NL2035768A - Method for hydroxylation of bile acids or analogues thereof - Google Patents

Method for hydroxylation of bile acids or analogues thereof Download PDF

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Publication number
NL2035768A
NL2035768A NL2035768A NL2035768A NL2035768A NL 2035768 A NL2035768 A NL 2035768A NL 2035768 A NL2035768 A NL 2035768A NL 2035768 A NL2035768 A NL 2035768A NL 2035768 A NL2035768 A NL 2035768A
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Prior art keywords
formula
compound
peroxide
enzyme
upo
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NL2035768A
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Dutch (nl)
Inventor
I Konovalov Andrey
Martinus Cernelis Harald Van Schie Morten
Sinha Tumkur Srinivasamurthy Vishnu
Barrero Peña Juan-José
Süss Philipp
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Centrient Pharmaceuticals Netherlands B V
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Priority to NL2035768A priority Critical patent/NL2035768A/en
Publication of NL2035768A publication Critical patent/NL2035768A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/06—Hydroxylating
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • C07J41/0061—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives one of the carbon atoms being part of an amide group
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/02—Oxidoreductases acting on a peroxide as acceptor (1.11) with H2O2 as acceptor, one oxygen atom of which is incorporated into the product (1.11.2)
    • C12Y111/02001—Unspecific peroxygenase (1.11.2.1)

Abstract

a method for hydroxylation of bile acids or analogues thereof Aspects of the disclosure provide method for 7B-hydroxylation of Formula I compound or analogue or salt thereof catalyzed by an enzyme in the presence of a peroxide. Additional aspects also relate to method for converting Formula I compound or analogue or salt thereof to Formula 11 compound or analogue or salt thereof in the presence of an enzyme and a peroxide; a corresponding reaction mixture; and the associated use of the enzyme to catalyze 7B-hydroxylation of Formula I compound or analogue or salt thereof

Description

“METHOD FOR HYDROXYLATION OF BILE ACIDS OR ANALOGUES
THEREOF”
FIELD OF THE INVENTION
The present disclosure relates to hydroxylation of bile acids and/or analogues thereof. Particularly provided is the enzymatic production of Formula II compound or analogue or salt thereof by 7f3- hydroxylation of Formula I compound or analogue or salt thereof.
BACKGROUND
Bile acids such as ursodeoxycholic acid (UDCA also known as Ursodiol) and derivatives thereof are powerful medicines used for the treatment of chronic liver disease and gallstones. Existing manufacturing of UDCA is based on animal derived cholic Acid (CA) with multi-step chemo- enzymatic processes. Cholic acid is recovered from bovine gallbladder at time of slaughter. As with any animal based raw materials, there are ethical concems, availability/shortage issue and there 1s also an increased risk regarding the purity of the product. Alternatively, plant based raw materials (e. g., phytosterols) can be used for the synthesis of UDCA. However, it typically requires more chemical steps. A purely chemical process (either from animal or plant-based steroids) is therefore complex, environmentally unfriendly, and potentially costly.
The main challenge is the (regio-, stereo-, and chemo-) selective hydroxylation of the starting steroids. Bile acids such as UDCA or its derivatives could be synthesized from lithocholic acid (LCA) or its derivatives by such a reaction, particularly by 7B-hydroxylation reaction of the LCA molecule. While enzymes can be used to perform such a reaction, the currently known enzymatic processes are complex, costly and resource intensive. Hence, there is a need for efficient, cost- effective, and simpler approaches to produce bile acids such as UDCA or its derivatives.
SUMMARY
Aspects of the present disclosure relate to a method for hydroxylating a compound of Formula I, or analogue or salt thereof, comprising contacting the compound of Formula I or analogue or salt thereof: 1
X “4 R 1 !
RO"
Y
Formula I with an enzyme in the presence of a peroxide, to produce a compound of Formula IT or analogue or salt thereof’ 4
X A R
1 1 1
R10 OH
Y
Formula II wherein, 2
R is independently selected from the group consisting of (CH2)nC=O(O)nR', alkyl, alkenyl,
CH:0H, CHO, (CH2):2CONH(CH:).SO:H, (CH:):CONH(CH:)COOH, -CH=CH-CO:-alkyl, -
CH=CH-CO:-aryl, -CH=CH-CO:H and -CH=CH-alkyl, wherein R' is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and NRa4Rs, wherein R, and Ry, are independently selected from the group consisting of hydrogen, alkyl, aryl, -C(=0)-alkyl, -C(=0)-aryl and (CH;).SOsH;
R; 1s independently selected from the group consisting of hydrogen, alkyl, acyl, mesityl, tosyl, -C(=0)-alkyl, -C(=0)-aryl, -S(=0):-alkyl, -S(=0);-aryl, -S(=0),-OH and -P(=0)-(OR2)2, wherein R; is independently hydrogen or alkyl; nis O0 or an integer ranging from 1 to 5; mis Oorl;
Y is hydrogen;
X 1s hydrogen, hydroxy or oxygen; wherein R, R’, R,, Ry, Ry, R; or X are optionally independently substituted, == represents either a single bond or a double bond; and with the proviso that: both Rings A and B do not simultaneously contain double bond inside the rings; when === is a double bond on the Ring A, Rj is absent; when === is a single bond on the Ring A, then R, 15 present; when = is a double bond inside either the Ring A or Ring B, Y is absent; and when === is a single bond inside both the Rings A and B simultaneously, Y is present.
Further aspects of the present disclosure relate to a method for converting the compound of
Formula I or analogue or salt thereof to the compound of Formula IT or analogue or salt thereof, comprising contacting the compound of Formula I or analogue or salt thereof with an enzyme in the presence of a peroxide, to produce the compound of Formula IT or analogue or salt thereof.
Additional aspects of the present disclosure relate to a method of manufacturing a compound of
Formula Il or analogue or salt thereof, the method comprising contacting the compound of Formula 3
I or analogue or salt thereof with an enzyme in the presence of a peroxide, to produce said compound of Formula TI or analogue or salt thereof.
In some embodiments, said method comprises 7B-hydroxylation of the compound of Formula I or analogue or a salt thereof to produce the compound of Formula II or analogue or salt thereof which has a B-hydroxyl group at position C7.
In some embodiments of the method, the enzyme 1s capable of using peroxide as a co-substrate, and the enzyme 1s a peroxygenase.
In some embodiments of the method, the enzyme is unspecific peroxygenase (UPO).
In some embodiments of the method, the peroxide is hydrogen peroxide (H202).
Insome embodiments, the method comprises converting lithocholic acid (LCA) or taurolithocholic acid (TLCA) into ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDCA) respectively, comprising contacting the LCA or TLCA with an enzyme in the presence of a peroxide, to produce the UDCA or TUDCA.
Insome embodiments, the method comprises converting lithocholic acid (LCA) or taurolithocholic acid (TLCA) into ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDCA) respectively, comprising contacting the LCA or TLCA with unspecific peroxygenase (UPO) in the presence of H2O:, to produce the UDCA or TUDCA.
Further aspects of the disclosure relate to use of an enzyme for: 1) 7B-hydroxylation of a compound of Formula I or analogue salt thereof to the compound of Formula II or analogue or salt thereof, or 11) converting a compound of Formula I or analogue or salt thereof to the compound of Formula
IT or analogue or salt thereof, wherein the enzyme requires peroxide as a co-substrate.
In some embodiments of the use, the enzyme is unspecific peroxygenase (UPO). 4
In some embodiments of the use, the Formula I or analogue or salt thereof is LCA or TLCA; the
Formula II or analogue or salt thereof is UDCA or TUDCA; the enzyme is unspecific peroxygenase (UPO); and the peroxide is hydrogen peroxide (H203).
Further aspects of the disclosure relate to a reaction mixture or reaction system comprising: (1) a compound of Formula I or analogue or salt thereof, (i1) an enzyme, and (111) a peroxide.
BRIEF DESCRIPTION OF FIGURES
Figure 1 depicts a reaction scheme representing one embodiment of the present method including a method for hydroxylation of LCA to produce UDCA.
Figure 2 shows HPLC results demonstrating the production of UDCA from LCA catalyzed by
UPO in accordance with Example 1.
Figure 3 shows HPLC results demonstrating the production of UDCA from LCA catalyzed by
UPO in accordance with Example 2.
Figure 4 shows HPLC results demonstrating the production of TUDCA from TLCA catalyzed by
UPO in accordance with Example 3.
Figure 5 shows HPLC results demonstrating the production of TUDCA from TLCA catalyzed by
UPO in accordance with Example 4.
DESCRIPTION OF THE INVENTION
In view of the limitations discussed above, and to remedy the need in the art for an efficient way to produce bile acids or its derivatives, the present disclosure provides an efficient and viable enzymatic method. Particularly, the present disclosure describes enzymatic production of Formula
IT compound or analogue or salt thereof including bile acids such as ursodeoxycholic acid (UDCA) or analogues or salts thereof. Preferably, the disclosure provides enzymatic production of Formula
II compound or analogue or salt thereof including bile acids such as ursodeoxycholic acid (UDCA) or its analogues or salts by 7B-hydroxylation of Formula I compound or analogue or salt thereof including bile acid precursors such as lithocholic acid (LCA) or its analogues or salts. Additionally provided are corresponding methods for conversion of Formula I compound or analogue or salt thereof to Formula II compound or analogue or salt thereof; a reaction mixture for production of 5
Formula IT compound or analogue or salt thereof; and use of enzyme(s) for production of Formula
IT compound or analogue or salt thereof.
The present methods, the corresponding reaction mixture and associated use of the present disclosure are further described in greater detail in the following embodiments. For the sake of brevity, identical embodiments may not be repeated for each of the different methods, the reaction mixture and the use described herein. However, any combination of an embodiment captured anywhere in this disclosure with any other embodiment captured elsewhere in this disclosure, fall wholly within the ambit of the present disclosure. Such combinations can therefore be considered to derive complete meaning of the aspects described herein.
Method for hydroxylation of Formula I or analogue or salt thereof
The present disclosure provides a method for hydroxylating a compound of Formula I or analogue or salt thereof, comprising contacting the compound of Formula I or analogue or salt thereof: 4
X “, R 1 .
I
I
I
H
> ” > ”
RO
Y
Formula I with an enzyme in the presence of a peroxide, to produce a compound of Formula II or analogue or salt thereof: 6
X “, R
GD
STE
R10% OH
Y
Formula IT wherein,
R is independently selected from the group consisting of (CH:)nC=O(O)nR', alkyl, alkenyl,
CH20H, CHO, (CH:):2CONH(CH:)250;H, (CH2)CONH(CH:)COOH, -CH=CH-CO:-alkyl, -
CH=CH-CO:-aryl, -CH=CH-CO:H and -CH=CH-alkyl, wherein R' is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and NRR, wherein R, and Ry, are independently selected from the group consisting of hydrogen, alkyl, aryl, -C(=0)-alkyl, -C{=O)-aryl and (CH:),SO:H,;
R; is independently selected from the group consisting of hydrogen, alkyl, acyl, mesityl, tosyl, -C(=0)-alkyl, -C(=0)-aryl, -S(=0),-alkyl, -S(=0)-aryl, -S(=0),-OH and -P(=0)-(OR2)2, wherein Rj is independently hydrogen or alkyl; nis O or an integer ranging from 1 to 5; misOorl;
Y is hydrogen;
X is hydrogen, hydroxy or oxygen; wherein R, R’, R,, Ry, Ry, R; or X are optionally independently substituted, === represents either a single bond or a double bond; and with the proviso that: 7 both Rings A and B do not simultaneously contain double bond inside the rings; when === is a double bond on the Ring A, R is absent; when === is a single bond on the Ring A, then R; is present; when === is a double bond inside either the Ring A or Ring B, Y is absent; and when = 1s a single bond inside both the Rings A and B simultaneously, Y is present.
In some embodiments of the method, the alkyl in Formula I or Formula II 1s C1-C2 alkyl, the alkenyl in Formula I or Formula II 1s C2-C2 alkenyl, the alkynyl in Formula I or Formula II is C:-
C12 alkynyl, the cycloalkyl in Formula I or Formula II is a hydrocarbon ring system radical comprising C:-C2 carbon atoms, and the aryl in Formula I or Formula II is a hydrocarbon ring system radical comprising Cs-Cig carbon atoms and at least one aromatic ring.
In some embodiments of the method, the alkyl in Formula I or Formula II is C1-C4 alkyl, the alkenyl in Formula I or Formula II is C+-C4 alkenyl, the alkynyl in Formula I or Formula II is C»-
Cs alkynyl, the cycloalkyl in Formula I or Formula II is a hydrocarbon ring system radical comprising C3-Cgs carbon atoms, and the aryl in Formula I or Formula II is a hydrocarbon ring system radical comprising Cs-C12 carbon atoms and at least one aromatic ring.
In some embodiments of the method, the salt of Formula I or Formula II is selected from the group consisting of sodium salt, potassium salt, and quaternary ammonium salts (QAS). In some embodiments, the QAS is selected from the group consisting of tetramethylammonium, tetraethylammonium, n-tetrabutylammonium, and alkylbenzyldimethylammonium, or any combination thereof.
As used in this disclosure, ‘analogs’, ‘analogues’, ‘derivative’ or ‘derivatives’ are used interchangeably referring to compounds in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group; or a compound that is formed from a similar compound or a compound that can be imagined to arise from another compound, if one atom is replaced with another atom or group of atoms. 8
As used in this disclosure, “7B-hydroxylation” or “hydroxylation at C7 position in beta- orientation” which are used interchangeably, refers to hydroxylating a compound (substrate) at C7 position of the molecule in beta orientation to obtain a compound (product) having hydroxyl group.
Thus, in some embodiments, the product of the 7B-hydroxylation reaction comprises hydroxyl group in beta-orientation at the C7 position.
As used in this disclosure, the numbering of atoms in the organic compounds (such as cyclopentanoperhydrophenanthrene/Sterane compounds or analogues or derivatives or salts thereof) follows the International Union of Pure and Applied Chemistry (IUPAC) numbering system.
In some embodiments, the method comprises 7B-hydroxylation of the compound of Formula I or a salt thereof to produce the compound of Formula II or a salt thereof which has a B-hydroxyl group at position C7.
