Patent Application
20230212532
The present invention relates to novel recombinant Bacillus megaterium cytochrome P450-monooxygenase (P450-BM3) variants for the C19 hydroxylation of steroids and derivatives thereof or for improved BM3 protein expression. In particular, the present invention also relates to methods and processes using P450-BM3 variants for the production of estrone and estradiol. The invention further relates to nucleotide sequences, constructs and vectors for the expression of these P450-BM3 variants.
The steroid hormones estrone and estradiol and their derivatives are used as medications, for instance in menopausal hormone therapy, contraception and oncology or are important intermediates for the synthesis of further steroid products. Estrone and estradiol can be generated in several steps starting from phytosterols. The different degradation products of phytosterols contain a methyl group in position C19 of the steroid ring systems which needs to be removed in order to synthesize estrone and estradiol and their derivatives under aromatization of the A ring of the steroid ring system. Inhoffen describes this aromatization by thermolysis for the synthesis of estradiol (Inhoffen 1947) and Hershberg et al describe this aromatization by thermolysis for the synthesis of estrone (Hershberg 1950). However, yields are not optimal and various side products are formed. Templeton et al. and Numazawa et al. describe this aromatization through steroid products which have been oxidized in position C19 (F. Templeton 1997), (Mitsuteru Numazawa 2009). Therefore, processes hydroxylating the methyl group in position C19 and thus allowing for further derivatization and eventually removal of the substituent in position 19 can pave the way for novel and improved syntheses of estrone and estradiol and their derivatives.
Enzymes as catalysts are typically characterized by an excellent regio- and/or stereoselectivity for the natural substrates but may show low thermal and solvent stabilities. Due to a narrow substrate spectrum, enzymes are often very specialized compared to chemical catalyst, which limits their broad applicability. It is therefore a challenging endeavor to find or build an enzyme which catalyzes a specific reaction with commercially exploitable yield and sufficient purity.
Cytochrome P450 monooxgenases (P450s) comprise a large group of heme enzymes that are ubiquitous in the natural world. Cytochrome P450 BM3 (P450-BM3, BM3), obtained from Bacillus megaterium has previously been described to catalyze the NADPH-dependent hydroxylation of long-chain fatty acids, alcohols, and amides, as well as the epoxidation of unsaturated fatty acids (see e.g. (Narhi 1986) and (Capdevila 1996)). For P450-BM3 the reductase (65 kDa) and monooxygenase (55 kDa) domains of the enzyme are fused and produced as a catalytically self-sufficient 120 kDa enzyme. In consequence, P450-BM3 enzymes exhibit the highest rate of catalysis amongst P450 monooxygenases due to the efficient electron transfer between the fused reductase and heme domains, see e.g., (Noble MA 1999); and (Munro 1996).
BM3 variants have been described for various biotransformation processes. Wild-type and mutant P450 BM3 were applied as biocatalysts in the production of chemicals, including pharmaceuticals (Hazel M Girvan 2016). For example, (WO200107630A1) discloses processes for the microbiological oxidation of different organic substrates, such as N-heterocyclic aromatic compounds, particularly methods for the preparation of indigo and indian ruby using special cytochrome P450-monooxygenases with altered substrate specificity. (EP1196603A1) claims a P450 BM3 variant with an altered profile in enzymatic hydroxylation of aliphatic carboxylic acids, owing to site-specific mutagenesis of its substrate-binding region, wherein F87 is replaced by Val, Ala or Leu, and L188 is replaced by Asn, Gln, Arg, Lys, Ala, Gly, Ser or Trp, and optionally at least one of amino acid positions 26, 47, 72, 74 and 354 is altered. (WO2016007623A1) discloses cytochrome P450 BM3 variants to obtain improved activity with regard to substrates selected from nifedipine, propranolol, verapamil and diclofenac. (EP1131440A2) discloses a process for oxidising a substrate which is an acyclic or cyclic terpene selected from monoterpenes, sesquiterpenes and diterpenes, or a cycloalkene; or a substituted derivative thereof, which process comprises oxidising said compound with a mutant P450 BM3 enzyme, the mutant comprising the substitution of an amino acid in the active site by an amino acid with a less polar side-chain.
None of the mentioned documents discloses the specific structure of the BM3 variants according to the current invention. Furthermore, none of the above indicated documents relates to the C19 hydroxylation of steroid derivatives, e.g. for the production of estradiol or estrone. Instead it has been described that BM3 wildtype (WT) does not accept testosterone nor steroids in general as a substrate.
(Kille Sabrina 2011), (Kille 2010) and (Acevedo-Rocha 2018) have described a library of P450 BM3 variants and the use of directed evolution of P450 BM3. Acevedo-Rocha et al. have reported the directed evolution of P450 BM3 for the selective hydroxylation of testosterone. With the resulting mutants, however, relevant amounts of hydroxylation of testosterone were obtained only at positions 16α/β. These findings discouraged further developments of BM3 variants for C19 hydroxylation, because there was a prejudice to obtain only trace amounts at the position C19 due to preferential hydroxylation at other positions (Acevedo-Rocha 2018).
According to the current invention it was nevertheless surprisingly found that specific novel variants of bacterial cytochrome P450 BM3 monooxygenases (CYPs) can be used to catalyze the efficient synthesis of estrone and estradiol and their derivatives through C19-hydroxylation, also in an efficient and commercially exploitable way. In particular, it was surprisingly found that C19-hydroxylation of substrates testosterone, delta-1-testosterone and androsta-1,4-dien-3,17-dione can be achieved using mutants of bacterial cytochrome P450 BM3 monooxygenases as biocatalysts.
These uses can be further supported by incorporating mutations into the BM3 varaints which improve the protein expression of BM3.
According to a first aspect of the invention there is provided a Cytochrome P450 BM3 monooxygenase (BM3) variant for catalyzing the C19 hydroxylation of a steroid or steroid derivative. According to a first embodiment of the first aspect, the BM3 variant comprises the mutation F87A and at least one and preferably two further mutations selected from (i) a mutation at position V78, preferably V78F, V78Y, V78M, V78l or V78L, (ii) a mutation at position A82, preferably A82E, A82Q or A82P. According to a second aspect of the current invention, there is provided a nucleic acid encoding for a Cytochrome P450 BM3 monooxygenase variant as described herein. According to a third aspect of the current invention, there is provided a host cell for the production of a Cytochrome P450 BM3 monooxygenase variant. In a preferred embodiment, the host cell comprises a nucleic acid encoding for a BM3 variant according to any of the aspects dercribed herein. According to a fourth aspect of the current invention, there is provided the use of a Cytochrome P450 BM3 monooxygenase (BM3) variant for the production of a compound according to formula I, wherein R1 and R2 form a six-membered ring as part of a steroid.
According to a fifth aspect of the current invention, there is provided the use of a BM3 variant for the C19-hydroxylation of a steroid or steroid derivative. According to a sixth aspect of the current invention, there is provided a process for C19-hydroxylation of a steroid or derivative thereof comprising (i) (a) culturing a recombinant Cytochrome P450 BM3 monooxygenase (BM3) variant producing microorganism in a culture medium, in the presence of an exogenous or intermediately formed substrate; or (b) incubating a substrate-containing reaction medium with a Cytochrome P450 BM3 monooxygenase; and (ii) isolating the oxidation product formed, or a secondary product thereof, from the medium; said process being further characterized in that said BM3 variant is a BM3 variant as described herein. According to a seventh aspect there is provided a method for obtaining optimized BM3 variants for the C19 hydroxylation of steroids, said method comprising (i) (a) culturing a recombinant microorganism expressing a BM3 variant (test variant) in a culture medium, in the presence of an exogenous or intermediately formed steroid or steroid derivative; or (b) incubating a steroid or steroid derivative-containing reaction medium with a BM3 variant (test variant); and (ii) comparing the obtained product yield and/or selectiviy for the C19 hydroxylation product formed by the test variant or a secondary product thereof with the respective value obtained for a parent variant of the test variant capable of catalyzing the C19 hydroxylation of a steroid, and (iii) selecting the test variant as optimized for the C19 hydroxylation of steroids, if the test variant has an improved product yield/and or selectivity compared to the parent variant. According to a further aspect, there are provided BM3 variants characterized by an improved BM3 protein expression relative to BM3 wildtype (WT), wherein the BM3 variant comprise specific mutations.
The Sequence Listing associated with this application is filed in electronic format and hereby incorporated by reference into the specification in its entirety.
Unless otherwise defined, all scientific and technical terms used in the description, figures and claims have their ordinary meaning as commonly understood by one of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control. The materials, methods, and examples are illustrative only and not intended to be limiting. Unless stated otherwise, the following terms used in this document, including the description and claims, have the definitions given below.
