423

56.4

BR2 25 μM

998132

1149619

935073

1027608

994522

77.5

BR2 50 μM

1657600

1788604

1619334

1688513

1655427

129.0

SR1 25 μM

579565

3067153

4212718

2619812

2586726

201.6

SR1 50 μM

2064420

3541782

2654102

2753435

2720349

212.1

METHODS FOR THE PRODUCTION OF RHAMNOSYLATED FLAVONOIDS

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