As used in this disclosure, “enzyme capable of using peroxide” or “enzyme requiring presence of a peroxide” or the likes which are used interchangeably, refer to enzyme(s) which utilize peroxide or peroxide containing compounds as a source of oxygen and electrons for catalyzing an enzymatic reaction. In some embodiments, the present disclosure defines said enzyme(s) by their Enzyme
Commission number (EC number). Such enzyme nomenclature based on EC number that classify the enzymes based on the enzyme-catalyzed reaction(s) is developed by the International Union of Biochemistry and Molecular Biology (IUBMB) and is well known to a person skilled in the art.
In some embodiments of the method, the enzyme is capable of using the compound of Formula I ora salt thereof as a substrate and peroxide as a co-substrate for catalyzing the hydroxylation of the compound of Formula I or a salt thereof to produce the compound of Formula II or a salt thereof.
In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate is an enzyme from subclass EC 1.11 (subclass of oxidoreductases) which acts on peroxide as acceptor 9 molecule. More particularly, the enzyme capable of using peroxide as a co-substrate is an enzyme from EC 1.11: Acting on a peroxide as acceptor.
In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate is a peroxygenase.
In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate is a peroxygenase from sub subclass EC 1.11.2. More particularly, the enzyme capable of using peroxide as a co-substrate is an enzyme from EC 1.11.2: Peroxygenases. In some embodiments, the peroxygenase of EC 1.11.2 comprises unspecific peroxygenase (EC 1.11.21), myeloperoxidase (EC 1.11.2.2), plant seed peroxygenase (EC 1.11.2.3), fatty-acid peroxygenase (EC 1.11.2.4), 3-methyl-L-tyrosine peroxygenase (EC 1.11.2.5), or L-tyrosine peroxygenase (EC 1.11.2.6).
In some embodiments of the method, the peroxygenase is a fungal peroxygenase.
In some embodiments of the method, the peroxygenase is a peroxygenase enzyme from Proto- fungi or pseudo-fungi.
In some embodiments of the method, the enzyme is unspecific peroxy genase (UPO).
In the present disclosure, the enzyme “unspecific peroxygenase” or “UPO” is defined based on the currently known classifications considering criteria such as their source, origin, size, EC number description etc. which is non-exhaustive, and thereby includes any UPO enzyme. In some embodiments, this also includes enzymes from other EC classes which are modified in such a way that they are covered by EC 1.11.2.1. In some embodiments, the present methods encompass any
UPO enzyme which is capable of using peroxide as a co-substrate for catalyzing 7B-hydroxylation of the compound of Formula I or a salt thereof to produce the compound of Formula II or a salt thereof. 10
In some embodiments of the method, the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1 belonging to the sub-subclass of oxidoreductases. More particularly, the enzyme of the present method 1s an enzyme from EC 1.11.2.1: Unspecific peroxy genase.
Insome embodiments of the method, the enzyme is unspecific peroxy genase (UPO) of EC 1.11.2.1 that act with H»O; or peroxide as acceptor molecule, one oxygen atom of which is incorporated into the product.
In some embodiments of the method, the unspecific peroxygenase (UPO) is a wild-type UPO obtained from fungi or proto-fungi, or both.
In some embodiments of the method, the unspecific peroxygenase (UPO) is a wild-type UPO obtained from fungi or proto-fungi.
In some embodiments, the UPO enzyme obtained from fungi can be divided into two families: 1) Family I UPOs — the ‘short’ UPO sequences which include representatives of UPO- containing phylogenetic groups of fungi selected from Ascomycota, Cryptomycota,
Basidiomycota, Chytridiomycota, Mucoromycotina, Oomycetes, Zoopagomycota, and
Peronosporomycetes, 11) Family IT UPOs — the ‘long’ UPO sequences which include representatives of UPO- containing phylogenetic groups of fungi selected from Ascomycota and Basidiomycota.
In some embodiments of the method, the UPO is Family I UPOs having a mean size of around 30 kDa. In some embodiments of the method, the UPO is Family IT UPOs having a mean size of around 44.4 kDa.
In some embodiments of the method, the UPO is a wild-type or native UPO obtained from the fungi selected from the group consisting of Agrocybe sp., Aspergillus sp., Coprinopsis sp.,
Chaetomium sp., Leptoxyphium sp., Coprinellus sp., Collariella sp., Daldinia sp., Humicola sp.,
Hypoxylon sp., Marasmius sp., Myceliophthora sp., Candolleomyces sp., Pestalotiopsis sp.,
Thielavia sp., fungi from the same family or class thereof, and combinations thereof. 11
In some embodiments of the method, the UPO is a wild-type or native UPO obtained from the fungi selected from the group consisting of Agrocybe aegerita, Aspergillus niger, Coprinopsis cinerea, Coprinopsis verticillate, Chaetomium globosum, Leptoxyphium fumago, Coprinellus radians, Collariella virescens, Daldinia caldariorum, Humicola insolens, Hypoxylon sp. EC38,
Marasmius rotula, Myceliophthora wettsteinii, Myceliophthora fergusii, Myceliophthora hinnulea, Candolleomyces aberdarensis, Pestalotiopsis virgatula, Thielavia hyrcaniae, and combinations thereof.
In some embodiments of the method, the UPO is a wild-type or native UPO obtained from the proto-fungi selected from the group consisting of proto-fungi from class Oomycetes (or
Peronosporomycetes), class Hyphochytriomycetes, and a combination thereof.
As used in this disclosure, “wild-type UPO” or “native UPO” or “wild-type or native UPO” which are used interchangeably, refer to an UPO enzyme which is expressed or produced in the native organism (i.e, an organism naturally producing the UPO enzyme). For instance, the UPO gene or polynucleotide is expressed in a native organism (e.g., fungi or proto-fungi) and the produced UPO enzyme is employed in the present methods. In some embodiments, said UPO gene or polynucleotide 1s not modified or mutated before expression in the native organism. In some embodiments, said UPO gene or polynucleotide is modified or mutated before expression in the native organism.
In some embodiments of the method, the unspecific peroxygenase (UPO) is a recombinant UPO enzyme expressed in a host cell. In some embodiments, the host cell is selected from fungi and bacteria.
As used in this disclosure, “fungi” refers to organisms of the Fungi kingdom which includes yeasts, rusts, smuts, mildews, molds, and mushrooms.
In some embodiments of the method, the host cell is yeast. 12
As used in this disclosure, “proto-fungr” and “pseudo-fungi” are used interchangeably and refer to organisms from phylum Oomycota and phylum Hyphochytriomycota.
As used 1n this disclosure, “recombinant UPO” or “recombinant unspecific peroxygenase” which are used interchangeably, refer to an UPO enzyme which is expressed or produced in a non-native organism (also referred as host cell) using recombinant DNA techniques. For instance, the UPO gene or polynucleotide sourced from a native organism (e.g., fungi or proto-fungi) is subjected to heterologous expression in the host cells to produce recombinant UPO enzyme(s). In some embodiments, said UPO gene or polynucleotide is not modified or mutated before expression in host cells. In some embodiments, said UPO gene or polynucleotide is modified or mutated before expression in host cells. In another instance, the UPO gene or polynucleotide is a synthetic or artificially synthesized UPO which is subjected to heterologous expression in the host cells to produce recombinant UPO enzyme(s).
As used in this disclosure, “host cell” refers to a cell or organism that can be used to express heterologous polynucleotide or gene, such as the peroxy genase described herein.
In some embodiments of the method, the UPO is a recombinant UPO expressed in a host cell which is yeast. In some embodiments, the yeast host cell is an industrial strain. Numerous industrial strains of yeast are known and are suitable for expressing or producing the recombinant UPO described herein.
In some embodiments of the method, the UPO is a recombinant UPO expressed in a host cell which is a yeast selected from the group consisting of Pichia sp., Saccharomyces sp.,
Leptoxyphium sp., Agrocybe sp., Candida sp., Hansenula sp., Schizosaccharomyces Sp.
Kliyveromyces sp., and Yarrowia sp..
In some embodiments of the method, the UPO 1s a recombinant UPO expressed 1n a host cell which is a yeast selected from the group consisting of Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans,
Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Saccharomyces cerevisiae, Saccaromyces carlsbergensis, Saccharomyces diastaticus, 13
Saccharomyces norbensis, Saccharomyces kluyveri, Leptoxyphium fumago, Agrocybe aegerita,
Schizosaccharomyces pombe, Hansenula polymorpha, Kluyveromyces lactis, Candida albicans, and Yarrowia lipolytica.
In some embodiments of the method, the UPO is a recombinant UPO expressed in Pichia pastoris.
In some embodiments of the method, the UPO is a recombinant UPO expressed in Pichia pastoris, which is Komagataella pastoris or Komagataella phaffii. In some embodiments of the method, the
UPO is a recombinant UPO expressed in all industrial strains of Pichia pastoris, which is
Komagataella pastoris or Komagataella phaffii.
In some embodiments of the method, the UPO is a recombinant UPO expressed in Saccharomyces cerevisiae. In some embodiments of the method, the UPO is a recombinant UPO expressed in all industrial strains of Saccharomyces cerevisiae.
In some embodiments of the method, the UPO is a recombinant UPO expressed in a fungal host cell. In some embodiments, the fungal host cell is an industrial strain. Numerous fungal industrial strains are known and are suitable for expressing or producing the recombinant UPO described herein.
In some embodiments of the method, the UPO is a recombinant UPO expressed 1n a host cell which is a fungi selected from the group consisting of Aspergillus sp., Penicillium sp., Fusarium sp., Myceliophthora sp., Rhizopus sp., Acremonium sp., Neurospora sp., Sordaria sp.,
Magnaporthe sp., Allomyces sp., Ustilago sp., Botrytis sp., and Trichoderma sp..
In some embodiments of the method, the UPO is a recombinant UPO expressed in a host cell which 1s a fungi selected from the group consisting of Aspergillus niger, Aspergillus oryzae,
Aspergillus nidulans, Fusarium venenatum, Fusarium graminearum and Myceliophthora thermophila.
In some embodiments of the method, the UPO is a recombinant UPO expressed in bacterial host cell. In some embodiments, the bacterial host cell 1s an industrial strain. Numerous bacterial 14 industrial strains are known and are suitable for expressing or producing the recombinant UPO described herein.
In some embodiments of the method, the UPO 1s a recombinant UPO expressed in bacteria selected from the group consisting of Escherichia sp., Streptomyces sp., Lactobacillus sp., and Bacillus sp.
In some embodiments of the method, the UPO is a recombinant UPO expressed in bacteria which 1s Escherichia coli. In some embodiments of the method, the UPO is a recombinant UPO expressed in all industrial strains of Escherichia coli.
In some embodiments of the method, the UPO is a recombinant UPO expressed in bacteria which 1s Bacillus subtilis. In some embodiments of the method, the UPO is a recombinant UPO expressed in all industrial strains of Bacillus subtilis.
In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate is any wild-type enzyme that can use peroxide as a source of oxygen and electrons and catalyze 7B- hydroxylation of the compound of Formula I or a salt thereof.
In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate 1s any variant or mutant of the UPO enzyme that can use peroxide as a source of oxygen and electrons and catalyze 7B-hydroxylation of the compound of Formula I or a salt thereof enzyme.
In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate 1s any variant or mutant enzyme that can use peroxide as a source of oxygen and electrons and catalyze 7B-hydroxylation of the compound of Formula I or a salt thereof.
Mutations can be made in a nucleotide or amino acid sequence of enzyme by a variety of methods known to one of ordinary skill in the art. For example, mutations can be made by PCR-directed mutation, site-directed mutagenesis, by chemical synthesis of a gene encoding a polypeptide, by gene editing tools, by any-gene-any-plasmid (AGAP) cloning, by codon optimization, or by insertions, such as insertion of a tag (e.g., a HIS tag or a GFP tag). Mutations can include, for 15 example, substitutions, deletions, and translocations, generated by any method known in the art.
Methods for producing mutations may be found in in references such as Molecular Cloning: A
Laboratory Manual, J. Sambrook, et al, eds., 4 Edition, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, etal, eds., John Wiley & Sons, Inc., New York, 2010. As will be understood by one of ordinary skill in the art, a mutation within a codon may or may not change the amino acid that is encoded by the codon due to degeneracy of the genetic code.
As will be understood by one of ordinary skill in the art, a mutation within a codon may or may not change the amino acid that is encoded by the codon due to degeneracy of the genetic code. In some embodiments, the one or more mutations in the coding nucleotide sequence do not alter the amino acid sequence and/or activity of the enzyme mutant relative to the reference enzyme before mutation. In some embodiments, the one or more mutations alter the amino acid sequence and/or activity of the enzyme mutant relative to the reference enzyme before mutation.
In some embodiments of the method, the enzyme described herein is provided in the form of an enzyme powder or enzyme solution. In some embodiments of the method, the peroxygenase enzyme described herein is provided in the form of an enzyme powder or enzyme solution. In some embodiments of the method, the UPO enzyme described herein is provided in the form of an enzyme powder or enzyme solution. In some embodiments of the method, the enzyme described herein 1s provided in the form of a lyophilized enzyme powder.
In some embodiments of the method, the enzyme described herein is provided in the form of an immobilized enzyme. In some embodiments of the method, the peroxygenase enzyme described herein is provided in the form of an immobilized enzyme. In some embodiments of the method, the UPO enzyme described herein 1s provided in the form of an immobilized enzyme.
Immobilization techniques which result in imprisonment of whole cells or enzymes in a support or matrix are known to an ordinary skilled in the art and any such immobilization techniques can be employed herein. For instance, immobilization techniques can include one or more of adsorption, covalent, anionic, or cationic binding, entrapment, copolymerization, encapsulation, active inclusion bodies and cross-linked enzyme aggregates (CLEA). For the sake of brevity, each 16 of these immobilization techniques are not being detailed herein. In some embodiments, the enzyme can be put on a matrix, synthetic or natural, via absorption, covalent, anionic, or cationic binding, or immobilized via encapsulation, active inclusion bodies or CLEA, or any other immobilization method known to an ordinary skilled in the art.
In some embodiments of the method, the enzyme described herein is provided in the form of whole cells expressing the enzyme. In some embodiments, the whole cells are organisms (e.g., fungi or proto-fungi) expressing wild-type or native peroxygenase. In some embodiments, the whole cells are organisms (e.g, fungi or proto-fungi) expressing wild-type or native UPO. In some embodiments, the whole cells are host cells (e.g., fungi, yeast, or bacteria) expressing recombinant peroxygenase. In some embodiments, the whole cells are host cells (e.g, fungi, yeast, or bacteria) expressing recombinant UPO.