The terms “comprising”, “including”, “containing”, “having” etc. shall be read expansively or open-ended and without limitation. Singular forms such as “a”, “an” or “the” include plural references unless the context clearly indicates otherwise. Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. The terms “at least one” and “at least one of” include for example, one, two, three, four, five, six, seven, eight, nine, ten or more elements.
It is furthermore understood that slight variations above and below a stated range can be used to achieve substantially the same results as a value within the range. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values.
Where protein or amino acid sequences are provided throughout the application it is also understood by the skilled person that single or multiple amino acids may be exchanged by amino acids with similar properties to achieve substantially the same effect, i.e.an equivalent result. The skilled person furthermore knows that a defined protein or amino acid sequence may be encoded by various nucleic acid sequences. For a given amino acid sequence as defined herein, each of the countable nucleic acid sequences encoding the specific amino acid sequence shall be deemed to be disclosed herein. Where nucleic acid sequences are provided throughout the application it is furthermore understood that silent mutations may be introduced.
The terms “peptide”, “polypeptide”, and “protein” are used interchangeably herein, and refer to a compound which comprises at least two amino acid residues covalently linked by at least one peptide bond. No limitation is placed on the maximum number of amino acids that can comprise a peptide’s sequence. A “peptide” may comprise without limitation modified amino acids, non naturally-occuring amino acids and/or D amino acids. Unless otherwise indicated, a particular peptide sequence also encompasses variants wherein at least one amino acid has been replaced by an amino acid which is characterized by similar structural properties. A “peptide” may be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof. A “peptide” may be, for example, a biologically active fragment, an oligopeptide, a homodimer, a heterodimer, a peptide variant, a modified peptide, a peptide derivative, a peptide analog, a fusion protein, among others.
The term “amino acid” or “amino acid residue” (“aa”) as used herein typically refers to a naturally-occuring amino acid but may also refer to a non naturally-occuring amino acid. The term typically refers to an L-amino acid but may also encompass a D-amino acid. An amino acid may or may not be modified as described elsewhere herein. The one letter code is used herein to refer to the respective amino acid. As used herein, a “charged amino acid” is an amino acid which is negatively charged or positively charged. “Negatively charged amino acids” are aspartic acid (D) and glutamic acid (E). “Positively charged amino acids” are arginine (R) lysine (K) and histidine (H). “Polar amino acids” are all amino acids that form hydrogen bonds as donors or acceptors. These are all charged amino acids and asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y) and cysteine (C). “Polar uncharged amino acids” are asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y) and cysteine (C). “Amphiphatic amino acids” are tryptophan (W), tyrosine (Y) and methionine (M). “Aromatic amino acids” are phenylalanine (F), tyrosine (Y), and tryptophan (W). “Hydrophobic amino acids” are glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M) and cysteine. “Small amino acids” are glycine (G), alanine (A), serine (S), proline (P), threonine (T), aspartic acid (D) and asparagine (N). Two amino acids are “characterized by similar structural properties” if (a) both are charged amino acids, preferably both are negatively charged amino or both are positively charged amino acids, (b) both are polar amino acids, (c) both are polar uncharged amino acids, (d) both are amphiphatic amino acids, (e) both are aromatic amino acids, (f) both are hydrophobic amino acids, or (g) both are small amino acids. As understood by the skilled person, where an amino acid sequence is provided throughout the disclosure, amino acids characterized by similar structural properties may be exchanged with each other to achieve substantially the same or an equivalent result.
The terms “cytochrome P450 BM3 monooxygenase”, “cytochrome P450-BM3”, “P450-BM3” (also BM3, BM-3) refer to the cytochrome P450 enzyme obtained from Bacillus megaterium that catalyzes the hydroxylation of long-chain fatty acids, alcohols, and amides, as well as the epoxidation of unsaturated fatty acids. Without being bound by theory, P450-BM3 is naturally expressed by Bacillus megateriumstrains ATCC 14581, DSM 32, JCM 2506, NBRC 15308, NCIMB 9376, NCTC 10342, VKM B-512. Alternative names comprise “Bifunctional cytochrome P450/NADPH--P450 reductase”. The P450-BM3 protein is encoded by the gene cyp102A1. Sequence(s) are accessible via UniProt Identifier P14779 (CPXB_BACMB). Different isoforms and variants may exist for the different strains and are all comprised by the term. Where a specific mutation can be exchanged without changing the described catalytic properties of the intitial sequence, it is clear that the sequence having such a functionally silent mutation is equivalent with regard to the initial sequence. In addition, the protein may furthermore be subject to various modifications, e.g, synthetic or naturally occurring modifications.
The gene encoding the cytochrome P450-monooxygenases (P450-BM3) from Bacillus megaterium was originally amplified from genomic DNA by PCR and subsequently cloned using restriction endonucleases Ncol and Sacl into the polylinker sequence of plasmid pETM11, resulting in pETM11-BM3 (Kille 2010). This procedure adds a hexa-histidine tag and an 18 amino acid linker sequence to the N-terminus of the resulting protein, which is however not essential for function. Introducing the Ncol site at the 5′ end of the gene sequence resulted in alteration of the second codon of the P450-BM3 gene and to an amino acid exchange in the resulting protein (T1A).
An amino acid position is typically given by specifying the number of the respective position behind the amino acid of the wildtype using the one letter code (e.g. V78 where the wildtype has a valine at position 78). A list of comma separated letters in squared brackets specifies the mutation or amino acid exchange. For example, where at position 78 of the wildtype variant the valine is replaced by any one of isoleucine, leucine, or methionine, or is not mutated at all, this is abbreviated as V78[I, L, M, V].
Throughout this application counting of amino acid positions of the P450-BM3 amino acid sequence starts with the first amino acid that appears in the protein sequence underlying the crystal structure of a P450-BM3 variant (PDB ID: 2UWH) that was used for designing protein libraries. This is also common practice in P450-BM3-related literature, see e.g. (Kille 2010), (Kille Sabrina 2011), (Acevedo-Rocha 2018). The N-terminal methionine is ignored such that counting starts with threonine 1, which was converted to an alanine in the cloning process of pETM11-BM3 and which is codon 28 according to the SEQ IDs listed herein.
The term “P450-BM3 variant” refers to a P450-BM3 comprising at least one mutation relative to the wildtype sequence. In particular, the term refers to proteins having at least 60%, 70%, 80% or 90% sequence identity with the P450-BM3 wildtype (SEQ ID No. 1). Preferably, P450-BM3 variants are functional, i.e. catalytically active with regard to at least one substrate. Where mutations of a variant are specified, these mutations are restrictive, i.e. a BM3-268; M177Y, A184Y (SEQ ID No. 15) variant having up to six mutations is required to have the mutations M177Y and A184Y.
A “daughter variant” of a “parent variant” is a variant having the sequence of its parent variant and at least one further mutation. A daughter variant can have multiple parent variants.
According to the IUPAC definition “steroids” are naturally occurring compounds and synthetic analogues, which are based on the cyclopenta[a]phenanthrene carbon skeleton, and can be partially or completely hydrogenated. Usually but not necessarily, there are methyl groups at C-10 and C-13, and often but not necessarily there is an alkyl group at C-17. By extension, one or more bond scissions, ring expansions and/or ring contractions of the skeleton may or may not have occurred (IUPAC kein Datum). Throughout this application, steroids are numbered and rings are lettered as in the following formula:
When the rings of a steroid are denoted as projections onto the plane of the paper, the formula is normally to be oriented as in the follwing formula:
An atom or group attached to a ring depicted as in this orientation is termed “alpha” if it lies below the plane of the paper or “beta” if it lies above the plane of the paper. Examples for steroids include androgens, estrogens, and progestogens, corticosteroids, glucocorticoids, mineralocorticoids, cholesterol, estradiol, testosterone, dexamethasone, lanosterol, progesterone, medrogestone, β-sitosterol.
The term steroid derivative refers to molecules derived from a steroid, i.e. molecules comprising a cyclopenta[a]phenanthrene carbon skeleton.
The term “C19 hydroxylation” refers to the introduction of a hydroxyl group (-OH) into a steroid or steroid derivative at the position C19.
The “selectivity” of a biotransformation process or enzyme can be determined based on the selectivity factor or target product ratio. Other methods to determine selectivity are known to the skilled person. In case of doubt the selectivity factor as defined herein shall control.