In some embodiments of the method, the enzyme described herein is provided in the form of an extract or a lysate of the whole cells expressing the enzyme. Preparation of an extract or lysate from whole cells obtained by culturing or fermentation process is known to an ordinary skilled in the art and any such preparation methods can be employed herein to obtain a cell extract or cell lysate comprising the enzyme described herein. For the sake of brevity, such methods for preparing cell extract or lysate are not being detailed herein. In some embodiments, the extract or lysate is prepared from whole cells which are organisms (e.g., fungi or proto-fungi) expressing wild-type or native peroxygenase. In some embodiments, the extract or lysate is prepared from whole cells which are organisms (e.g, fungi or proto-fungi) expressing wild-type or native UPO. In some embodiments, the extract or lysate is prepared from whole cells which are host cells (e.g., fungi, yeast, or bacteria) expressing recombinant peroxygenase. In some embodiments, the extract or lysate 1s prepared from whole cells which are host cells (e.g., fungi, yeast, or bacteria) expressing recombinant UPO.
In some embodiments of the method, the enzyme described herein is provided in the form of an enzyme extract which is prepared by processing the excrete of cell culture to obtain a supernatant and a pellet, wherein the supernatant is the enzyme extract. 17
In some embodiments of the method, the enzyme described herein is provided in the form of an enzyme extract which is prepared by subjecting the excrete of cell culture to solid liquid separation to obtain a supernatant and a pellet, wherein the supernatant is the enzyme extract. In some embodiments, the solid liquid separation is performed by methods selected from the group consisting of centrifugation, filtration, sedimentation and decantation, distillation, other methods known to an ordinary person skilled in the art, and combinations thereof.
In some embodiments, said supernatant is directly used as biocatalyst. In some embodiments, said supernatant is directly used as immobilized enzyme. In some embodiments, said supernatant is further purified via techniques such as chromatography (for instance size exclusion chromatography or ion exchange chromatography), or precipitation, or adsorption of other compounds in the mixture.
In some embodiments of the method, the enzyme is provided in the form of any combination of 1) an enzyme powder or solution, 11) immobilized enzyme, 11) whole cells expressing the enzyme, and 1v) an extract or lysate of the whole cells expressing the enzyme.
In some embodiments of the method, the peroxide is an inorganic peroxide, an organic peroxide, or both.
In some embodiments of the method, the peroxide is an inorganic peroxide selected from the group consisting of hydrogen peroxide (H:0,), persulfate (SOZ~ or S,037), potassium peroxymonosulfate (KHSOs), oxone (2KHSO: KHSO: K2S0y), peroxydisulfuric acid (H2S:0s), sodium peroxide (Na202), potassium peroxide (K203), barium peroxide (BaQ,), calcium peroxide (Ca0:), magnesium peroxide (MgO:)}, lithium peroxide (L120:), and combinations thereof.
In some embodiments of the method, the peroxide 15 hydrogen peroxide (H:02).
In some embodiments of the method, the peroxide is an organic peroxide selected from the group consisting of peracetic acid (CH3CO:H), tert-butyl hydroperoxide [(CH3):3COOH], magnesium monoperoxyphthalate (CisHioMgO1o), 3-chloroperoxybenzoic acid (3-CICsHsCOsH), urea hydrogen peroxide (NH;CONH::H:0:}, benzoyl peroxide (C1sH1004), tert-butyl peroxybenzoate 18
(CsHsCO:C(CHs)s), benzoyl peroxide (C1sHi004), di-tert-butyl peroxide [(CH3):COOC(CHs)s], methyl ethyl ketone peroxide (CsHisOs), diacetyl peroxide ((CH3COz)2), cumene hydroperoxide (CoH 1203), trifluoroperacetic acid (CF3CO3H), and combinations thereof.
In some embodiments of the method, all the peroxide is provided or dosed at the start of the reaction.
In some embodiments of the method, the peroxide is provided or dosed at various time points including at the start of the reaction followed by dosing the peroxide at different time-points of the reaction.
In some embodiments of the method, all the peroxide is provided or dosed during the progress of the reaction.
In some embodiments of the method, the peroxide is provided as a solution.
In some embodiments of the method, the peroxide 1s provided in a pure peroxide form.
In some embodiments of the method, the peroxide is generated by in situ peroxide generation system. /n situ peroxide generation using techniques such as biocatalytic, photochemical, or electrochemical methods or any other method are known to an ordinary skilled in the art. For the sake of brevity, such methods of in situ peroxide generation are not being detailed herein.
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(a) to produce a compound of Formula TI(a) 19
“4, Oo i | R"
H
Ho
Y
Formula I(a) 4, 0
Gi | R" ww * i
HOW OH
Y
Formula TI(a) wherein,
R" is OH, NH(CH,),SOsH, NHCH>CO:H, O-alkyl, or O-aryl; comprising contacting the compound of Formula I(a) with an enzyme in the presence of a peroxide, to produce said compound of Formula (a).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(a) to produce a compound of Formula II{a)
CA Oo i | R"
H
Ho”
Y
Formula I(a)
CN 0 i | R" ww 8 +
HOW OH
Y
Formula TI(a) wherein,
R" is OH, NH(CH;):SOsH, NHCH:CO:H, O-alkyl, or O-aryl, comprising contacting the compound of Formula I(a) with unspecific peroxygenase (UPO) in the presence of a peroxide, to produce said compound of Formula II(a). 21
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(a) to produce a compound of Formula TI(a) “4, Oo i | R"
H vo”
Y
Formula I(a) %, 0
Gi | R" ww * i
HON OH
Y
Formula TI(a) wherein,
R" is OH, NH(CH,),SOsH, NHCH>CO:H, O-alkyl, or O-aryl; 22 comprising contacting the compound of Formula I(a) with unspecific peroxy genase (UPO) in the presence of hydrogen peroxide (H20:), to produce said compound of Formula l(a).
In some embodiments, when R" is OH in Formula I(a), the compound is LCA and when R" is
OH in Formula II(a), the compound is UDCA. Accordingly, in some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA 4 “, 0 i u ’
H
HO”
H
LCA
Z
“a, 7 8 iy u ’ ww
HON OH
H
UDCA
23 comprising contacting the LCA with an enzyme in the presence of a peroxide, to produce said UDCA.
In some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA 4 “, 0
Gi u ’
H
HO”
H
LCA
4 “a, 7 8 iy u ’ ww
HON OH
H
UDCA
24 comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said UDCA.
In some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA 4 “, 0
Gi u ’
H
HO”
H
LCA
4 “a, 9 8 iy u ’ ww
HON OH
H
UDCA comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O:) to produce said UDCA.
In some embodiments, when R'" is NH(CH:),SO:H in Formula Ka), the compound is taurolithocholic acid (TLCA) and when R" is NH(CH2)SO:H in Formula II(a), the compound is tauroursodeoxycholic acid (TUDCA). Accordingly, in some embodiments, the present disclosure provides a method for hydroxylation of TLCA to produce TUDCA
A Oo
TE H
Ho
H
TLCA
%, Oo
TE H
Ho” oH
H
TUDCA
26 comprising contacting the TLCA with an enzyme in the presence of a peroxide to produce said TUDCA.
In some embodiments, when R" is NH(CH.):SO;H in Formula I(a), the compound is taurolithocholic acid (TLCA) and when R"' is NH(CH2)SOsH in Formula II(a), the compound is tauroursodeoxycholic acid (TUDCA). Accordingly, in some embodiments, the present disclosure provides a method for hydroxylation of TLCA to produce TUDCA comprising contacting the
TLCA with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said TUDCA.
In some embodiments, when R" is NH(CH:);SOsH in Formula I(a), the compound is taurolithocholic acid (TLCA) and when R" is NH(CH2):SOsH in Formula II(a), the compound is tauroursodeoxycholic acid (TUDCA). Accordingly, in some embodiments, the present disclosure provides a method for hydroxylation of TLCA to produce TUDCA comprising contacting the
TLCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H;0:2) to produce said TUDCA.
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(b) to produce a compound of Formula II{b)
KN R
0
Formula I(b) 27
KN R
0 OH
Formula II(b) wherein,
R is as defined according to Formula I or Formula II above; comprising contacting the compound of Formula I(b) with an enzyme in the presence of a peroxide, to produce said compound of Formula TI(b).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(b) to produce a compound of Formula II{b) comprising contacting the compound of
Formula I(b) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(b).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(b) to produce a compound of Formula II(b) comprising contacting the compound of
Formula I(b) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H:0:) to produce said compound of Formula II(b).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(c) to produce a compound of Formula TI(c) 28
KA R
Ho”
Formula I(c) 4,
KA R ww * ©
HON OH
Formula II(c) wherein,
R is as defined according to Formula I or Formula II above; comprising contacting the compound of Formula I(c) with an enzyme in the presence of a peroxide to produce said compound of Formula I1(c).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(c) to produce a compound of Formula II(¢c) comprising contacting the compound of 29
Formula I(c) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(c).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(c) to produce a compound of Formula II(c) comprising contacting the compound of
Formula I(c) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H202) to produce said compound of Formula T(c).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(d) to produce a compound of Formula II(d)
X KN R i i i o
Formula I(d)
X * R i i i o OH
Formula TI(d) wherein,
R and X are as defined according to Formula I or Formula II above; comprising contacting the compound of Formula I(d) with an enzyme in the presence of a peroxide to produce said compound of Formula II(d).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(d) to produce a compound of Formula II{d) comprising contacting the compound of
Formula I(d) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(d).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(d) to produce a compound of Formula II(d) comprising contacting the compound of
Formula I(d) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H:0:) to produce said compound of Formula II(d).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(e) to produce a compound of Formula TI(e) 31
X KN R
I
1 !
R40"
Formula I(e)
X KN R
!
R10 OH
Formula 1l{e) wherein,
R, R; and X are as defined according to Formula I or Formula II above; comprising contacting the compound of Formula I(e) with an enzyme in the presence of a peroxide to produce said compound of Formula II{e). 32
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(e) to produce a compound of Formula II{e) comprising contacting the compound of
Formula I(e) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula TI(e).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(e) to produce a compound of Formula TI{(e) comprising contacting the compound of
Formula I(e) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H202) to produce said compound of Formula Il(e).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(f) to produce a compound of Formula TI(f)
OH o
Formula I(f) 33
OH oO OH
Formula II(f) comprising contacting the compound of Formula I(f) with an enzyme in the presence of a peroxide to produce said compound of Formula TI(f).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(f) to produce a compound of Formula II(f) comprising contacting the compound of
Formula I(f) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(f).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(f) to produce a compound of Formula II(f) comprising contacting the compound of
Formula I(f) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H202) to produce said compound of Formula II(f).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(g) to produce a compound of Formula II(g) 34
HO
Formula I(g) “, \
HO OH
Formula II(g) comprising contacting the compound of Formula I(g) with an enzyme in the presence of a peroxide to produce said compound of Formula II(g).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(g) to produce a compound of Formula II{g) comprising contacting the compound of
Formula I(g) with unspecific peroxy genase (UPO) in the presence of a peroxide to produce said compound of Formula TI(g).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(g) to produce a compound of Formula II(g) comprising contacting the compound of
Formula I(g) with unspecific peroxy genase (UPO) in the presence of hydrogen peroxide (H202) to produce said compound of Formula II(g).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(h) to produce a compound of Formula II(h) o
Formula I(h) 36 o OH
Formula Il(h) comprising contacting the compound of Formula I(h) with an enzyme in the presence of a peroxide to produce said compound of Formula TI(h).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(h) to produce a compound of Formula II(h) comprising contacting the compound of
Formula I(h) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(h).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(h) to produce a compound of Formula II(h) comprising contacting the compound of
Formula I(h) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H20:2) to produce said compound of Formula II(h).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(1) to produce a compound of Formula II(1) 37
KN \ o oO
Formula I(i)
A \ o oO OH
Formula II(i) comprising contacting the compound of Formula I(1) with an enzyme in the presence of a peroxide to produce said compound of Formula II(1).
In some embodiments, the present disclosure provides a method for hydroxylation a compound of
Formula I(1) to produce a compound of Formula II(1}) comprising contacting the compound of
Formula I(1) with unspecific peroxy genase (UPO) in the presence of a peroxide to produce said compound of Formula II(1). 38
In some embodiments, the present disclosure provides a method for hydroxylation a compound of
Formula I(i) to produce a compound of Formula II(1) comprising contacting the compound of
Formula I(1) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H202) to produce said compound of Formula II(1).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(j) to produce a compound of Formula HG)
CN 0
GD) D 0
Formula I(j)
KA 0
GD) D 0 OH
Formula I1(j) 39 wherein,
R; is hydrogen, alkyl, or aryl; comprising contacting the compound of Formula I(j) with an enzyme in the presence of a peroxide to produce said compound of Formula T1(}).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(j) to produce a compound of Formula II(j) comprising contacting the compound of
Formula I(j) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(j).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(j) to produce a compound of Formula II(j) comprising contacting the compound of
Formula I(j) with unspecific peroxy genase (UPO) in the presence of hydrogen peroxide (H202) to produce said compound of Formula I1(j).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(k) to produce a compound of Formula II{k) %, 0
N
) ° or
Ho
H
Formula I(k)
%, 0
N
BD or
Ho OH
H
Formula TI(k) comprising contacting the compound of Formula I(k) with an enzyme in the presence of a peroxide to produce said compound of Formula II(k).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(k) to produce a compound of Formula II(k) comprising contacting the compound of
Formula I(k) with unspecific peroxy genase (UPO) in the presence of a peroxide to produce said compound of Formula II(k).
In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(k) to produce a compound of Formula II(k) comprising contacting the compound of
Formula I(k) with unspecific peroxy genase (UPO) in the presence of hydrogen peroxide (H:0:) to produce said compound of Formula II(k).
In some embodiments, the method is carried out at a temperature of about 5°C to about 80°C.
In some embodiments, the method is carried out at a temperature of about 10°C to about 60°C.
In some embodiments, the method 1s carried out at a temperature of about 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 41
39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, or 80°C, including any values therebetween.
In some embodiments, the method is carried out for a time period ranging from about 15 minutes to about 150 hours.
In some embodiments, the method is carried out for a time period ranging from about 30 minutes to about 48 hours.
In some embodiments, the method is carried out for a time period ranging from about 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hour, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours, 75 hours, 80 hours, 85 hours, 90 hours, 95 hours, 100 hours, 105 hours, 110 hours, 115 hours, 120 hours, 125 hours, 130 hours, 135 hours, 140 hours, 145 hours, or 150 hours, including any values or ranges therebetween.
In some embodiments, the method is carried out at a pH of about 2 to about 10.
In some embodiments, the method is carried out at a pH of about 4 to about 9.
In some embodiments, the method 1s carried out at a pH of about 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5,7,7.5,8,85,9,9.5 or 10, including any values therebetween.
In some embodiments of the method, the molar ratio of the compound of Formula I or analogue or a salt thereof and the enzyme is from about 100:1 to about 10,000,000:1.
In some embodiments of the method, the molar ratio of the compound of Formula I or analogue or a salt thereof and the enzyme 1s about 1000:1 to about 10,000,000:1. 42
In some embodiments of the method, the molar ratio of the compound of Formula I or analogue or a salt thereof and the peroxide is from about 1:0.1 to about 1:10.
In some embodiments of the method, the molar ratio of the compound of Formula I or analogue or a salt thereof and the peroxide is from about 1:0.8 to about 1:2.
In some embodiments of the method, the molar ratio of the enzyme and the peroxide is from about 1:100 to about 1:100,000,000.
In some embodiments of the method, the molar ratio of the enzyme and the peroxide is from about 1:1000 to about 1: 2,000,000.
In some embodiments, the method is carried out in the presence of one or more of: 1) a solvent, 11) a surfactant, 111) solubilizing agent,
Iv) an antioxidant, v) a buffer, and vi) a monovalent salt.
In some embodiments of the method, the solvent 1s a polar solvent, or a non-polar solvent, or a mixture thereof.
In some embodiments, the solvent 1s selected from the group consisting of water, dimethyl sulfoxide, methanol, ethanol, acetone, acetonitrile, ethyl acetate, tetrahydrofuran, dimethylformamide, propylene carbonate, isopropanol, butanol, octanol, dimethylacetamide, hexane, chloroform, diethyl ether, cyclopentyl methyl ether, ethyl laurate, phthalates, toluene, petroleum ether, carbon tetrachloride, benzene, cyclohexane, xylene, heptane, pentane, decane, dodecane, isopropyl ether, dichloromethane, methyl tert-butyl ether, oleates, and combinations thereof 43
In some embodiments of the method, the surfactant is selected from the group consisting of cationic surfactant, anionic surfactant, non-ionic surfactant, and combinations thereof
In some embodiments, the cationic surfactant is selected from the group consisting of cetyltrimethylammonium bromide, benzalkonium chloride, cetrimonium chloride, lauryl trimethyl ammonium chloride, dodecylbenzenesulfonic acid, octadecyltrimethylammonium bromide, stearalkonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, hexadecylpyridmium chloride, dodecylpyridinium chloride, cetylpyridinium chloride, and combinations thereof.
In some embodiments, the anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, sodium lauryl sulfate, sodium alkylbenzenesulfonate, sodium dioctyl sulfosuccinate, sodium oleate, sodium stearate, sodium palmitate, sodium cocoyl isethionate, sodium lauroyl sarcosinate, sodium dodecylbenzenesulfonate, sodium naphthalenesulfonate, sodium decyl sulfate, sodium lauroyl lactylate, and combinations thereof.
In some embodiments, the non-ionic surfactant is selected from the group consisting of octylphenol ethoxylate (Triton X-100), octylphenol ethoxylate (Triton X-100), polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), sorbitan monooleate (Span 80), polyethylene glycol dodecyl ether (Brij 35), polyethylene glycol cetyl ether (Brij 58), polyethylene glycol-block- polypropylene glycol-block-polyethylene glycol (Pluronic F68), sorbitan monooleate ethoxylated (Polysorbate 80), octylphenol ethoxylate (Nonidet P-40), octylphenol ethoxylate (octylphenol ethoxylate), poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Poloxamer 188), poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Poloxamer 407), nonylphenol ethoxylate (Tergitol NP-10), nonylphenol ethoxylate (Tergitol NP- 40), octylphenol ethoxylate (Igepal CO-630), and combinations thereof.
In some embodiments of the method, the solubilizing agent is selected from the group consisting of cyclodextrins, cucurbit[n]urils, pillararenes, lecithin, and combinations thereof. 44
In some embodiments of the method, the cyclodextrins is selected from the group consisting of « (alpha)-cyclodextrin, B (beta)-cyclodextrin, y (gamma)-cyclodextrin, modified cyclodextrin, and combinations thereof. In some embodiments, the modified cyclodextrins are native cyclodextrins (e.g., a-cyclodextrin, B-cyclodextrin, or y-cyclodextrin) which serve as scaffolds on which functional groups and/or other substituents can be assembled. In some embodiments, the modified cyclodextrin is methyl-B- cyclodextrin.
In some embodiments of the method, the antioxidant is selected from the group consisting of ascorbic acid, tocopherol, polyphenol, flavonoids, and combinations thereof.
In some embodiments of the method, the buffer is selected from the group consisting of potassium phosphate buffer, sodium phosphate buffer, citric acid buffer, acetic acid buffer, 2-(N- morpholino)ethanesulfonic acid [MES] buffer, BisTris buffer, piperazine-N,N'-bis(2- ethanesulfonic acid) [PIPES] buffer, N,N-Bis(hydroxyethyl)-2aminoethanesulfonic acid [BES] buffer, (3-(N-morpholino)propanesulfonic acid) [MOPS] buffer, (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) [HEPES] buffer, tris(hydroxymethyl)aminomethane [Tris] buffer,
Tris-acetate-EDTA [TAE] buffer, carbonate buffer, and combinations thereof.
In some embodiments of the method, the monovalent salt 1s selected from the group consisting of sodium salt, potassium salt, lithium salt, quaternary ammonium salt (QAS), and combinations thereof.
In some embodiments, the method comprises agitation of the reaction mixture comprising the compound of Formula I or analogue or a salt thereof, the enzyme and the peroxide. In some embodiments, the agitation is performed by techniques selected from the group consisting of stirring, shaking, bubbling of gas, overhead shaking, and combinations thereof.
In some embodiments, the method comprises agitation of the reaction mixture to increase mass transfer rates. In some embodiments, agitation is performed for enhancing or lowering the reaction rates as desirable and understood by an ordinary skilled in the art. In some embodiments, agitation
1s performed until mass transfer rates are achieved as desirable and understood by an ordinary skilled in the art.
In some embodiments, the method comprises agitation of a reaction mixture comprising the LCA or TLCA, the UPO and the H;0: to achieve desirable reaction rate or mass transfer rate.
In some embodiments of the method, the compound of Formula II or analogue or salt thereof is recovered and/or purified from an extract or lysate using techniques selected from the group consisting of single or multiple solvent extractions, precipitation, chromatography, any other downstream method known to an ordinary skilled in the art, and combinations thereof.
In some embodiments of the method, the conversion of Formula I, or analogues or salts thereof to
Formula II, or analogues or salts thereof ranges from about 1% to 100%.
In some embodiments of the method, the conversion of Formula I, or analogues or salts thereof to
Formula II, or analogues or salts thereof ranges from about 80% to 100%.
In some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA 4 Oo ft u ’ -
HOY
H
LCA
46
%, 0
Gi ) wr b
HON OH
H
UDCA comprising contacting the LCA with an enzyme in the presence of a peroxide to produce said UDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the LCA and the enzyme is from about 100:1 to about 10,000,000: 1, the molar ratio of the LCA and the peroxide is from about 1:0.1 to about 1:10, and the molar ratio of the enzyme and the peroxide is from about 1:100 to about 1:100,000,000.
In some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA as represented by Figure 1.
In some embodiments, the present disclosure provides a method for hydroxylating LCA to produce
UDCA
47
A 2 8 fy u ’ ww
HON
H
LCA
4, 9 i u ’
H
=
HO OH
H
UDCA comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said UDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, 48 and wherein the molar ratio of the LCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the LCA and the peroxide is from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the peroxide is from about 1:100 to about 1:100,000,000.
In some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA 4 %, 0 8 fy u ’
Ho
H
LCA
A 9 8 iy u ’ ww
HOY OH
H
UDCA
49 comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H202) to produce said UDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the LCA and the UPO is from about 100:1 to about 10,000,000: 1, the molar ratio of the LCA and the H20: is from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the H20» is from about 1:100 to about 1:100,000,000.
In some embodiments of the method, UDCA is recovered and/or purified from an extract or lysate using techniques selected from the group consisting of single or multiple solvent extractions, precipitation, chromatography, any other downstream method known to an ordinary skilled in the art, and combinations thereof.
In some embodiments of the method, the conversion of LCA to UDCA ranges from about 1% to 100%.
In some embodiments of the method, the conversion of LCA to UDCA ranges from about 80% to 100%.
In some embodiments, the present disclosure provides a method for hydroxylation of TLCA to produce TUDCA
%, Oo
TE H
Ho”
H
TLCA
%, Oo
TE A
Ho OH
H
TUDCA comprising contacting the TLCA with unspecific peroxy genase (UPO) in the presence of hydrogen peroxide (H202) to produce said TUDCA, wherein the method 1s carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the TLCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the TLCA and the HO: 1s from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the H:0» is from about 1:100 to about 1:100,000,000. 51
In some embodiments of the method, TUDCA is recovered and/or purified from an extract or lysate using techniques selected from the group consisting of single or multiple solvent extractions, precipitation, chromatography, any other downstream method known to an ordinary skilled in the art, and combinations thereof.
In some embodiments of the method, the conversion of TLCA to TUDCA ranges from about 1% to 100%.
In some embodiments of the method, the conversion of TLCA to TUDCA ranges from about 80% to 100%.
Biocatalysts/enzymes such as cytochrome P450 monooxygenases (P450) which can catalyse 7B- hydroxylation of substrates such as bile acid or their analogues (e.g., LCA) often rely on complex regeneration systems to deliver the required redox equivalents to the active site to form the active ‘Compound TI’, activating molecular oxygen and performing the reaction. Required redox equivalents come in the form of NAD(P)H and are often transferred using other proteins like cytochrome P450 reductase (CPR). Furthermore, especially in fungi, these reaction systems are membrane bound. This restricts the use of such biocatalysts to whole cells, introducing transfer limitations over the cell wall for both oxygen and substrate, potential side reactions and extra need of substrates. Additional issues with enzymes such as P450 are uncoupling of activated oxygen, the slow dissolution of molecular oxygen in the aqueous phase and the relative instability of these proteins. In general, employing enzymes such as P450 in the reaction is considered complex and there is no or very limited industrial application yet of such enzyme classes towards production of bile acids or their analogues (e.g., UDCA) due to the limitations discussed above. Therefore, using enzymes such as P450 is expected to be resource and time intensive and increase the overall cost of the process.
On the other hand, without wishing to be bound by any theory, the present inventors surprisingly found that employing enzyme(s) capable of using peroxide as the source of both oxygen and electrons such as peroxygenases (e.g., UPO) would circumvent the above discussed issues, thereby 52 allowing for a simple cost-effective process for 7B-hydroxylation of substrates such as bile acid or their derivatives. For mstance, UPO are heme containing enzymes able to perform various oxyfunctionalization reactions. The actual reaction takes place via an oxoferryl-heme species at the active site called ‘Compound I’. However, unlike enzymes such as P450, for UPO, the oxygen and electrons required to form the active compound I do not come from molecular oxygen and expensive cofactors like NAD(P)H. Nor are there complicated regeneration systems required for said biocatalyst. Instead, UPO can directly accept HO: or other inorganic/organic peroxides as the source of oxygen and electrons to form the active intermediate. Additionally, UPO also conveniently function in solution or as immobilized biocatalyst. Altogether, compared to P450 based systems, employing enzymes capable of using peroxide, such as unspecific peroxygenases (e.g., UPO) result in much less complicated reaction schemes, more stable proteins and most importantly, the additional flexibility to apply these enzymes conveniently outside a living organism. The ability to apply these enzymes independently of their host cells or native organisms, and their general higher stability as compared to enzymes such as P450 along with being economical are great advantages in large scale applications in production of bile acids or their analogues.
Method for converting Fornula I or analogue or salt thereof to Fornuila Il or analogue or salt thereof
The present disclosure further relates to a method for converting the compound of Formula I or analogue or a salt thereof to the compound of Formula IT or analogue or a salt thereof, comprising contacting the compound of Formula I or analogue or a salt thereof: 53
X “a, R
I
I
I
R,0"
Y
Formula I with an enzyme in the presence of a peroxide, to produce a compound of Formula II or analogue or salt thereof’ “a,
X “a, R
I
I
I
R10" OH
Y
Formula IT wherein,
R is independently selected from the group consisting of (CH2),C=0(O)uR', alkyl, alkenyl,
CH»OH, CHO, (CH:2):CONH(CH:):SOsH, (CH:2)>CONH(CH:)COOH, -CH=CH-CO:-alkyl, -
CH=CH-CO:-aryl, -CH=CH-CO:H and -CH=CH-alkyl, 54 wherein R' is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and NR.Rw, wherein R, and Ry, are independently selected from the group consisting of hydrogen, alkyl, aryl, -C(=0)-alkyl, -C(=0)-aryl and (CH:)25$0:H;
R; is independently selected from the group consisting of hydrogen, alkyl, acyl, mesityl, tosyl, -C(=0)-alkyl, -C(=0)-aryl, -S(=0):-alkyl, -S(=0)2-aryl, -S(=0)2-OH and -P(=0)-(OR2)., wherein Rj 1s independently hydrogen or alkyl; n is Ó or an integer ranging from 1 to 5; misOorl;
Y is hydrogen;
Xs hydrogen, hydroxy or oxygen; wherein R, R’, R,, Ry, Ry, R; or X are optionally independently substituted; === represents either a single bond or a double bond; and with the proviso that: both Rings A and B do not simultaneously contain double bond inside the rings; when === is a double bond on the Ring A, Ri is absent; when == is a single bond on the Ring A, then R; is present; when === is a double bond inside either the Ring A or Ring B, Y 1s absent; and when == is a single bond inside both the Rings A and B simultaneously, Y is present.