The “selectivity factor” of a reaction is a measure for the selectivity of the reaction and is calculated by dividing the measured product concentration by the consumed substrate concentration (which is the difference between the initial substrate concentration and the measured substrate concentration after the biotransformation process). Product and substrate concentration can be measured as known in the art and as described in the examples. Usual methods comprise HPLC analyses using substrate and product standards as references and for quantification. If not specified otherwise, the selectivity factor for the C19 hydroxylation of at least one steroid or derivative thereof is calculated based on the steroid or derivative thereof (for the consumed substrate concentration) and the C19 hydroxylated steroid or derivative thereof (for the product concentration). Where the C19 hydroxylated steroid is further converted into a secondary product, selectivity factor may also be calculated with regard to the commercially relevant product.
The “target product ratio” (TPR) of a reaction is calculated as integrated area (in HPLC chromatogram) for the desired product divided by the sum of all integrated areas for products. The product may be the C19 hydroxylated steroid. Where the C19 hydroxylated steroid is further converted into a secondary product, target product ratio may also be calculated with regard to the commercially relevant product. For example, with regard to the conversion from ADD to the product estrone, the area under the curve for estrone was divided by the area under the curve for all other products (i.e. not for the educt ADD). In case of doubt, reference is made to the various examples described throughout the disclosure.
The “product yield” is calculated by dividing the measured product concentration by the initial substrate concentration in the experiment. The product yield for a given P450-BM3 variant is therefore specific for a given substrate and a given product. For example, the substrate may be a steroid and the product may be the C19 hydroxylated steroid. Where the C19 hydroxylated steroid is further converted into a secondary product, product yield may also be calculated with regard to the commercially relevant product.
The “product titer” is provided in mg/L and is an alternative measure to describe the obtainable yields for a biotransformation product. If not specified elsewhere herein, product titer is determined under standard conditions as described in Example 2.
The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. For example and without limitation nucleic acids may occur in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., (1991); Ohtsuka et al., (1985); Rossolini et al., (1994)).
“Sequence identity”, “percent identity” or “percent (%) sequence identity” describes how similar a query sequence is to a target sequence, more precisely how many characters in each sequence are identical after alignment. Sequence identity can be calculated using BLAST (basic local alignment search tool, NCBI), which performs comparisons between pairs of sequences, searching for regions of local similarity. Suitable alignment methods are known in the art, e.g. Needleman-Wunsch algorithm for global-global alignment, using BLOSUM62 matrix, with gap opening penalty of 11 and a gap extension penalty of 1. Afterwards, the pairs of aligned identical residues can be counted and then divided by the total length of the alignment (including gaps, internal as well as external) to arrive at the percent identity value.
A “host cell” is a cell that is used in to receive, maintain, reproduce and amplify a vector. A host cell can also be used to express the polypeptide encoded by the vector. The nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acids.
The term “expression system” refers to any protein production system known in the art and suitable for production of P450-BM3 variants according to the current invention. Suitable expression systems are well known in the art and example systems comprise E. coli strains such as DH5alpha, BL21 (DE3), or Rosetta (DE3), but also various other bacterial and non bacterial systems. Suitable expression systems include gram positive bacteria, such as Bacillus or Rhodococcus, such as Bacillus megaterium or Bacillus subtilis.
The term “vector”, as used herein, is known in the art and refers to a nucleic acid molecule capable of propagating a nucleic acid molecule to which it is linked. The term further comprises plasmids (non-viral) and viral vectors.
According to a first aspect of the invention there is provided a Cytochrome P450 BM3 monooxygenase (BM3) variant for catalyzing the C19 hydroxylation of a steroid or steroid derivative.
According to a first embodiment of the first aspect, the BM3 variant comprises the mutation F87A and at least one and preferably two further mutations selected from
These BM3 may or may not comprise further mutations.
For example the mutation according to (i) can be V78F and the mutation according to (ii) can be A82E, A82Q or A82P. For example the mutation according to (i) can be V78Y and the mutation according to (ii) can be A82E, A82Q or A82P. For example the mutation according to (i) can be V78M and the mutation according to (ii) can be A82E, A82Q or A82P. For example the mutation according to (i) can be V78I and the mutation according to (ii) can be A82E, A82Q or A82P. For example the mutation according to (i) can be V78L and the mutation according to (ii) can be A82E, A82Q or A82P. For example the mutation according to (ii) can be A82E and the mutation according to (i) can be V78F, V78Y, V78M, V78l or V78L. For example the mutation according to (ii) can be A82Q and the mutation according to (i) can be V78F, V78Y, V78M, V78l or V78L. For example the mutation according to (ii) can be A82P and the mutation according to (i) can be V78F, V78Y, V78M, V78l or V78L.
The optional further mutations may comprise a mutation at positon M177 and/or A184. For example the mutations at positon M177 and/or A184 can be M177Y and/or A184Y.
Generation of enzyme variants according to the current invention may be performed as known in the art. For example, mutations can be introduced into the nucleic acid sequences which encode the BM3 wildtype or BM3 variants by any means appropriate for replacing nucleotides in nucleic acid sequences. A useful method for preparing a mutated nucleic acid sequence according to the invention and the corresponding protein comprises carrying out site-directed mutagenesis on codons encoding one or more amino acids which are selected in advance, thereby changing the selected codons in a way that they encode for different amino acids. The methods for obtaining these site-directed mutations are well known to the skilled person and widely described in the literature (in particular: (McPherson 1991)). Various kits are commercially available, for example the QUIKCHANGE™ lightening mutagenesis kit from Qiagen or Stratagene.
According to a second embodiment of the first aspect, there are provided variants which may or may not be variants according to the first embodiment. The P450-BM3 variants according to the second embodiment comprise
In some highly preferred of these second embodiments, the P450-BM3 variant comprises S72G. In some highly preferred of these second embodiments, the P450-BM3 variant comprises T146F. In some highly preferred of these second embodiments, the P450-BM3 variant comprises M177Y. In some highly preferred of these second embodiments, the P450-BM3 variant comprises V178W. In some highly preferred of these second embodiments, the P450-BM3 variant comprises V178P. In some highly preferred of these second embodiments, the P450-BM3 variant comprises L181Y. In some highly preferred of these second embodiments, the P450-BM3 variant comprises A184Y. In some highly preferred of these second embodiments, the P450-BM3 variant comprises L188F. In some highly preferred of these second embodiments, the P450-BM3 variant comprises G457S. In some highly preferred of these second embodiments, the P450-BM3 variant comprises 1458G. In some highly preferred of these second embodiments, the P450-BM3 variant comprises A477N. Except where obviously incompatible with each other, these highly preferred embodiments can be combined.
The BM3 variants according to the second embodiment may or may not comprise further mutation(s).
Some preferred of the variants according to the second embodiment of the first aspect comprise at least one further mutation, preferably at least one, two, three, four, five, six, seven, eigth, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more further mutations.
While the P450-BM3 wild type as well as hundreds of tested variants from literature or generated de novo were not able to catalyze the C19 hydroxylation of steroids or steroid derivatives, the variants according to the first aspect were surprisingly found to be suitable catalysts for the efficient C19 hydroxylation of steroids or derivatives thereof.
Without being bound by theory, the described variants according to the aspect at hand, in particular the variants according to SEQ ID No. 2 to 28 and 117 to 119 are technically related, such that they induce a tertiary protein structure which enables not only the binding of steroids but also their efficient C19 hydroxylation, without supporting side reactions, such as other oxidation reactions, e.g. as mentioned herein.
In brief, the P450-BM3 variants according to the first aspect recognize steroids as substrates, in particular steroids comprising a 1,4-dien-3-one-A-ring, or steroids comprising a 4-en-3-one-A-ring. Furthermore, the P450-BM3 variants according to the first aspect can catalyze the C19 hydroxylation of these steroids.
Without being bound by theory, for steroids with a 1,4-dien-3-one-A-ring such as (17beta)-17-hydroxyandrosta-1,4-dien-3-one (synonyms: 1-dehydrotestosterone, Δ1-testosterone, boldenone) or androsta-1,4-dien-3,17-dione (synonym: ADD) a C19-hydroxylation is believed to lead to an instable intermediate undergoing immediate aromatization and formation of estradiol or estrone and their derivatives.
Example 8, Table E2 show the obtained screening yields of estradiol based on delta-1-testosterone as a substrate for P450-BM3 variants BM3-254 (SEQ ID No. 2), BM3-261 (SEQ ID No. 3), BM3-263 (SEQ ID No. 4) and BM3-268 (SEQ ID No. 5).
Without being bound by theory, for steroids with a 4-en-3-one-A-ring like (17beta)-17-hydroxyandrost-4-en-3-one (synonym: testosterone) the product of C19-hydroxylation is typically stable and can be isolated. These kind of products can then by further functionalization under dehydrogenation form the same instable intermediate and subsequently under aromatization estradiol and estradiol derivatives (as shown for similar estrone derivatives by (F. Templeton 1997).