The present disclosure additionally relates to a method for converting the compound of Formula
I(a) or analogue or a salt thereof to the compound of Formula II(a) or analogue or a salt thereof, comprising contacting the compound of Formula I(a) or analogue or a salt thereof:
“4, Oo i | R"
H
Ho
Y
Formula I(a) with an enzyme 1n the presence of a peroxide, to produce the compound of Formula (a) or analogue or a salt thereof:
KA 0
KN i | R" - ~ i
HOY OH
Y
Formula II(a) wherein,
R'" is OH, NH(CH:),SO:H, NHCH;CO:H, O-alkyl, or O-aryl.
In some embodiments, the enzyme is capable of using peroxide as a co-substrate and is an enzyme from class EC 1.11 which acts on peroxide as acceptor. 56
In some embodiments, the enzyme is peroxygenase.
In some embodiments, the enzyme is a peroxygenase from class EC 1.11.2.
In some embodiments, the peroxygenase is a fungal peroxygenase or a peroxygenase obtained from proto-fungi.
In some embodiments, the enzyme is unspecific peroxygenase (UPO).
In some embodiments, the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1 belonging to the sub-subclass of oxidoreductases. More particularly, the enzyme in the present method is an enzyme from EC 1.11.2.1: Unspecific peroxygenase.
In some embodiments, the UPO is a wild-type UPO obtained from fungi, or proto-fungi, or a combination thereof.
In some embodiments, the UPO is a recombinant unspecific peroxygenase (UPO) expressed in a host cell selected from fungi and bacteria. In some embodiments, the fungi 1s yeast.
In some embodiments, the enzyme is in the form of: 1) an enzyme powder or solution, 11) immobilized enzyme, 111) whole cells expressing the enzyme, iv) an extract or lysate of the whole cells expressing the enzyme, or v) any combination of 1 to Iv.
In some embodiments, the peroxide is an organic peroxide, an inorganic peroxide, or both.
In some embodiments of the method, the peroxide 1s an inorganic peroxide selected from the group consisting of hydrogen peroxide (H20:), persulfate (SO2~ or S,0%7), potassium peroxymonosulfate (KHSOs), oxone (2ZKHSOs KHSO: KSO), peroxydisulfuric acid (H:S20s), sodium peroxide (Na:0:), potassium peroxide (K20:}, barium peroxide (Ba0:), calcium peroxide (CaO), magnesium peroxide (MgO), lithium peroxide (L120), and combinations thereof. 57
In some embodiments of the method, the peroxide is hydrogen peroxide (H20:).
In some embodiments of the method, the peroxide is an organic peroxide selected from the group consisting of peracetic acid (CH3CO:H), tert-butyl hydroperoxide [(CH3);COOH], magnesium monoperoxyphthalate (CisHioMgO1o), 3-chloroperoxybenzoic acid (3-CICsHsCOsH), urea hydrogen peroxide (NH>CONH:'H;0:), benzoyl peroxide (C1sH1004), tert-butyl peroxybenzoate (CsHsCO5C(CHa)z), benzoyl peroxide (C1aH1004), di-tert-butyl peroxide [(CH;);:COOC(CHz3)3], methyl ethyl ketone peroxide (CgHisOg), diacetyl peroxide ((CH3CO2)2), cumene hydroperoxide (CsHi202), trifluoroperacetic acid (CF:5CO:H), and combinations thereof.
In some embodiments of the method, the conversion of Formula I, or analogues or salts thereof to
Formula II, or analogues or salts thereof ranges from about 1% to 100%.
In some embodiments of the method, the conversion of Formula I, or analogues or salts thereof to
Formula II, or analogues or salts thereof ranges from about 80% to 100%.
In some embodiments, the method comprises converting lithocholic acid (LCA):
KA o
Gi ) no
H
LCA
58 to ursodeoxycholic acid (UDCA): 4
KA 0 8 fy u ’
Ww
HOW OH
H
UDCA
In some embodiments, the method comprises converting lithocholic acid (LCA) to ursodeoxycholic acid (UDCA), comprising contacting the LCA with an enzyme in the presence of a peroxide to produce the UDCA.
In some embodiments, the method comprises converting lithocholic acid (LCA) to ursodeoxycholic acid (UDCA), comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of a peroxide to produce the UDCA.
In some embodiments, the method comprises converting lithocholic acid (LCA) to ursodeoxycholic acid (UDCA), comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H20:), to produce the UDCA.
In some embodiments, the method comprises converting lithocholic acid (LCA) to ursodeoxycholic acid (UDCA), comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H20:), to produce the UDCA, 59 wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the LCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the LCA and the H20: is from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the HO: is from about 1:100 to about 1:100,000,000.
In some embodiments of the method, the conversion of LCA to UDCA ranges from about 80% to 100%.
In some embodiments, the method comprises converting taurolithocholic acid (TLCA):
A 5
TE H
Ho”
H
TLCA to tauroursodeoxycholic acid (TUDCA): 60
A 0
HO OH
H
TUDCA .
In some embodiments, the method comprises converting taurolithocholic acid (TLCA) to tauroursodeoxycholic acid (TUDCA), comprising contacting the TLCA with an enzyme in the presence of a peroxide to produce the TUDCA.
In some embodiments, the method comprises converting TLCA to TUDCA, comprising contacting the TLCA with unspecific peroxygenase (UPO) in the presence of a peroxide to produce the
TUDCA.
In some embodiments, the method comprises converting TLCA to TUDCA, comprising contacting the TLCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H20:2) to produce the TUDCA.
In some embodiments, the method comprises converting TLCA to TUDCA, comprising contacting the TLCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H202) to produce the TUDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, 61 and wherein the molar ratio of the TLCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the TLCA and the H2021s from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the H20» is from about 1:100 to about 1:100,000,000.
In some embodiments of the method, the conversion of TLCA to TUDCA ranges from about 80% to 100%.
In all embodiments of the method for converting a compound of Formula I or analogue or salt thereof to a compound of Formula IT or analogue or salt thereof, all the additional features are as described by any of the embodiments mentioned above under ‘Method for hydroxylation of
Formula I or analogue or salt thereof’. For the sake of brevity, and avoiding repetition, each of those embodiments are not being reiterated here again. However, each of the said embodiments, completely fall within the purview of the method for converting a compound of Formula I or a salt thereof to a compound of Formula II or a salt thereof.
Reaction Mixture or Reaction System
The present disclosure also provides a reaction mixture or reaction system comprising: 1) a compound of Formula I or analogue or salt thereof, as described above; 11) an enzyme; and 111) a peroxide.
In some embodiments, the reaction mixture or reaction system comprises the compound of
Formula I(a) or analogue or a salt hereof which is LCA or TLCA.
In some embodiments, the enzyme 1n the reaction mixture or reaction system is capable of using peroxide as a co-substrate and 1s an enzyme from class EC 1.11 which acts on peroxide as acceptor.
In some embodiments, the reaction mixture or reaction system comprises the enzyme which is peroxygenase.
In some embodiments, the reaction mixture or reaction system comprises the enzyme which is a peroxygenase from class EC 1.11.2. 62
In some embodiments, the enzyme of the reaction mixture or reaction system is unspecific peroxygenase (UPO).
In some embodiments, the enzyme of the reaction mixture or reaction system is unspecific peroxygenase (UPO) of EC 1.11.2.1 belonging to the sub-subclass of oxidoreductases. More particularly, the enzyme of the reaction mixture or reaction system is an enzyme from EC 1.11.2. 1:
Unspecific peroxygenase.
In some embodiments, the UPO is a wild-type UPO obtained from fungi, or proto-fungi, or a combination thereof. In some embodiments, the UPO is a recombinant unspecific peroxygenase (UPO) expressed in a host cell selected from fungi and bacteria. In some embodiments, the fungi is yeast.
In some embodiments, the enzyme of the reaction mixture or reaction system is in the form of: 1) an enzyme powder or solution, 11) immobilized enzyme, ii1) whole cells expressing the enzyme, iv) an extract or lysate of the whole cells expressing the enzyme, or v) any combination of i to iv.
In some embodiments, the peroxide in the reaction mixture or reaction system is an organic peroxide, an inorganic peroxide, or both.
In some embodiments, the peroxide in the reaction mixture or reaction system is an inorganic peroxide selected from the group consisting of hydrogen peroxide (H:02), persulfate (SO? or
S,037), potassium peroxymonosulfate (KHSOs), oxone (2KHSO:KHSO,4K;SO:), peroxydisulfuric acid (H:S20s), sodium peroxide (Na202), potassium peroxide (K20:2), barium peroxide (Ba0:}), calcium peroxide (Ca0O:), magnesium peroxide (MgO:), lithium peroxide (L1202), and combinations thereof.
In some embodiments of the method, the peroxide in the reaction mixture or reaction system is hydrogen peroxide (H:02). 63
In some embodiments, the peroxide in the reaction mixture or reaction system is an organic peroxide selected a group comprising peracetic acid (CH;COsH), tert-butyl hydroperoxide [(CH3);COOH], magnesium monoperoxyphthalate (CisHioMgO10), 3-chloroperoxybenzoic acid (3-CICsH4CO3H), urea hydrogen peroxide (NH;CONH;H;0:2), benzoyl peroxide (C1sH 1004), tert- butyl peroxybenzoate (CsHsCO:C(CHs)s), benzoyl peroxide (Ci14Hi004), di-tert-butyl peroxide [(CH3);:COOC(CHs)3], methyl ethyl ketone peroxide (CsHisOs), diacetyl peroxide ((CH5CO2)2), cumene hydroperoxide (C5Hi20:), trifluoroperacetic acid (CF;COsH), and combinations thereof.
In some embodiments, the reaction mixture or reaction system comprises: 1) LCA; 11) unspecific peroxygenase (UPO); and 11) hydrogen peroxide (H20:). In some embodiments, the reaction mixture or reaction system comprises: 1) TLCA; 11) unspecific peroxygenase (UPO); and iii) hydrogen peroxide (H20-).
In all embodiments of the reaction mixture or reaction system, all the additional features are as described by any of the embodiments mentioned above under ‘Method for hydroxylation of
Formula I or analogue or salt thereof”. For the sake of brevity, and avoiding repetition, each of those embodiments are not being reiterated here again. However, each of the said embodiments, completely fall within the purview of the reaction mixture or reaction system.
Use
The present disclosure also relates to use of an enzyme for hydroxylation of a compound of
Formula I or analogue or salt thereof, wherein the Formula I is as defined in the preceding embodiments, and wherein the enzyme requires peroxide as a co-substrate. In some embodiments of the use, the hydroxylation 15 7B-hydroxylation.
The present disclosure additionally relates to use of an enzyme for converting a compound of
Formula I or analogue or salt thereof to the compound of Formula IT or analogue or salt thereof, wherein the Formula I and Formula II are as defined in the preceding embodiments, and wherein the enzyme requires peroxide as a co-substrate. 64
In some embodiments of the use, the enzyme is capable of using peroxide as a co-substrate and is an enzyme from class EC 1.11 which acts on peroxide as acceptor.
In some embodiments of the use, the enzyme is peroxygenase.
In some embodiments of the use, the enzyme is a peroxygenase from class EC 1.11.2.
In some embodiments of the use, the peroxygenase is a fungal peroxygenase or a peroxygenase obtained from proto-fungi.
In some embodiments of the use, the enzyme is unspecific peroxygenase (UPO).
In some embodiments of the use, the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1 belonging to the sub-subclass of oxidoreductases. More particularly, the enzyme is an enzyme from EC 1.11.2.1: Unspecific peroxy genase.
In some embodiments of the use, the UPO is a wild-type UPO obtained from fungi, or proto-fungi, or a combination thereof.
In some embodiments of the use, the UPO is a recombinant unspecific peroxygenase (UPO) expressed in a host cell selected from fungi and bacteria. In some embodiments, the fungi is yeast.
In some embodiments of the use, the hydroxylation or the conversion is carried out in the presence of a peroxide which is an organic peroxide, an inorganic peroxide, or both. In some embodiments of the use, the hydroxylation or the conversion is carried out in the presence of hydrogen peroxide (H202).
In some embodiments, the present disclosure provides use of unspecific peroxygenase (UPO) for converting lithocholic acid (LCA) to ursodeoxycholic acid (UDCA). In some embodiments, the present disclosure provides use of UPO for converting taurolithocholic acid (TLCA) to tauroursodeoxycholic acid (TUDCA). 65
In all embodiments of the use, all the additional features are as described by any of the embodiments mentioned above under ‘Method for hydroxylation of Formula I or analogue or salt thereof’. For the sake of brevity, and avoiding repetition, each of those embodiments are not being reiterated here again. However, each of the said embodiments, completely fall within the purview of the use described herein.
Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well- known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating one or more of the above-described embodiments. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.
Example 1 7B-hydroxylation of LCA to produce UDCA
Fungal unspecific peroxygenase (UPO) was employed for UDCA production by 7B-hydroxylation of LCA. About 2 gm Ll! of substrate (LCA, approx. 5.0 mM), about 0.1% (w/v) methyl-p- cyclodextrin, about 4% (v/v) DMSO, about 10 mM ascorbic acid, about 3.0 mM H>O2 and about 1 to 2 mg of lyophilized enzyme powder (fungal UPO) were added in about 100 mM KP: buffer pH 6.0 at about 0.5 mL reaction volume. The reaction mixture was kept at about 30°C at about 1000 rpm for about 65 hours.
HPLC results demonstrating the production of UDCA from LCA catalyzed by UPO 1s provided as Figure 2.
Example 2 7B-hydroxylation of LCA to produce UDCA 66
Fungal unspecific peroxygenase (UPO) was employed for UDCA production by 7B-hydroxylation of LCA. About 1 gm LL! of substrate (LCA, approx. 2.5 mM), about 0.1% (w/v) methyl-p- cyclodextrin, about 4% (v/v) DMSO, about 10 mM ascorbic acid, about 0.3 mM H:0: and about 1 to 2 mg of lyophilized enzyme powder (fungal UPO) were added in about 100 mM KP: buffer pH 6.0 at about 0.5 mL reaction volume. The reaction mixture was kept at about 30°C at about 1000 rpm for about 65 hours.
HPLC results demonstrating the production of UDCA from LCA catalyzed by UPO is provided as Figure 3.
Example 3 7B-hvdroxylation of TLCA to produce TUDCA
Fungal unspecific peroxygenase (UPO) was employed for TUDCA production by 7p- hydroxylation of TLCA. About 2 gm L™! of substrate (TLCA, approx. 4.0 mM), about 0.1% (w/v) methyl-B-cyclodextrin, about 4% (v/v) DMSO, about 10 mM ascorbic acid, about 3.0 mM H;0: and about 1 to 2 mg of lyophilized enzyme powder (fungal UPO) were added in about 100 mM
KP1 buffer pH 6.0 at about 0.5 mL reaction volume. The reaction mixture was kept at about 30°C at about 1000 rpm for about 65 hours.
HPLC results demonstrating the production of TUDCA from TLCA catalyzed by UPO is provided as Figure 4.
Example 4 7B-hydroxylation of TLCA to produce TUDCA
Fungal unspecific peroxygenase (UPO) was employed for TUDCA production by 7p- hydroxylation of TLCA. About 1 gm L™ of substrate (TLCA, approx. 2.5 mM), about 0.1% (w/v) methyl-f-cyclodextrin, about 4% (v/v) DMSO, about 10 mM ascorbic acid, about 0.3 mM H:0: and about 1 to 2 mg of lyophilized enzyme powder (fungal UPO) were added in about 100 mM
KPi buffer pH 6.0 at about 0.5 mL reaction volume. The reaction mixture was kept at about 30°C at about 1000 rpm for about 65 hours. 67
HPLC results demonstrating the production of TUDCA from TLCA catalyzed by UPO is provided as Figure 5.
In Figures 2 to 5, LCA, TLCA and TUDCA are detected using a photodiode array (PDA) at 215 nm for LCA or at 210 nm for TLCA and TUDCA. UDCA is detected using a refractive index detector. In Figures 2 to 5, X-axis corresponds to the retention time of any given molecule in the system and the unit is minutes. Y-axis is either milli-absorbance units (mAU) or micro-Refractive
Index Units (uRIU) depending on the detection method.
The present invention is further defined by the following numbered embodiments: 1) A method for hydroxylating a compound of Formula I or analogue or a salt thereof, comprising contacting the compound of Formula I or analogue or salt thereof:
X “, R 1 !
R/0°
Y
Formula I with an enzyme in the presence of a peroxide, to produce a compound of Formula
II or analogue or salt thereof: 68
A
X “, R
CIEL
R07 OH
Y
Formula IT wherein,
R is independently selected from the group consisting of (CH2)4C=O(O)mR', alkyl, alkenyl,
CH:0H, CHO, (CH:)CONH(CH;):SO:H, (CH) CONH(CH;)COOH, -CH=CH-CO:- alkyl, -CH=CH-COz-aryl, -CH=CH-CO;H and -CH=CH-alkyl, wherein R' is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and NRaRs, wherein R; and Ry, are independently selected from the group consisting of hydrogen, alkyl, aryl, -C(=0)-alkyl, -C(=0)-aryl and (CH2)2S0;H;
R; is independently selected from the group consisting of hydrogen, alkyl, acyl, mesityl, tosyl, -C(=0)-alkyl, -C(=0)-aryl, -S(=0)2-alkyl, -S(=0)2-aryl, -S(=0),-OH and -P(=0)-(OR2), wherein R; 1s independently hydrogen or alkyl; n is 0 or an integer ranging from 1 to 5; misOorl;
Y is hydrogen;
X is hydrogen, hydroxy or oxygen; wherein R, R’, R;, Ry, Ry, R; or X are optionally independently substituted; === represents either a single bond or a double bond; 69 and with the proviso that: both Rings A and B do not simultaneously contain double bond inside the rings; when === is a double bond on the Ring A, Ri is absent; when === is a single bond on the Ring A, then R; is present; when === is a double bond inside either the Ring A or Ring B, Y 1s absent; and when === is a single bond inside both the Rings A and B simultaneously, Y is present.
2) The method of embodiment 1, wherein the method comprises 7B-hydroxylation of the compound of Formula I or analogue or a salt thereof to produce the compound of Formula II or analogue or a salt thereof which has a B-hydroxyl group at position 7.
3) The method of embodiment 1 or embodiment 2, wherein the enzyme is capable of using the compound of Formula I or analogue or a salt thereof as a substrate and peroxide as a co- substrate for catalysing the hydroxylation of said compound of Formula I or analogue or a salt thereof to produce the compound of Formula TI or analogue or a salt thereof.
4) The method of any of the embodiments 1 to 3, wherein the enzyme capable of using peroxide as a co-substrate is an enzyme from Enzyme Commission (EC) class 1.11 which acts on peroxide as acceptor.
5) The method of any of the embodiments 1 to 4, wherein the enzyme capable of using peroxide as a co-substrate is a peroxygenase.
6) The method of any of the embodiments 1 to 5, wherein the peroxygenase is a fungal peroxygenase or a peroxygenase obtained from proto-fungi.
7) The method of any of the embodiments 1 to 6, wherein the enzyme is unspecific peroxygenase
(UPO); or the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1.
8) The method of any of the embodiments 5 to 7, wherein the unspecific peroxygenase (UPO) is a wild-type UPO obtained from fungi, or proto-fungi, or both. 70
9) The method of any of the embodiments 5 to 8, wherein the unspecific peroxygenase (UPO) is a recombinant unspecific peroxygenase (UPO) expressed in a host cell selected from fungi and bacteria. 10) The method of any of the embodiments 5 to 9, wherein the unspecific peroxygenase (UPO) is a recombinant unspecific peroxygenase (UPO) expressed in fungi or yeast selected from the group consisting of Pichia sp., Saccharomyces sp., Aspergillus sp., Fusarium sp., and
Myceliophthora sp, or wherein the unspecific peroxygenase (UPO) is a recombinant unspecific peroxygenase (UPO) expressed in bacteria which is Escherichia sp., Streptomyces sp.,
Lactobacillus sp., and Bacillus sp. 11) The method of any of the embodiments 1 to 10, wherein the enzyme is in the form of: 1) an enzyme powder or solution, 11) immobilized enzyme, 111) whole cells expressing the enzyme,
Iv) an enzyme extract or lysate of the whole cells expressing the enzyme, or v) any combination of 1 to Iv. 12) The method of any of the embodiments 1 to 11, wherein the peroxide is an inorganic peroxide, or an organic peroxide, or both. 13) The method of embodiment 12, wherein the peroxide is an inorganic peroxide selected from the group consisting of hydrogen peroxide (H:0:), persulfate (S02~ or S, 037), potassium peroxymonosulfate (KHSOs), oxone (2ZKHSOs KHSO::K;S04}), peroxydisulfuric acid (H:S:Os), sodium peroxide (Na20:}, potassium peroxide (K20:), barium peroxide (Ba0:), calcium peroxide (CaO;), magnesium peroxide (MgO:), lithium peroxide (L1;02), and combinations thereof. 14) The method of embodiment 12, wherein the peroxide is an organic peroxide selected from the group consisting of peracetic acid (CH3CO:H), tert-butyl hydroperoxide [(CH3):COOH], magnesium monoperoxyphthalate (Ci6HioMgOio), 3-chloroperoxybenzoic acid (3-
CICsH4CO:H), urea hydrogen peroxide (NH:CONH:;:H:0:2), benzoyl peroxide (C14H190:), 71 tert-butyl peroxybenzoate (CsHsCO:C(CHs)s), benzoyl peroxide (Ci14H1004), di-tert-butyl peroxide [(CH3):COOC(CHs)s], methyl ethyl ketone peroxide (CsHisOs), diacetyl peroxide ((CH3CO:)2), cumene hydroperoxide (CsHi202), trifluoroperacetic acid (CF:CO:H), and combinations thereof. 15) The method of any of the embodiments 1 to 14, wherein the peroxide is provided as a pure peroxide, a peroxide solution or peroxide generated by in situ peroxide generation system. 16) The method of any of the embodiments 1 to 15, wherein the alkyl is C1-C12 alkyl, the alkenyl 1s C7-C12 alkenyl, the alkynyl is C+-C12 alkynyl, the cycloalkyl is a hydrocarbon ring system radical comprising C:3-C29 carbon atoms, and the aryl is a hydrocarbon ring system radical comprising Cs-C 5 carbon atoms and at least one aromatic ring. 17) The method of any of the embodiments 1 to 16, wherein the salt of Formula I or Formula II is selected from the group consisting of sodium salt, potassium salt, and quaternary ammonium salt (QAS). 18) The method of any of the embodiments 1 to 17, wherein the method comprises: a) hydroxylation of a compound of Formula I(a) to produce a compound of Formula (a)
KN 0 i | R" no
Y
Formula I(a) 72
“4, Oo
Gi | R" ww * ©
HOW OH
Y
Formula Il(a) wherein,
R" is OH, NH(CH:):S0:H, NHCH;CO:H, O-alkyl, or O-aryl; or b) hydroxylation of a compound of Formula I(b) to produce a compound of Formula II(b) ,
KN R oO
Formula I(b) 73
KN R oO OH
Formula II(b) wherein,
R is as defined in claim 1; or c) hydroxylation of a compound of Formula I(c) to produce a compound of Formula II(c)
KN R
H
HO”
Formula I{c) 74
KN R al * ©
HOW OH
Formula II(c) wherein,
Ris as defined in claim 1; or d) hydroxylation of a compound of Formula I(d) to produce a compound of Formula II(d)
X “, R i i i o
Formula I(d) 75
X KN R i i i oO OH
Formula II(d) wherein,
R and X are as defined in claim 1; or e) hydroxylation of a compound of Formula I(e) to produce a compound of Formula II(e)
X Ka R
I
I
R,0°
Formula I{e) 76
X “, R 1 !
R,0 OH
Formula Il(e) wherein,
R, R; and X are as defined in claim 1; or f) hydroxylation of a compound of Formula I(f) to produce a compound of Formula II(f)
OH
0
Formula I(f) 77
OH o OH
Formula II(f) ; or g) hydroxylation of a compound of Formula I(g) to produce a compound of Formula TI(g)
HO
Formula I(g) 78
“ey \
HO OH
Formula II(g) ; or h) hydroxylation of a compound of Formula I(h) to produce a compound of Formula II(h) o
Formula I(h) 79 oO OH
Formula Il(h) ; or 1) hydroxylation of a compound of Formula I(1) to produce a compound of Formula TI(1)
AY oO
Formula I(i) 80
N\, oO OH
Formula I1(i) ; or |) hydroxylation of a compound of Formula I(j) to produce a compound of Formula H(j)
CA 0
GD) ë oO
Formula I(j) 81
4, Oo
GD) ë oO OH
Formula I1(j) wherein,
R; is hydrogen, alkyl, or aryl; or, k) hydroxylation of a compound of Formula I(k) to produce a compound of Formula II(k) %, O
N
BD or no
H
Formula I(k) 82
CN 0
N
7 ° or
HO OH
H
Formula II(k) 19) The method of any of the embodiments 1 to 18, wherein the method is carried out at a temperature of from about 5°C to about 80°C. 20) The method of any of the embodiments 1 to 19, wherein the method is carried out for a time period ranging from about 15 minutes to about 150 hours. 21) The method of any of the embodiments 1 to 20, wherein the method is carried out at a pH of about 2 to about 10. 22) The method of any of the embodiments 1 to 21, wherein the molar ratio of the compound of
Formula I or a salt thereof and the enzyme 1s from about 100:1 to about 10,000,000:1; or wherein the molar ratio of the compound of Formula I or a salt thereof and the peroxide is from about 1:0.1 to about 1:10 or wherein the molar ratio of the enzyme and the peroxide is from about 1:100 to about 1:100,000,000. 23) The method of any of the embodiments 1 to 22, wherein the method is carried out in the presence of one or more of: 1} a solvent selected from the group consisting of water, dimethyl sulfoxide, methanol, ethanol, acetone, acetonitrile, ethyl acetate, tetrahydrofuran, dimethylformamide, propylene carbonate, isopropanol, butanol, octanol, dimethylacetamide, hexane, 83 chloroform, diethyl ether, cyclopentyl methyl ether, ethyl laurate, phthalates, toluene, petroleum ether, carbon tetrachloride, benzene, cyclohexane, xylene, heptane, pentane, decane, dodecane, isopropyl ether, dichloromethane, methyl tert-butyl ether, oleates, and combinations thereof; 11) a surfactant selected from the group consisting of cationic surfactant, anionic surfactant, non-ionic surfactant, and combinations thereof, wherein the cationic surfactant is selected from the group consisting of cetyltrimethylammonium bromide, benzalkonium chloride, cetrimonium chloride, lauryl trimethyl ammonium chloride, dodecylbenzenesulfonic acid, octadecyltrimethylammonium bromide, stearalkonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, hexadecylpyridinium chloride, dodecylpyridinium chloride, cetylpyridinium chloride, and combinations thereof, the anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, sodium lauryl sulfate, sodium alkylbenzenesulfonate, sodium dioctyl sulfosuccinate, sodium oleate, sodium stearate, sodium palmitate, sodium cocoyl isethionate, sodium lauroyl sarcosinate, sodium dodecylbenzenesulfonate, sodium naphthalenesulfonate, sodium decyl sulfate, sodium lauroyl lactylate, and combinations thereof, and the non-ionic surfactant 1s selected from the group consisting of octylphenol ethoxylate (Triton X-100), octylphenol ethoxylate (Triton X-100), polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), sorbitan monooleate (Span 80), polyethylene glycol dodecyl ether (Brij 35), polyethylene glycol cetyl ether (Brij 58), polyethylene glycol- block-polypropylene glycol-block-polyethylene glycol (Pluronic F68), sorbitan monooleate ethoxylated (Polysorbate 80), octylphenol ethoxylate (Nonidet P-40), octylphenol ethoxylate (octylphenol ethoxylate), poly(ethylene oxide)-block- poly(propylene oxide)-block-poly(ethylene oxide) (Poloxamer 188), poly(ethylene oxide)- block-poly(propylene oxide)-block-poly(ethylene oxide) (Poloxamer 407), nonylphenol ethoxylate (Tergitol NP-10), nonylphenol ethoxylate (Tergitol NP-40), octylphenol ethoxylate (Igepal CO-630), and combinations thereof; 84 i)solubilizing agent is selected from the group consisting of cyclodextrins, modified cyclodextrins, cucurbit[n]urils, pillararenes, lecithin, and combinations thereof;
1v)an antioxidant selected from the group consisting of ascorbic acid, tocopherol, polyphenol, flavonoids, and combinations thereof.
v) a buffer selected from the group consisting of potassium phosphate buffer, sodium phosphate buffer, citric acid buffer, acetic acid buffer, 2-(N-morpholino)ethanesulfonic acid [MES] buffer, BisTris buffer, piperazine-N,N'-bis(2-ethanesulfonic acid) [PIPES] buffer, N,N-Bis(hydroxyethyl)-2aminoethanesulfonic acid [BES] buffer, (3-(N- morpholino)propanesulfonic acid) [MOPS] buffer, (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid) [HEPES] buffer, tris(hydroxymethyl)aminomethane [Tris]
buffer, Tris-acetate-EDTA [TAE] buffer, carbonate buffer, and combinations thereof; and vi) monovalent salt selected from the group consisting of sodium salt, potassium salt, lithium salt, quaternary ammonium salts (QAS) and combinations thereof.