Example 9, Table E5 show the obtained screening yields for C19 hydroxylated products based on testosterone as a substrate for P450-BM3 variants BM3-254 (SEQ ID No. 2), BM3-261 (SEQ ID No. 3), BM3-263 (SEQ ID No. 4) and BM3-268 (SEQ ID No. 5).
In consequence, the described P450-BM3 variants are suitable catalysts for the C19-hydroxylation of steroids, thereby enabling production of compounds of formula (I)
wherein R1 and R2 form a six-membered ring as part of a steroid. In particular, the described P450-BM3 variants according to the first aspect are suitable catalysts for the C19-hydroxylation of steroids, thereby enabling production of compounds of formula (II)
wherein R3 is a beta-hydroxy group (-OH) or an oxo group (=O).
According to some highly preferred embodiments the P450-BM3 variants according to the first aspect catalyze the C19-hydroxylation of steroids for the production of compounds of formula (I) wherein the formula (I) is preferably formula (II).
Without being bound by theory, by using the P450-BM3 variants as catalysts, the compounds of formula (I) and (II) can thus be synthesized in the following ways:
Estradiol and estradiol derivatives can be oxidized to estrone and estrone derivatives, e.g. as described by (Kawahara R 2012).
According to a third embodiment of the first aspect, there are provided variants which are characterized by a product yield for at least one C19 hydroxylated steroid or derivative thereof or a secondary product thereof which is higher than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 92, 92, 93, 94 or 95%.
For example, said product yield for the at least one C19 hydroxylated steroid or derivative thereof, or a secondary product thereof can be > 5%, preferably > 8%, >10%, or > 12%, even more preferably > 15%, >20%, >25%, > 30%, >35%, > 40%, > 45%, or > 50%, most preferably > 55%, > 60%, > 65%, > 70% or > 75%. For example, said product yield can be the product yield for the product estradiole or estrone, or derivatives thereof. For example, said steroid or steroid derivative can be a a steroid or steroid derivative comprising a 1,4-dien-3-one-A-ring or a 4-en-3-one-A-ring, such as d1-testosterone or ADD.
The third embodiment according to the first aspect can be and is suggested to be combined with each embodiment or example according to the first aspect and in particular the first, second, fourth, fifth or sixth embodiment of the first aspect.
For example there is provided a BM3 variant comprising the mutation F87A and at least one and preferably two further mutations selected from
For example, the BM3 variant may comprise the mutations M177Y and/or A184Y.
For example there is provided a BM3 variant comprising
Table A1 shows estradiol product yields for d1-testosterone to estradiol conversion from 50 mg/L substrate for variants derived from the parent type V78L, A82E, F87A (SEQ ID No. 5) having further mutations as depicted.
Table A2 shows estradiol product yields for Δ1-testosterone to estradiol conversion from 100 mg/L substrate for variants derived from the parent type V78L, A82E, F87A, M177Y, A184Y (SEQ ID No. 15) having further mutations as depicted.
Table A3 shows product yield for estradiol obtained in biotransformation reactions with BM3-268; M177Y, A184Y (SEQ ID No. 15) variants having up to six mutations. 100 mg/L Δ1-testosterone were used as substrate.
Table A4 shows estrone product yields for ADD to estrone conversion from 100 mg/L substrate for variants derived from the parent type BM3-268; M177Y, A184Y (SEQ ID No. 15) having further mutations as depicted.
Table A5 shows the product yield for estrone obtained in biotransformation reactions with SEQ ID No. 15 derived variants having up to six mutations. 100 mg/L ADD were used as substrate.
Table A6 shows estrone product titers for ADD to estrone conversion from 2000 mg/L substrate for variants derived from the parent type BM3-268; M177Y, A184Y; S72G, V178P, L181Y, L188F (SEQ ID No. 117) having further mutations as depicted.
According a fourth embodiment of the first aspect, there are provided variants which are characterized by a selectivity factor for the C19 hydroxylation of at least one steroid or derivative thereof, which is higher than 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98 or 0.99.
For example, said selectivity factor for the C19 hydroxylation of a steroid or steroid derivative can be > 0.1, preferably > 0.2 or > 0.3, even more preferably > 0.4 or > 0.5, most preferably > 0.5, > 0.6, > 0.7, > 0.8 or > 0.9. For example, said steroid or steroid derivative can be a a steroid or steroid derivative comprising a 1,4-dien-3-one-A-ring or a 4-en-3-one-A-ring, such as d1-testosterone or ADD.
The fourth embodiment according to the first aspect can be and is suggested to be combined with each embodiment according to the first aspect and in particular with the first, second, third, fifth, or sixth embodiment of the first aspect.
For example there is provided a BM3 variant comprising the mutation F87A and at least one and preferably two further mutations selected from
Preferably, the BM3 variant comprises the mutations M177Y and/or A184Y.
For example there is provided a BM3 variant comprising
The P450-BM3 parent variants, in particular the variants according to SEQ ID No. 2 to 5 and variants derived thereof catalyze not only C19 hydroxylation but also further oxidative reactions. Significant generation of products with C19-hydroxylation and an additional oxidation in other positions of the steroid molecule was observed. With testosterone as substrate (2beta)-2,19-dihydroxytestosterone and (15beta)-15,19-dihydroxytestosterone were isolated, with delta-1-testosterone as substrate (1beta,2beta)-1,2-epoxy-19-hydroxytestosterone was isolated.
Thus, for example the P450-BM3 variants may catalyze formation of (6beta)-OH, (15beta)-OH or (1 beta,2beta)-1 ,2-epoxy derivatives either instead of or in combination with C19 hydroxylation (see Examples 8, 9, Tables E3, E5). The formation of these side products leads to an impaired selectivity factor. However, for the listed BM3 parent variants it was surprisingly found that even where side reactions occur, the resulting C19 hydroxylation (overoxidation) products may still be relevant for the commercial steroid synthesis. Compared with their respective parent variant the variants according to the fourth embodiment of the first aspect were superior with regard to selectivity.
Table B1 shows estradiol selectivity factors for d1-testosterone to estradiol conversion from 50 mg/L substrate for variants derived from the parent type BM3-268 (SEQ ID No. 5) having further mutations as depicted.
Table B2 shows estradiol selectivity factors for Δ1-testosterone to estradiol conversion from 100 mg/L substrate for variants derived from the parent type BM3-268; M177Y, A184Y (SEQ ID No. 15) having further mutations as depicted.
Table B3 shows selectivity factors for estradiol obtained in biotransformation reactions from 100 mg/L Δ1-testosterone as substrate with BM3-268; M177Y, A184Y (SEQ ID No. 15) variants having up to six mutations.
Table B4 shows estrone selectivity factors for ADD to estrone conversion from 100 mg/L substrate for variants derived from the parent type BM3-268; M177Y, A184Y (SEQ ID No. 15) having further mutations as depicted.
Table B5 shows the selectivity factor for estrone obtained in biotransformation reactions from 100 mg/L ADD with BM3-268; M177Y, A184Y (SEQ ID No. 15) derived variants having up to six mutations.
Table B6 shows the target product ratio (TPR) for ADD to estrone conversion from 2000 mg/L substrate for variants derived from the parent type BM3-268; M177Y, A184Y; S72G, V178P, L181Y, L188F (SEQ ID No. 117) having further mutations as depicted.
Provided are variants according to a fifth embodiment according to the first aspect, wherein said P450-BM3 variant comprises further mutation(s) at at least one, two, three, four, five, six, seven, eight, nine, ten or more position(s). In particular, the variants according to the fifth embodiment can be variants according to the first or second embodiment of the first aspect, such as variants derived from variants according to SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 117, 118 or 119.
Preferrably, said position(s) are selected from (A184), (A191), (A221), (A264), (A321), (A328), (A33), (A330), (A399), (A74), (D168), (D208), (D222), (D363), (E13), (E143), (E267), (E352), (E64), (E82), (E93), (F173), (F205), (F261), (F331), (F393), (F77), (F81), (G240), (G271), (G402), (G415), (G570), (G677), (G85), (H266), (H659), (I153), (I174), (I258), (I259), (I263), (I401), (K210), (K224), (L150), (L181), (L188), (L20), (L262), (L272), (L29), (L324), (L333), (L356), (L437), (L75), (L78), (L86), (M118), (M177), (M185), (M212), (M354), (N70), (P243), (P326), (P329), (P392), (P9), (R147), (R161), (R190), (R203), (R255), (R323), (R47), (R50), (S164), (S176), (S54), (S72), (S89), (T146), (T149), (T260), (T268), (T269), (T327), (T365), (T436), (T438), (T49), (T88), (V178), (V26), (V299), (V314), (V371), (V48), (W325), (W367), (W90), (Y51), (A225), (A44), (D182), (D80), (E140), (E183), (E337), (E409), (G114), (G227), (G46), (H236), (H92), (K187), (K440), (K76), (M5), (N186), (P172), (Q189), (Q397), (Q403), (Q404), (Q73), (R179), (R79), (S270), (S53) and (T175).