24) The method of any of the embodiments 1 to 23, wherein the method comprises agitation of the reaction mixture comprising the compound of Formula I or a salt thereof, the enzyme and the peroxide by techniques selected from the group consisting of stirring, shaking, bubbling of gas, overhead shaking, and combinations thereof.
25) A method for converting a compound of Formula I(a) or analogue or salt thereof to a compound of Formula II(a) or analogue or salt thereof, comprising contacting the compound of Formula I(a) or analogue or salt thereof:
85
CN Oo i | R"
H
Ho
Y
Formula I(a) with an enzyme in the presence of a peroxide, to produce the compound of Formula II(a) or analogue or salt thereof:
A 0
A
Gi | R" ww * ©
HOW OH
Y
Formula II(a) wherein,
R" is OH, NH(CH:),SOsH, NHCH:CO:H, O-alkyl, or O-aryl. 86
26) The method of embodiment 25, wherein the method comprises converting lithocholic acid (LCA): 4, 0 8 fy u ’
HON
H
LCA to ursodeoxycholic acid (UDCA): 4, 0 i u ’
H
=
HO OH
H
UDCA
27) The method of embodiment 25, wherein the method comprises converting taurolithocholic acid (TLCA): 87
A 5
TE H
Ho”
H
TLCA to tauroursodeoxycholic acid (TUDCA): %, Oo
TE A
HON OH
H
TUDCA . 28) The method of any of the embodiments 25 to 27, wherein the enzyme is peroxygenase. 29) The method of any of the embodiments 25 to 28, wherein the enzyme is unspecific peroxygenase (UPO). 88
30) The method of any of the embodiments 25 to 29, wherein the peroxide is an organic peroxide, an inorganic peroxide, or both. 31) The method of any of the embodiments 25 to 30, wherein the peroxide is an inorganic peroxide selected from the group consisting of hydrogen peroxide (H>0>), persulfate (S02~ or S, 027), potassium peroxymonosulfate (KHSOs), oxone (2KHSOs- KHSO: KSO), peroxydisulfuric acid (H2S:0s), sodium peroxide (Na:02}), potassium peroxide (K:02), barium peroxide (Ba0:), calcium peroxide (Ca0:), magnesium peroxide (MgO:), lithium peroxide (L1203), and combinations thereof, or the peroxide is an organic peroxide selected a group comprising peracetic acid (CHs;CO:H), tert-butyl hydroperoxide [(CH;3)sCOOH], magnesium monoperoxyphthalate (CisHioMgO1o), 3-chloroperoxybenzoic acid (3-CIC5H:CO:3H), urea hydrogen peroxide (NH;CONH::H:0:), benzoyl peroxide (CisHioO4), tert-butyl peroxybenzoate (CsHsCO3;C(CHs)s), benzoyl peroxide (Ci1sHi00s), di-tert-butyl peroxide [(CH3)sCOOC(CHs);], methyl ethyl ketone peroxide (CgHisOs), diacetyl peroxide
((CH3CO:)2), cumene hydroperoxide (CsHi202), trifluoroperacetic acid (CF:CO:H), and combinations thereof.
32) Use of an enzyme for 7B-hydroxylation of a compound of Formula I or analogue or salt thereof, or converting a compound of Formula I or analogue or salt thereof to the compound of Formula II or analogue or salt thereof,
wherein the enzyme requires peroxide as a co-substrate, and wherein the Formula I and the Formula II are as defined in the preceding embodiments.
33) A reaction mixture or reaction system comprising: (1) a compound of Formula I or analogue or a salt thereof, or a compound of Formula I(a) or analogue or a salt thereof, as defined in the preceding claims; (11) an enzyme; and (11) a peroxide.
34) The use of embodiment 32 or reaction mixture of embodiment 33, wherein the Formula I or a salt thereof, or the compound of Formula I(a) or analogue or a salt thereof is LCA or TLCA.
89
35) The use of embodiment 32 or reaction mixture of embodiment 33, wherein the enzyme is peroxygenase. 36) The use or reaction mixture of any of the embodiments 32 to 35, wherein the enzyme is unspecific peroxygenase (UPO). 37) The use or reaction mixture of any of the embodiments 32 to 35, wherein the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1. 38) The use or reaction mixture of any of the embodiments 32 to 37, wherein the peroxide is an organic peroxide, an inorganic peroxide, or both. 39) The use or reaction mixture of any of the embodiments 32 to 38, wherein the inorganic peroxide selected from the group consisting of hydrogen peroxide (H;0:), persulfate (SOZ~ or S047), potassium peroxymonosulfate (KHSOs), oxone (2KHSO5KHSO: KSO), peroxydisulfuric acid (H2S:0z), sodium peroxide (Na>0.), potassium peroxide (K+0:), barium peroxide (Ba0:}, calcium peroxide (C402}), magnesium peroxide (MgO;), lithium peroxide (L102), and combinations thereof; or the organic peroxide selected from the group consisting of peracetic acid (CH3COzH), tert-butyl hydroperoxide [(CH:)3COOH], magnesium monoperoxyphthalate (Ci6HioMgOi0), 3-chloroperoxybenzoic acid (3-CICsHsCOsH), urea hydrogen peroxide (NH;CONH:H:0:), benzoyl peroxide (CisH1004), tert-butyl peroxybenzoate (CsHsCO3C(CHz)3), benzoyl peroxide (C14Hi003), di-tert-butyl peroxide [(CH;):3COOC(CHs):], methyl ethyl ketone peroxide (CgHisOs), diacetyl peroxide ((CH3CO2)2), cumene hydroperoxide (CsHi202), trifluoroperacetic acid (CF3CO:H), and combinations thereof. 40) The reaction mixture of any of the embodiments 32 to 39, comprising: (1) LCA or TLCA; (n1) unspecific peroxy genase (UPO); and (111) hydrogen peroxide (H202).
41) The method of any of the embodiments 1 to 24, wherein the method comprises 7P- hydroxylation of LCA to produce UDCA 90
4 Oo i u ’
H
H oo
H
LCA
4, 0 8 fy u ’
Ww
HOW OH
H
UDCA comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H>O:) to produce said UDCA, 91 wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the LCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the LCA and the HO» is from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the H:20» is from about 1:100 to about 1:100,000,000.
The foregoing description of the embodiments reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It 1s to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “including” or “includes” or “such as” or “have” or “having” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Throughout this specification, the term ‘combinations thereof’ or ‘and combinations thereof’ or ‘any combination thereof’ or ‘any combinations thereof’ are used interchangeably and are intended to have the same meaning, as regularly known in the field of patents disclosures.
As used herein, the term “comprising” when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and/or after the recited steps. For example, a method comprising steps a, b, and ¢ encompasses a method of steps 92 a, b, x, and c; a method of steps a, b, ¢, and x, as well as a method of steps x, a, b, and c.
Furthermore, the term “comprising” when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, ¢, and b; the order of steps ¢, b, and a; and the order of steps ¢, a, and b, etc.
As used in this specification and the appended claims, the singular forms “a,” “an” and “the” includes both singular and plural references unless the content clearly dictates otherwise. The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The suffix “(s)” at the end of any term 1n the present disclosure envisages in scope both the singular and plural forms of said term.
Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.
The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed. 93
As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and L it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H: AAD, I, AE GA E HAE, IAF, G AF, HAF, 1; B, D,
G:B,D,H;B,D,I,B,E,G;B.EH;B,E, IBF, GBF. H,B,F,I,CD GC DH CDL
C.E, GC EH; CEIC,F,G;C,F, H; CF, L unless specifically mentioned otherwise.
Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application. 94

Claims (15)

CONCLUSIESCONCLUSIONS 1. Werkwijze voor hydroxylering van een verbinding met formule I of een zout daarvan, omvattende het in contact brengen van de verbinding met formule I of een zout daarvan: 4 X “4, R I 1 ” CF 5 > R;0 : : Y Formule I met een enzym in aanwezigheid van een peroxide om een verbinding met formule II of een zout daarvan: X “, R1. Process for hydroxylation of a compound of formula I or a salt thereof, comprising contacting the compound of formula I or a salt thereof: 4 X “4, R I 1 ” CF 5 > R;0 : : Y Formula I with an enzyme in the presence of a peroxide to form a compound of formula II or a salt thereof: 1 . H ”, ~~ > ” R;0 OH Y Formule II te produceren, waarbij R onafhankelijk is gekozen uit de groep bestaande uit (CH:}4C=O(O)nR", alkyl. alkenyl, CH,OH, CHO, (CH2}:CONH(CH:):S0:H, (CH:):CONH(CH:)COOH, -CH=CH-CO:-alkyl, -CH=CH-CO:-aryl, - CH=CH-CO:H en -CH=CH-alkyl, 95 waarbij R' onafhankelijk is gekozen uit de groep bestaande uit waterstof, alkyl. alkenyl, alkynyl, cycloalkyl, aryl en NRR; waarbij Ra en Ry onafhankelijk zijn gekozen uit de groep bestaande uit waterstof, alkyl, aryl, -C(=O)-alkyL -C{=0O)-aryl en (CH:)2S0:H; R; onafhankelijk is gekozen uit de groep bestaande uit waterstof, alkyl, acyl, mesityl, tosyl, -C(=0)- alkyl, -C(=0)-arvl, -S(=0);-alkyl, -S(=0)-arvl. -S(=0),-OH en -P(=0)-(OR;)2, waarbij Ra onafhankelijk waterstof of alkyl is; n 0 of een geheel getal is dat in het bereik ligt van 1 tot 5; m 0 of lis: Y waterstof is; X waterstof, hydroxy of zuurstof is; waarbij R. R’, Rs, Ry, Ri, R2 of X optioneel onafhankelijk gesubstitueerd zijn; —— of een enkele binding of een dubbele binding voorstelt: en op voorwaarde dat: beide ringen A en B niet tegelijkertijd een dubbele binding in de ringen bevatten; wanneer —— een dubbele binding in de ring A is, R; ontbreekt: wanneer ——— een enkele binding in de ring A is, dan R; aanwezig is: wanneer —— een dubbele binding in of de ring A of ring B is. Y ontbreekt; en wanneer —— tegelijkertijd een enkele binding in beide ringen A en B is, Y aanwezig is.1. H ", ~~ > " R;0 OH Y Formula II, where R is independently selected from the group consisting of (CH:}4C=O(O)nR", alkyl. alkenyl, CH,OH, CHO, (CH2}:CONH(CH:):S0:H, (CH:):CONH(CH:)COOH, -CH=CH-CO:-alkyl, -CH=CH-CO:-aryl, - CH=CH -CO:H and -CH=CH-alkyl, 95 where R' is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and NRR; wherein Ra and Ry are independently selected from the group consisting of hydrogen, alkyl, aryl, -C(=O)-alkyl -C{=0O)-aryl and (CH:)2S0:H; R; is independently selected from the group consisting of hydrogen, alkyl, acyl, mesityl, tosyl , -C(=0)-alkyl, -C(=0)-arvl, -S(=0);-alkyl, -S(=0)-arvl. -S(=0),-OH and -P (=0)-(OR;)2, where Ra is independently hydrogen or alkyl; n is 0 or an integer ranging from 1 to 5; m 0 or lis: Y is hydrogen; is oxygen; where R. R', Rs, Ry, Ri, R2 or contain in the rings; when —— is a double bond in the ring A, R; missing: if ——— is a single bond in the ring A, then R; is present: when —— is a double bond in either ring A or ring B. Y missing; and when —— is a single bond in both rings A and B at the same time, Y is present. 2. Werkwijze volgens conclusie 1, waarbij de werkwijze 7B-hydroxylering van de verbinding met formule I of een zout daarvan omvat om de verbinding met formule II of een zout daarvan die een f- hydroxylgroep op positie 7 heeft te produceren; en waarbij het enzym in staat is de verbinding met formule I of een zout daarvan als een substraat en peroxide als een cosubstraat te gebruiken voor het katalyseren van de hydroxylering van de verbinding met formule 1 of een zout daarvan om de verbinding met formule TI of een zout daarvan te produceren.A method according to claim 1, wherein the method comprises 7β-hydroxylation of the compound of formula I or a salt thereof to produce the compound of formula II or a salt thereof having an f-hydroxyl group at position 7; and wherein the enzyme is capable of using the compound of formula I or a salt thereof as a substrate and peroxide as a co-substrate to catalyze the hydroxylation of the compound of formula I or a salt thereof to form the compound of formula T1 or a salt thereof to produce salt from it. 3. Werkwijze volgens conclusie 1 of conclusie 2, waarbij het enzym dat in staat is peroxide als een cosubstraat te gebruiken een enzym van enzymcommissie-klasse (EC-klasse) 1.11 is dat werkt met peroxide als acceptor; of waarbij het enzym dat in staat is peroxide als een cosubstraat te gebruiken een peroxy genase van EC-klasse 1.11.2 is.A method according to claim 1 or claim 2, wherein the enzyme capable of using peroxide as a co-substrate is an enzyme of enzyme committee class (EC class) 1.11 that works with peroxide as an acceptor; or wherein the enzyme capable of using peroxide as a co-substrate is a peroxygenase of EC class 1.11.2. 4. Werkwijze volgens een van de conclusies 1 tot 3, waarbij het enzym niet-specifieke peroxy genase (UPO) is.A method according to any one of claims 1 to 3, wherein the enzyme is non-specific peroxygenase (UPO). 5. Werkwijze volgens conclusie 4, waarbij het enzym niet-specificke peroxygenase (UPO) van ECThe method of claim 4, wherein the enzyme is non-specific peroxygenase (UPO) from EC 1.11.2.1 is.1.11.2.1 is. 6. Werkwijze volgens conclusie 4 of conclusie 5, waarbij de niet-specifieke peroxy genase (UPO) een wildtype-UPO is die wordt verkregen uit schimmels of protoschimmels; of waarbij de niet-specifieke peroxygenase (UPO) een recombinante niet-specifieke peroxygenase (UPO) is die tot expressie wordt gebracht in een gastheercel gekozen uit schimmels en bacteriën. 96A method according to claim 4 or claim 5, wherein the non-specific peroxygenase (UPO) is a wild-type UPO obtained from fungi or protofungi; or wherein the non-specific peroxygenase (UPO) is a recombinant non-specific peroxygenase (UPO) expressed in a host cell selected from fungi and bacteria. 96 7. Werkwijze volgens een van de conclusies 1 tot 6, waarbij het enzym in de vorm is van: i) een enzympoeder of oplossing, ii) geïmmobiliseerd enzym. iii) hele cellen die het enzym tot expressie brengen, iv) een enzymextract of -lysaat van de hele cellen die het enzym tot expressie brengen of v) een willekeurige combinatie van i tot iv.A method according to any one of claims 1 to 6, wherein the enzyme is in the form of: i) an enzyme powder or solution, ii) immobilized enzyme. (iii) whole cells expressing the enzyme, iv) an enzyme extract or lysate from the whole cells expressing the enzyme, or v) any combination of i to iv. 8. Werkwijze volgens een van de conclusies 1 tot 7, waarbij het peroxide een anorganisch peroxide, een organisch peroxide of beide is; of waarbij het peroxide waterstofperoxide (H;0:) is; of waarbij het peroxide wordt verschaft als een zuiver peroxide, een peroxideoplossing of een peroxide dat wordt gegenereerd door een in-situ-peroxidegeneratiesysteem.A method according to any one of claims 1 to 7, wherein the peroxide is an inorganic peroxide, an organic peroxide or both; or wherein the peroxide is hydrogen peroxide (H;0:); or wherein the peroxide is provided as a pure peroxide, a peroxide solution or a peroxide generated by an in-situ peroxide generation system. 9. Werkwijze volgens een van de conclusies 1 tot 8, waarbij het alkyl C:-C::-alkyl is, het alkenyl C:- Cn-alkenyl is, het alkynyl C:-C;2-alkynyl is, het cycloalkyl een koolwaterstofringsysteemradicaal. omvattende C:3-C:9 koolstofatomen, is en het arvl een koolwaterstofringsysteemradicaal. omvattende Ce-Cis koolstofatomen en ten minste één aromatische ring, is. en waarbij het zout met formule I of formule II is gekozen uit de groep bestaande uit natriumzout, kaliumzout en quaternair ammoniumzout (QAS).A process according to any one of claims 1 to 8, wherein the alkyl is C: -C:: alkyl, the alkenyl is C: -C 1 -alkenyl, the alkynyl is C: -C 12 -alkynyl, the cycloalkyl is a hydrocarbon ring system radical. comprising C:3-C:9 carbon atoms, and the arvl is a hydrocarbon ring system radical. comprising Ce-C18 carbon atoms and at least one aromatic ring. and wherein the salt of formula I or formula II is selected from the group consisting of sodium salt, potassium salt and quaternary ammonium salt (QAS). 10. Werkwijze volgens een van de conclusies 1 tot 9, waarbij de werkwijze omvat: a) hydroxylering van een verbinding met formule I(a) om een verbinding met formule TI(a) te produceren, KA 0 . Gi : u Ho Y Formule I(a) 97A method according to any one of claims 1 to 9, wherein the method comprises: a) hydroxylation of a compound of formula I(a) to produce a compound of formula TI(a), KA 0 . Gi : u Ho Y Formula I(a) 97 % 0 A i H ww HON OH Y Formule TI(a) waarbij R" OH, NH(CH:):S0;H.% 0 A i H ww HON OH Y Formula TI(a) where R" OH, NH(CH:):S0;H. NHCH;CO:H, O-alkyl of O-arvl is; of b) hydroxylering van een verbinding met formule I(b) om een verbinding met formule II(b) te produceren, G “, KN R o Formule I(b) 98NHCH;CO:H, O-alkyl or O-arvl; or b) hydroxylation of a compound of formula I(b) to produce a compound of formula II(b), G “, KN R o Formula I(b) 98 “, “, R 0] OH Formule II(b) waarbij R zoals gedefinieerd in conclusie 1 is; of Cc) hydroxylering van een verbinding met formule I{c) om een verbinding met formule II{c) te produceren, 2 9, 2, R a BS EB Ho Formule I(c) 99“, “, R 0] OH Formula II(b) where R is as defined in claim 1; or Cc) hydroxylation of a compound of formula I{c) to produce a compound of formula II{c), 2 9, 2, R a BS EB Ho Formula I(c) 99 1, R H al HOY OH Formule II{c) waarbij R zoals gedefinieerd in conclusie 1 is; of d) hydroxylering van een verbinding met formule I(d) om een verbinding met formule I1(d) te produceren, 4 X Go R i i t . : o Formule I(d) 1001, R H al HOY OH Formula II{c) where R is as defined in claim 1; or d) hydroxylation of a compound of formula I(d) to produce a compound of formula I1(d), 4 X Go R i i t . : o Formula I(d) 100 “, X KN R t t i : : oO OH Formule II(d) waarbij Ren X zoals gedefinieerd in conclusie 1 zijn; of €) hydroxylering van een verbinding met formule I(e) om een verbinding met formule TI(e) te produceren, 4, X , R i A I H > ” pi ” RO Formule I(e) 101“, or €) hydroxylation of a compound of formula I(e) to produce a compound of formula TI(e), 4, % X “, > R H ” ” 5 5 R10 OH Formule Ile) waarbij R, Ri en X zoals gedefinieerd in conclusie 1 zijn; of f) hydroxylering van een verbinding met formule I(f) om een verbinding met formule II(f) te produceren 4 4 OH : : o Formule If) 102% X “, > R H ” ” 5 5 R10 OH Formula Ile) where R, Ri and or f) hydroxylation of a compound of formula I(f) to produce a compound of formula II(f) 4 4 OH : : o Formula If) 102 “a, OH : : o OH Formule II(f) of 2) hydroxylering van een verbinding met formule I(g) om een verbinding met formule II(g) te produceren ó 4 74 : : HO Formule I(g) 103“a, OH : : o OH Formula II(f) or 2) hydroxylation of a compound of formula I(g) to produce a compound of formula II(g) ó 4 74 : : HO Formula I(g) 103 : : HO OH Formule l(g) : of h) hydroxylering van een verbinding met formule I(h) om een verbinding met formule II(h) te produceren 4 7 o Formule I(h) 104: : HO OH Formula I(g) : or h) hydroxylation of a compound of formula I(h) to produce a compound of formula II(h) 4 7 o Formula I(h) 104 % : : o OH Formule I(h} of 1) hydroxylering van een verbinding met formule I(i) om een verbinding met formule II(i) te produceren 4 oO : : oO Formule Ii) 105 o o OH Formule II{i) : of » hydroxylering van een verbinding met formule I(j) om een verbinding met formule II) te produceren, ‘4 “4, 0 Gi BD D oO Formule Ij) 4 A 0 i ®- D oO OH 106% : : o OH Formula I(h} or 1) hydroxylation of a compound of formula I(i) to produce a compound of formula II(i) 4 oO : : oO Formula Ii) 105 o o OH Formula II{i) : or » hydroxylation of a compound of formula I(j) to produce a compound of formula II), '4 “4.0 Gi BD D oO Formula Ij) 4 A 0 i ®- D oO OH 106 Formule II(j) waarbij Rs waterstof, alkyl of aryl is; of k) hydroxylering van een verbinding met formule I(k) om een verbinding met formule II(k) te produceren KA 0 Gi TN BD H SO, Na* CJ ao H Formule I(k) “a, 0 i TN ®- H $0, Na* CIEL HO” OH H Formule TI{k) .Formula II(j) where R5 is hydrogen, alkyl or aryl; or k) hydroxylation of a compound of formula I(k) to produce a compound of formula II(k) KA 0 Gi TN BD H SO, Na* CJ ao H Formula I(k) “a, 0 i TN ®- H $0, Na* CIEL HO” OH H Formula TI{k) . 11. Werkwijze volgens een van de conclusies 1 tot 10, waarbij de werkwijze wordt uitgevoerd bij een temperatuur van ongeveer 5 °C tot ongeveer 80 °C en gedurende een tijdsduur in het bereik van ongeveer minuten tot ongeveer 150 uur; of waarbij de werkwijze wordt uitgevoerd bij een pH van ongeveer 2 tot ongeveer 10; 15 of waarbij de molverhouding van de verbinding met formule I of een zout daarvan en het enzym ongeveer 100:1 tot ongeveer 10.000.000:1 is, de molverhouding van de verbinding met formule I of een zout daarvan en het peroxide ongeveer 1:0,1 tot ongeveer 1:10 is, of de molverhouding van het enzym en het peroxide ongeveer 1:100 tot ongeveer 1:100.000.000 is.The method of any one of claims 1 to 10, wherein the method is carried out at a temperature of about 5°C to about 80°C and for a period of time in the range of about minutes to about 150 hours; or wherein the process is carried out at a pH of about 2 to about 10; 15 or wherein the molar ratio of the compound of formula I or a salt thereof and the enzyme is about 100:1 to about 10,000,000:1, the molar ratio of the compound of formula I or a salt thereof and the peroxide is about 1:0 .1 to about 1:10, or the mole ratio of the enzyme to the peroxide is about 1:100 to about 1:100,000,000. 12. Wetkwijze volgens een van de conclusies 1 tot 11, waarbij de werkwijze 7B-hydroxylering van LCA om UDCA te produceren omvat, 107The method of any one of claims 1 to 11, wherein the method comprises 7β-hydroxylation of LCA to produce UDCA, 107 2 0 A © u ) H ww Ww HON H LCA 4 %, oO KA GL ) H ww ow HO OH H UDCA omvattende het in contact brengen van het LCA met niet-specifieke peroxygenase (UPO) in aanwezigheid van waterstofperoxide (H:0:) om het UDCA te produceren, waarbij de werkwijze wordt uitgevoerd bij een temperatuur van ongeveer 5 °C tot ongeveer 80 °C gedurende een tijdsduur in het bereik van ongeveer 15 minuten tot ongeveer 150 uur en bij een pH van ongeveer 2 tot ongeveer 10, en waarbij de molverhouding van het LCA en de UPO ongeveer 100:1 tot ongeveer 10.000.000:1 is, de molverhouding van het LCA en het H20: ongeveer 1:0,1 tot ongeveer 1:10 is, en de molverhouding van de UPO en het H;0: ongeveer 1:100 tot ongeveer 1:100.000.000 is.2 0 A © u ) H ww Ww HON H LCA 4%, oO KA GL ) H ww ow HO OH H UDCA comprising contacting the LCA with non-specific peroxygenase (UPO) in the presence of hydrogen peroxide (H:0 :) to produce the UDCA, the process being carried out at a temperature of about 5°C to about 80°C for a period of time in the range of about 15 minutes to about 150 hours and at a pH of about 2 to about 10 , and wherein the mole ratio of the LCA and the UPO is about 100:1 to about 10,000,000:1, the mole ratio of the LCA and the H 2 O: is about 1:0.1 to about 1:10, and the mole ratio of the UPO and the H;0: about 1:100 to about 1:100,000,000. 13. Werkwijze voor het omzetten van een verbinding met formule I(a) of een zout daarvan tot een verbinding met formule II(a) of een zout daarvan, omvattende het in contact brengen van de verbinding met formule I(a) of een zout daarvan: 10813. Process for converting a compound of formula I(a) or a salt thereof into a compound of formula II(a) or a salt thereof, comprising contacting the compound of formula I(a) or a salt of which: 108 4 O Z Gi 0 R" H ww HON Y Formule I(a) met een enzym in aanwezigheid van een peroxide om de verbinding met formule TI(a) of een zout daarvan: 4 O 4 i ~ R" H ow HOY OH Y Formule II{a) te produceren, waarbij R" OH, NH(CH:)2S0:H, NHCH:CO:H, O-alkyl of O-aryl is.4 O Z Gi 0 R" H ww HON Y Formula I(a) with an enzyme in the presence of a peroxide to form the compound of formula TI(a) or a salt thereof: 4 O 4 i ~ R" H ow HOY OH Y Formula II{a) where R" is OH, NH(CH:)2SO:H, NHCH:CO:H, O-alkyl or O-aryl. 14. Werkwijze volgens conclusie 13, waarbij: a) de werkwijze het omzetten van lithocholzuur (LCA): 109A method according to claim 13, wherein: a) the method converts lithocholic acid (LCA): 109 4 oO 0 u ’ Ww HOW | : H LCA tot ursodeoxycholzuur (UDCA): A oO Gi u ) H aw HON OH H UDCA omvat; of b) de werkwijze het omzetten van taurolithocholzuur (TLCA): 1104 oO 0 h ’ Ww HOW | : H LCA to ursodeoxycholic acid (UDCA): A oO Gi u ) H aw HON OH H UDCA includes; or b) the process of converting taurolithocholic acid (TLCA): 110 “, 0 Gi) i TN so H Ho” H TLCA tot tauroursodeoxycholzuur (TUDCA): 4 A 0 Gi) No H 7 HO OH H TUDCA omvat; of Cc) het enzym niet-specifieke peroxy genase (UPO) is; of d) het peroxide waterstofperoxide (H:0:) is.“, 0 Gi) i TN so H Ho” H TLCA to tauroursodeoxycholic acid (TUDCA): 4 A 0 Gi) No H 7 HO OH H TUDCA includes; or Cc) the enzyme is non-specific peroxygenase (UPO); or d) the peroxide is hydrogen peroxide (H:0:). 15. Gebruik van een enzym voor 7f-hydroxylering van een verbinding met formule I of een zout daarvan 19 om een verbinding met formule II of een zout daarvan te produceren, waarbij het enzym niet-specifieke peroxy genase (UPO) is, en waarbij de formule I en de formule II zoals gedefinieerd in conclusie 1 zijn. 11115. Use of an enzyme for 7β-hydroxylation of a compound of formula I or a salt thereof 19 to produce a compound of formula II or a salt thereof, wherein the enzyme is non-specific peroxygenase (UPO), and wherein the formula I and formula II are as defined in claim 1. 111
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