Preferably said BM3 variant has an improved selectivity and/or product yield for C19 hydroxylation compared with its corresponding parent BM3 variant. Preferably, the BM3 variant comprises the mutations M177Y and/or A184Y.
It was surprisingly found that targeting the(se) position(s) and introducing a “suitable” amino acid exchange could further improve either product yield (cf. Tables A1, A2, A3, A4, A5, and A6) or selectivity (cf. Tables B1, B2, B3, B4, B5 and B6) of the C19 hydroxylation, thereby further improving the efficiency of the C19 hydroxylation process. Evaluation which amino acid is “suitable” for a position to obtain the improvement is easily feasible where the targeting position is disclosed herein. To evaluate which of the 20 amino acids is “suitable” for the given position, one simply has to mutate the given position by introducing the amino acid as known in the art (see also Example 1) and evaluate the performance of the obtained variant for C19 hydroxylation, e.g in comparison with the parent variant and/or in comparison with the remaining 18 amino acids.
There is provided a first preferred subset comprising variants according to the fifth embodiment, wherein the P450-BM3 variants have an improved product yield or product titer for C19 hydroxylation in comparison with the parent variant. Comprised within this subset are variants wherein said position(s) are selected from (A184), (A191), (A221), (A264), (A321), (A33), (A330), (A399), (A74), (D208), (D222), (D363), (E13), (E143), (E267), (E352), (E64), (E82), (E93), (F173), (F205), (F261), (F331), (F393), (F77), (F81), (G240), (G271), (G402), (G415), (G677), (G85), (H266), (H659), (I153), (I174), (I258), (I259), (I263), (I401), (K210), (K224), (L150), (L181), (L188), (L20), (L262), (L272), (L29), (L324), (L356), (L437), (L75), (L78), (L86), (M118), (M177), (M185), (M212), (M354), (N70), (P243), (P329), (P392), (P9), (R147), (R161), (R190), (R203), (R255), (R47), (R50), (S176), (S54), (S72), (S89), (T146), (T149), (T260), (T268), (T269), (T327), (T365), (T436), (T438), (T49), (T88), (V178), (V26), (V314), (V371), (V48), (W325), (W367), (W90), (Y51), (A225), (A44), (D182), (D80), (E140), (E183), (E337), (G114), (G227), (H236), (H92), (K187), (K440), (K76), (M5), (N186), (P172), (Q189), (Q397), (Q403), (Q404), (Q73), (R179), (R79), (S270), (S53), (T175) and (V299).
There is provided a second preferred subset comprising variants according to the fifth embodiment, wherein the P450-BM3 variants have an improved selectivity (as determined by SF or TPR) for C19 hydroxylation in comparison with the parent variant (see
There is provided a third preferred subset comprising variants according to the fifth embodiment, wherein the P450-BM3 variants have an improved selectivity (as determined by SF or TPR) and an improved product yield or product titer for C19 hydroxylation in comparison with the parent variant. Comprised within this subset are variants wherein said position(s) are selected from (A184), (A191), (A221), (A264), (A321), (A33), (A330), (A399), (A74), (D208), (D222), (D363), (E13), (E143), (E267), (E352), (E64), (E82), (E93), (F173), (F205), (F261), (F331), (F393), (F77), (F81), (G240), (G271), (G402), (G677), (G85), (H266), (H659), (I153), (I174), (I258), (I259), (I263), (I401), (K210), (K224), (L150), (L181), (L188), (L20), (L262), (L272), (L29), (L324), (L356), (L437), (L75), (L78), (L86), (M118), (M177), (M185), (M212), (M354), (N70), (P243), (P329), (P392), (P9), (R147), (R161), (R190), (R203), (R255), (R47), (R50), (S176), (S54), (S72), (S89), (T146), (T149), (T268), (T269), (T327), (T365), (T436), (T438), (T49), (T88), (V178), (V26), (V314), (V48), (W325), (W367), (W90), (Y51), (A225), (A44), (D182), (E140), (E337), (G114), (H236), (H92), (K187), (K440), (K76), (M5), (N186), (P172), (Q189), (Q403), (Q404), (Q73), (R179), (R79), (S270), (S53), (T175) and (V299).
The fifth embodiment and each of the subsets provided can be and is suggested to be combined with all previous embodiments, as understood by the skilled person, and may or may not be an embodiment according to the second embodiment of the first aspect.
Provided are variants according to a sixth embodiment according to the first aspect, wherein said P450-BM3 variant comprise at least one, two, three, four, five, six, seven, eight, nine, ten or more further mutations. In particular the variants according to the sixth embodiment can be variants according to the first or second embodiment of the first aspect, such as variants derived from variants according to SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 117, 118 or 119. The sixth embodiment can be and is suggested to be combined with all previous embodiments, and may or may not be an embodiment according to the fifth embodiment.
Preferrably, said further mutation(s) according to the sixth embodiment of the first aspect are (A184D) or (A184D, L188K) or (A184E) or (A184F) or (A184G) or (A184H) or (A184I) or (A184K) or (A184L) or (A184N) or (A184Q) or (A184R) or (A184W) or (A184W, L188F) or (A184Y) or (A184Y, L188K) or (A184Y, R50S) or (A221S) or (A264V) or (A321C) or (A321D) or (A321E) or (A321G) or (A321I) or (A321N) or (A321T) or (A321V) or (A328P) or (A330C) or (A330D) or (A330E) or (A330F) or (A330G) or (A330H) or (A330I) or (A330K) or (A330L) or (A330M) or (A330N) or (A330Q) or (A330R) or (A330S) or (A330T) or (A330T, E352A) or (A330T, F331V) or (A330V) or (A330W) or (A330Y) or (A330Y, W367C) or (A33V) or (A399C) or (A399E) or (A399G) or (A399I) or (A399L) or (A399M) or (A399N) or (A399Q) or (A399R) or (A399S) or (A399T) or (A399V) or (A74C) or (A74C, L75V) or (A74D) or (A74D, F81V) or (A74E) or (A74F) or (A74G) or (A74G, L75C) or (A74H) or (A74I) or (A74I, A184W, L188F) or (A74I, F77A, L181Y, A184W) or (A74I, L181Y, L188K) or (A74I, L181Y, L188R) or (A74I, L188F) or (A74I, L188K) or (A74I, L188R) or (A74I, M177Y, A184R, L188F) or (A74I, M177Y, V178P, A184G) or (A74I, T146F) or (A74I, T146F, L181Y) or (A74I, T146F, L181Y, L188K) or (A74I, T146F, L181Y, L188R) or (A74I, T146F, L188F) or (A74I, T146F, L188K) or (A74I, T146F, V178P) or (A74I, T146F, V178P, L181Y, L188F) or (A74I, T146F, V178P, L181Y, L188K) or (A74I, T146F, V178P, L188K) or (A74I, T146F, V178P, L188R) or (A74I, T146F, V178W) or (A74I, T146F, V178W, L181Y, L188F) or (A74I, T146F, V178W, L181Y, L188R) or (A74I, T146F, V178W, L188F) or (A74I, T146F, V178W, L188K) or (A74I, T146F, V178W, L188R) or (A74I, V178P) or (A74I, V178P, L181Y) or (A74I, V178P, L181Y, L188R) or (A74I, V178P, L188F) or (A74I, V178P, L188K) or (A74I, V178P, L188R) or (A74I, V178W, L188F) or (A74I, V178W, L188K) or (A74I, V178W, L188R) or (A74K) or (A74L) or (A74M) or (A74N) or (A74P) or (A74Q) or (A74R) or (A74R, L75V) or (A74S) or (A74T) or (A74T, A184D) or (A74T, F77A, L181Y, A184D, L188K) or (A74T, F77A, V178W, A184Y) or (A74T, F77L, L181Y, A184D, L188K) or (A74T, L181Y) or (A74T, L181Y, A184D, L188K) or (A74T, L181Y, A184Y, L188K) or (A74T, L181Y, L188K) or (A74T, L181Y, L188R) or (A74T, L188K) or (A74T, M177Y, L181Y, A184D, L188K) or (A74T, T146F, L181Y, A184D, L188K) or (A74T, T146F, L181Y, L188K) or (A74T, T146F, L181Y, L188R) or (A74T, T146F, L188F) or (A74T, T146F, L188K) or (A74T, T146F, L188R) or (A74T, T146F, V178P) or (A74T, T146F, V178P, L181Y) or (A74T, T146F, V178P, L188K) or (A74T, T146F, V178P, L188R) or (A74T, T146F, V178W) or (A74T, T146F, V178W, L181Y) or (A74T, T146F, V178W, L181Y, L188K) or (A74T, T146F, V178W, L188R) or (A74T, V178P) or (A74T, V178P, L188F) or (A74T, V178P, L188K) or (A74T, V178P, L188R) or (A74T, V178W, L181Y, L188K) or (A74T, V178W, L188K) or (A74T, V178W, L188R) or (A74V) or (A74V, F77A, A184N, L188F) or (A74V, L181Y) or (A74V, L181Y, A184D, L188K) or (A74V, L181Y, A184W, L188F) or (A74V, L181Y, L188F) or (A74V, L188F) or (A74V, L188K) or (A74V, L75V) or (A74V, T146F, L181Y, L188K) or (A74V, T146F, L181Y, L188R) or (A74V, T146F, L188K) or (A74V, T146F, L188R) or (A74V, T146F, V178W) or (A74V, T146F, V178W, L181Y, L188F) or (A74V, V178P, A184W, L188K) or (A74V, V178P, L181Y, L188F) or (A74V, V178P, L181Y, L188K) or (A74V, V178P, L181Y, L188R) or (A74V, V178P, L188F) or (A74V, V178W, L188K) or (A74W) or (A74Y) or (D168Y, F173D) or (D363G, T438C) or (E13D, R47L) or (E143A) or (E143C) or (E143D) or (E143F) or (E143G) or (E143I) or (E143K) or (E143L) or (E143M) or (E143N) or (E143P) or (E143Q) or (E143R) or (E143S) or (E143V) or (E143W) or (E143Y) or (E267A) or (E267C) or (E267D) or (E267G) or (E267K) or (E267P) or (E267R) or (E267S) or (E267T) or (E267Y) or (E352A) or (E352D) or (E352F) or (E352G) or (E352I) or (E352L) or (E352M) or (E352N) or (E352P) or (E352R) or (E352S) or (E352T) or (E352V) or (E352W) or (E352Y) or (E64A) or (E64G) or (E64H) or (E64I) or (E64K) or (E64L) or (E64M) or (E64N) or (E64Q) or (E64V) or (E64W) or (E64Y) or (E82P) or (E93G) or (F173C) or (F173C, F205G) or (F173D) or (F173I) or (F173K) or (F173L) or (F173M) or (F173N) or (F173P) or (F173Q) or (F173R) or (F173S) or (F173S, F205P) or (F173V) or (F173W) or (F173Y) or (F173Y, I174F) or (F205A) or (F205C) or (F205D) or (F205D, D208N) or (F205E) or (F205G) or (F205H) or (F205I) or (F205K) or (F205L) or (F205M) or (F205N) or (F205P) or (F205R) or (F205S) or (F205T) or (F205V) or (F205W) or (F205Y) or (F261A) or (F261C) or (F261D) or (F261G) or (F261I) or (F261L) or (F261M) or (F261N) or (F261Q) or (F261S) or (F261T) or (F261V) or (F261W) or (F261Y) or (F331C) or (F331H) or (F331I) or (F331L) or (F331M) or (F331N) or (F331P) or (F331T) or (F331V) or (F331W) or (F331Y) or (F393M) or (F393W) or (F77A) or (F77A, A184W) or (F77A, L181P, A184Y, L188K) or (F77A, M177Y) or (F77C) or (F77D) or (F77E) or (F77G) or (F77H) or (F77I) or (F77K) or (F77L) or (F77M) or (F77N) or (F77P) or (F77R) or (F77S) or (F77T) or (F77V) or (F77V, L86M) or (F77W) or (F77Y) or (F81A) or (F81C) or (F81D) or (F81I) or (F81L) or (F81P) or (F81R) or (F81S) or (F81T) or (F81V) or (F81W) or (F81Y) or (G271A) or (G271C) or (G271D) or (G271E) or (G271F) or (G271H) or (G271K) or (G271L) or (G271M) or (G271N) or (G271P) or (G271Q) or (G271R) or (G271S) or (G271T) or (G271V) or (G271W) or (G271Y) or (G402A) or (G415A) or (G415D) or (G415S) or (G415T) or (G415V) or (G85A) or (G85L) or (G85S) or (H266A) or (H266C) or (H266D) or (H266E) or (H266F) or (H266G) or (H266I) or (H266K) or (H266M) or (H266N) or (H266P) or (H266Q) or (H266R) or (H266S) or (H266T) or (H266V) or (H266W) or (H266Y) or (I153F, G271L) or (I153L) or (I153L, F173Y) or (I259A) or (I259C) or (I259D) or (I259F) or (I259G) or (I259H) or (I259K) or (I259L) or (I259M) or (I259N) or (I259Q) or (I259S) or (I259T) or (I259V) or (I259W) or (I259Y) or (I263A) or (I263C) or (I263E) or (I263F) or (I263G) or (I263H, A264G) or (I263K) or (I263L) or (I263M) or (I263N) or (I263Q) or (I263S) or (I263T) or (I263V) or (I263Y) or (I401A) or (I401L) or (1401M) or (I401T) or (I401V) or (K210E) or (K210T, G271V) or (K224C) or (K224E) or (K224F) or (K224H) or (K224I) or (K224L) or (K224M) or (K224P) or (K224Q) or (K224W) or (K224Y) or (L150A) or (L150C) or (L150D) or (L150E) or (L150F) or (L150G) or (L150H) or (L150I) or (L150K) or (L150M) or (L150N) or (L150Q) or (L150R) or (L150R, F205R) or (L150S) or (L150S, F173L) or (L150T) or (L150V) or (L150W) or (L150Y) or (L181H) or (L181I) or (L181M) or (L181P) or (L181V) or (L181Y) or (L181Y, A184D) or (L181Y, L188F) or (L181Y, L188K) or (L181Y, L188K, H659R) or (L188A) or (L188D) or (L188E) or (L188F) or (L188H) or (L188I) or (L188K) or (L188M) or (L188N) or (L188Q) or (L188R) or (L188S) or (L188W) or (L20C) or (L20D) or (L20E) or (L20F) or (L20G) or (L20G, R47L) or (L20I) or (L20M) or (L20N) or (L20P) or (L20R) or (L20S) or (L20T) or (L20V) or (L20W) or (L20Y) or (L262I) or (L262V) or (L262W) or (L262Y) or (L272A) or (L272C) or (L272E) or (L272F) or (L272G) or (L272I) or (L272K) or (L272M) or (L272N) or (L272Q) or (L272R) or (L272S) or (L272T) or (L272V) or (L272W) or (L272Y) or (L29A) or (L29C) or (L29D) or (L29F) or (L29H) or (L29I) or (L29M) or (L29M, R47G) or (L29P) or (L29Q) or (L29S) or (L29T) or (L29V) or (L29W) or (L29Y) or (L324F) or (L356C) or (L356F) or (L356H) or (L356I) or (L356M) or (L356N) or (L356Q) or (L356S) or (L356T) or (L356V) or (L356W) or (L437I) or (L437M) or (L75H, F81S) or (L75I) or (L75I, F81C) or (L75I, F81G) or (L75I, F81H) or (L75I, F81I) or (L75I, F81L) or (L75I, F81S) or (L75I, F81V) or (L75I, F81Y) or (L75V) or (L75V, F81C) or (L75V, F81H) or (L75V, F81I) or (L75V, F81L) or (L75V, F81V) or (L75V, F81Y) or (L78F) or (L78I) or (L78M) or (L78V) or (L78Y) or (L86A, S89T) or (L86I) or (L86I, S89T) or (L86M) or (L86M, S89T) or (L86N, S89T) or (L86V) or (L86V, S89T) or (M118A) or (M118E) or (M118F) or (M118G) or (M118H) or (M118I) or (M118K) or (M118L) or (M118N) or (M118P) or (M118Q) or (M118S) or (M118T) or (M118V) or (M118W) or (M118Y) or (M177A) or (M177C) or (M177C, V178Y) or (M177D) or (M177E) or (M177F) or (M177G) or (M177H) or (M177I) or (M177K) or (M177L) or (M177N) or (M177P) or (M177Q) or (M177R) or (M177T) or (M177V) or (M177Y) or (M177Y, A184W) or (M177Y, A184Y) or (M177Y, M 185V) or (M177Y, R50S) or (M177Y, V178P) or (M177Y, V178P, A184Y, L188F) or (M177Y, V178W) or (M177Y, V178W, A184Y) or (M177Y, V178W, A184Y, L188F) or (M185C) or (M185D) or (M185E) or (M185G) or (M185H) or (M185K) or (M185L) or (M185N) or (M185Q) or (M185R) or (M185V) or (M185Y) or (M212A) or (M212C) or (M212C, I259L) or (M212D) or (M212E) or (M212F) or (M212G) or (M212H) or (M212K) or (M212L) or (M212L, I259F) or (M212P) or (M212Q) or (M212R) or (M212S) or (M212T) or (M212V) or (M212W) or (M212Y) or (M354A) or (M354C) or (M354D) or (M354E) or (M354G) or (M354I) or (M354K) or (M354K, D363Y) or (M354L) or (M354N) or (M354Q) or (M354R) or (M354S) or (M354T) or (M354V) or (M354W) or (M354Y) or (N70A) or (N70C) or (N70F) or (N70G) or (N70H) or (N70K) or (N70R) or (N70W) or (N70Y) or (P243T, H266L) or (P326C) or (P326D) or (P326G) or (P326N) or (P326S) or (P326T) or (P329A) or (P329C) or (P329G) or (P329K) or (P329S) or (P329T) or (P392C) or (P392G) or (P392Q) or (P392R) or (P392V) or (P9S) or (R147C, H266G) or (R161C, G271D) or (R190L, F261V) or (R203C) or (R255A) or (R255F) or (R255G) or (R255I) or (R255K) or (R255L) or (R255M) or (R255N) or (R255P) or (R255T) or (R255W) or (R323C) or (R47A) or (R47C) or (R47D) or (R47E) or (R47F) or (R47G) or (R47H) or (R47H, A74W) or (R47I) or (R47K) or (R47L) or (R47M) or (R47N) or (R47P) or (R47Q) or (R47S) or (R47T) or (R47V) or (R47W) or (R50A) or (R50C) or (R50D) or (R50E) or (R50F) or (R50G) or (R50I) or (R50L) or (R50M) or (R50N) or (R50P) or (R50Q) or (R50S) or (R50S, A184W, L188F) or (R50S, A74I, A184W, L188F) or (R50S, A74M, A184Y, L188K) or (R50S, A74T, L181Y, A184D, L188K, G240R) or (R50S, A74V) or (R50S, M177Y, V178P, A184Y) or (R50S, M177Y, V178W, A184W) or (R50S, M177Y, V178W, A184Y) or (R50S, M177Y, V178W, A184Y, A330R) or (R50S, M177Y, V178W, A184Y, A74V, G677D) or (R50S, M177Y, V178W, A184Y, H266S) or (R50S, M177Y, V178W, A184Y, L181P) or (R50S, M177Y, V178W, A184Y, L181Y) or (R50S, M177Y, V178W, A184Y, L188K) or (R50S, M177Y, V178W, A184Y, L188R) or (R50S, M177Y, V178W, A184Y, T146F) or (R50S, M177Y, V178W, A184Y, V26N) or (R50S, M177Y, V178W, L181Y, A184Y) or (R50S, S72G, A74V) or (R50S, S72G, A74V, M177Y, A184Y) or (R50S, S72G, A74V, M177Y, V178P, A184Y) or (R50S, S72G, A74V, M177Y, V178W, A184Y) or (R50S, S72G, A74V, M177Y, V178W, A184Y, L188R) or (R50S, T146F, M177Y, V178W, A184Y) or (R50V) or (R50Y) or (S164N, F261L) or (S176A, A184Y, L188K) or (S176C) or (S176D) or (S176E) or (S176F) or (S176G) or (S176H) or (S176K) or (S176L) or (S176M) or (S176N) or (S176P) or (S176Q) or (S176R) or (S72C, A74C) or (S72C, A74I) or (S72C, A74L) or (S72C, A74V) or (S72C, A74Y) or (S72D, A74C) or (S72D, A74F) or (S72G) or (S72G, A74C) or (S72G, A74F) or (S72G, A74H) or (S72G, A74I) or (S72G, A74I, A184W, L188F) or (S72G, A74I, F77A, M177Y) or (S72G, A74I, F77L) or (S72G, A74I, F77L, M177Y) or (S72G, A74I, L181Y) or (S72G, A74I, L181Y, L188F) or (S72G, A74I, L181Y, L188K) or (S72G, A74I, L181Y, L188R) or (S72G, A74I, L188F) or (S72G, A74I, L188K) or (S72G, A74I, L188R) or (S72G, A74I, T146F) or (S72G, A74I, T146F, L181Y) or (S72G, A74I, T146F, L181Y, L188F) or (S72G, A74I, T146F, L181Y, L188K) or (S72G, A74I, T146F, L181Y, L188R) or (S72G, A74I, T146F, L188F) or (S72G, A74I, T146F, L188K) or (S72G, A74I, T146F, L188R) or (S72G, A74I, T146F, V178P) or (S72G, A74I, T146F, V178P, L181Y) or (S72G, A74I, T146F, V178P, L181Y, L188F) or (S72G, A74I, T146F, V178P, L181Y, L188K) or (S72G, A74I, T146F, V178P, L181Y, L188R) or (S72G, A74I, T146F, V178P, L188F) or (S72G, A74I, T146F, V178P, L188K) or (S72G, A74I, T146F, V178P, L188R) or (S72G, A74I, T146F, V178W) or (S72G, A74I, T146F, V178W, L181Y) or (S72G, A74I, T146F, V178W, L181Y, L188F) or (S72G, A74I, T146F, V178W, L181Y, L188K) or (S72G, A74I, T146F, V178W, L181Y, L188R) or (S72G, A74I, T146F, V178W, L188F) or (S72G, A74I, T146F, V178W, L188K) or (S72G, A74I, T146F, V178W, L188R) or (S72G, A74I, V178P) or (S72G, A74I, V178P, L181Y) or (S72G, A74I, V178P, L181Y, L188K) or (S72G, A74I, V178P, L181Y, L188R) or (S72G, A74I, V178P, L188F) or (S72G, A74I, V178P, L188K) or (S72G, A74I, V178P, L188R) or (S72G, A74I, V178W) or (S72G, A74I, V178W, L181Y, L188F) or (S72G, A74I, V178W, L181Y, L188K) or (S72G, A74I, V178W, L181Y, L188R) or (S72G, A74I, V178W, L188F) or (S72G, A74I, V178W, L188K) or (S72G, A74I, V178W, L188R) or (S72G, A74L) or (S72G, A74M, A184Y, L188K) or (S72G, A74S) or (S72G, A74T) or (S72G, A74T, L181Y, A184D, L188K) or (S72G, A74T, L181Y, L188F) or (S72G, A74T, L181Y, L188K) or (S72G, A74T, L181Y, L188R) or (S72G, A74T, L188F) or (S72G, A74T, L188K) or (S72G, A74T, L188R) or (S72G, A74T, T146F) or (S72G, A74T, T146F, L181Y) or (S72G, A74T, T146F, L181Y, L188K) or (S72G, A74T, T146F, L181Y, L188R) or (S72G, A74T, T146F, L188F) or (S72G, A74T, T146F, L188K) or (S72G, A74T, T146F, L188R) or (S72G, A74T, T146F, V178P) or (S72G, A74T, T146F, V178P, L181Y) or (S72G, A74T, T146F, V178P, L188F) or (S72G, A74T, T146F, V178P, L188K) or (S72G, A74T, T146F, V178P, L188R) or (S72G, A74T, T146F, V178W) or (S72G, A74T, T146F, V178W, L181Y) or (S72G, A74T, T146F, V178W, L181Y, L188F) or (S72G, A74T, T146F, V178W, L181Y, L188K) or (S72G, A74T, T146F, V178W, L181Y, L188R) or (S72G, A74T, T146F, V178W, L188F) or (S72G, A74T, T146F, V178W, L188K) or (S72G, A74T, T146F, V178W, L188R) or (S72G, A74T, V178P) or (S72G, A74T, V178P, L181Y) or (S72G, A74T, V178P, L181Y, L188F) or (S72G, A74T, V178P, L181Y, L188K) or (S72G, A74T, V178P, L188F) or (S72G, A74T, V178P, L188K) or (S72G, A74T, V178P, L188R) or (S72G, A74T, V178W) or (S72G, A74T, V178W, L181Y, L188F) or (S72G, A74T, V178W, L181Y, L188K) or (S72G, A74T, V178W, L188F) or (S72G, A74T, V178W, L188K) or (S72G, A74T, V178W, L188R) or (S72G, A74V) or (S72G, A74V, A184W) or (S72G, A74V, A221V) or (S72G, A74V, F77A) or (S72G, A74V, F77A, M177Y) or (S72G, A74V, F77L, M177Y) or (S72G, A74V, F77S) or (S72G, A74V, L181Y) or (S72G, A74V, L181Y, L188F) or (S72G, A74V, L181Y, L188K) or (S72G, A74V, L188F) or (S72G, A74V, L188K) or (S72G, A74V, L188R) or (S72G, A74V, T146F) or (S72G, A74V, T146F, L181Y) or (S72G, A74V, T146F, L181Y, L188F) or (S72G, A74V, T146F, L181Y, L188K) or (S72G, A74V, T146F, L181Y, L188R) or (S72G, A74V, T146F, L188F) or (S72G, A74V, T146F, L188K) or (S72G, A74V, T146F, L188R) or (S72G, A74V, T146F, V178P) or (S72G, A74V, T146F, V178P, L181Y) or (S72G, A74V, T146F, V178P, L181Y, L188F) or (S72G, A74V, T146F, V178P, L181Y, L188K) or (S72G, A74V, T146F, V178P, L181Y, L188R) or (S72G, A74V, T146F, V178P, L188F) or (S72G, A74V, T146F, V178P, L188K) or (S72G, A74V, T146F, V178P, L188R) or (S72G, A74V, T146F, V178W) or (S72G, A74V, T146F, V178W, L181Y) or (S72G, A74V, T146F, V178W, L181Y, L188K) or (S72G, A74V, T146F, V178W, L181Y, L188R) or (S72G, A74V, T146F, V178W, L188F) or (S72G, A74V, T146F, V178W, L188K) or (S72G, A74V, T146F, V178W, L188R) or (S72G, A74V, V178P) or (S72G, A74V, V178P, L181Y) or (S72G, A74V, V178P, L181Y, L188F) or (S72G, A74V, V178P, L181Y, L188K) or (S72G, A74V, V178P, L181Y, L188R) or (S72G, A74V, V178P, L188F) or (S72G, A74V, V178P, L188K) or (S72G, A74V, V178P, L188R) or (S72G, A74V, V178W) or (S72G, A74V, V178W, L181Y) or (S72G, A74V, V178W, L181Y, L188F) or (S72G, A74V, V178W, L181Y, L188K) or (S72G, A74V, V178W, L181Y, L188R) or (S72G, A74V, V178W, L188F) or (S72G, A74V, V178W, L188K) or (S72G, A74V, V178W, L188R) or (S72G, A74Y) or (S72G, L181Y) or (S72G, L181Y, L188F) or (S72G, L181Y, L188K) or (S72G, L181Y, L188R) or (S72G, L188F) or (S72G, L188K) or (S72G, L188R) or (S72G, M177Y, V178W) or (S72G, M177Y, V178W, L188K) or (S72G, T146F) or (S72G, T146F, L181Y) or (S72G, T146F, L181Y, L188F) or (S72G, T146F, L181Y, L188K) or (S72G, T146F, L181Y, L188R) or (S72G, T146F, L188F) or (S72G, T146F, L188K) or (S72G, T146F, L188R) or (S72G, T146F, V178P) or (S72G, T146F, V178P, L181Y) or (S72G, T146F, V178P, L181Y, L188F) or (S72G, T146F, V178P, L181Y, L188K) or (S72G, T146F, V178P, L181Y, L188R) or (S72G, T146F, V178P, L188F) or (S72G, T146F, V178P, L188K) or (S72G, T146F, V178P, L188R) or (S72G, T146F, V178W) or (S72G, T146F, V178W, L181Y) or (S72G, T146F, V178W, L181Y, L188F) or (S72G, T146F, V178W, L181Y, L188K) or (S72G, T146F, V178W, L188F) or (S72G, T146F, V178W, L188K) or (S72G, T146F, V178W, L188R) or (S72G, V178P) or (S72G, V178P, L181Y) or (S72G, V178P, L181Y, L188F) or (S72G, V178P, L181Y, L188K) or (S72G, V178P, L181Y, L188R) or (S72G, V178P, L188F) or (S72G, V178P, L188K) or (S72G, V178P, L188R) or (S72G, V178W) or (S72G, V178W, L181Y) or (S72G, V178W, L181Y, L188F) or (S72G, V178W, L181Y, L188K) or (S72G, V178W, L181Y, L188R) or (S72G, V178W, L188F) or (S72G, V178W, L188K) or (S72G, V178W, L188R) or (S72H, A74C) or (S72H, A74G) or (S72H, A74S) or (S72H, A74Y) or (S72N, A74C) or (S72N, A74I) or (S72N, A74N) or (S72N, A74V) or (S72T) or (S72W) or (S72W, A74I, A184W, L188F) or (S72W, A74V) or (S72W, L333R) or (S72W, W90Y, V299G) or (S72Y, A74V) or (S89C) or (S89E) or (S89G) or (S89I) or (S89L) or (S89M) or (S89N) or (S89Q) or (S89R) or (S89T) or (S89V) or (T146A) or (T146A, D222Y, 1263M) or (T146A, F173D) or (T146A, F173V) or (T146A, F205G) or (T146A, F261C) or (T146A, F261S) or (T146A, F261V) or (T146A, G271R) or (T146A, G271S) or (T146A, G271T) or (T146A, H266T) or (T146A, H266V) or (T146A, I258T, I259S) or (T146A, I259H) or (T146A, I263F) or (T146A, I263L) or (T146A, I263N) or (T146A, I263V) or (T146A, L150A) or (T146A, L150G) or (T146A, L150K) or (T146A, L150R, F173I) or (T146A, L272H) or (T146A, L272R) or (T146A, L272S) or (T146A, L272W) or (T146A, M212A) or (T146C) or (T146D) or (T146E) or (T146F) or (T146F, L181Y) or (T146F, L181Y, L188F) or (T146F, L181Y, L188K) or (T146F, L188K) or (T146F, L188R) or (T146F, V178P) or (T146F, V178P, L181Y, L188R) or (T146F, V178P, L188K) or (T146F, V178P, L188R) or (T146F, V178W) or (T146F, V178W, L181Y, L188R) or (T146F, V178W, L188F) or (T146F, V178W, L188K) or (T146F, V178W, L188R) or (T146G) or (T146H) or (T146L) or (T146M) or (T146N) or (T146P) or (T146R) or (T146S) or (T146V) or (T146V, A191T) or (T146Y) or (T149P) or (T260A) or (T260S) or (T260W) or (T268A) or (T268S) or (T269K) or (T327A) or (T327A, A330W) or (T327C) or (T327D) or (T327E) or (T327G) or (T327I) or (T327L) or (T327M) or (T327N) or (T327P) or (T327Q) or (T327S) or (T327V) or (T365A) or (T365C) or (T365D) or (T365F) or (T365G) or (T365H) or (T365I) or (T365K) or (T365L) or (T365N) or (T365P) or (T365Q) or (T365V) or (T365W) or (T365Y) or (T436A) or (T436C) or (T436D) or (T436E) or (T436F) or (T436G) or (T436H) or (T436I) or (T436K) or (T436L) or (T436M) or (T436N) or (T436P) or (T436Q) or (T436R) or (T436S) or (T436V) or (T436W) or (T436Y) or (T438A) or (T438C) or (T438G) or (T438I) or (T438L) or (T438S) or (T438V) or (T49A) or (T49C) or (T49D) or (T49E) or (T49F) or (T49G) or (T49H) or (T49I) or (T49K) or (T49L) or (T49L, S54N) or (T49M) or (T49N) or (T49P) or (T49Q) or (T49R) or (T49R, R50C) or (T49R, R50S, M177Y, V178W, A184Y) or (T49S) or (T49V) or (T49W) or (T49Y) or (T88A) or (T88C) or (T88S) or (T88V) or (V178A) or (V178D) or (V178F) or (V178H) or (V178I) or (V178K) or (V178L) or (V178M) or (V178N) or (V178P) or (V178P, L181Y) or (V178P, L181Y, L188R) or (V178P, L188F) or (V178P, L188K) or (V178P, L188R) or (V178Q) or (V178R) or (V178S) or (V178T) or (V178W) or (V178W, A184Y) or (V178W, L181Y, L188K) or (V178W, L181Y, L188R) or (V178W, L188R) or (V178W, R50S) or (V178Y) or (V26A) or (V26C) or (V26E) or (V26F) or (V26G) or (V26G, R47S) or (V26H) or (V26I) or (V26K) or (V26L) or (V26M) or (V26N) or (V26N, R50S, M177Y, V178W, A184Y) or (V26Q) or (V26R) or (V26S) or (V26T) or (V26W) or (V26Y) or (V314A) or (V314E) or (V314F) or (V314H) or (V314P) or (V314Q) or (V314S) or (V314Y) or (V371 P) or (V48A) or (V48D) or (V48E) or (V48F) or (V48G) or (V48H) or (V48I) or (V48L) or (V48M) or (V48P) or (V48Q) or (V48R) or (V48S) or (V48T) or (V48W) or (V48Y) or (W325A) or (W325C) or (W325D) or (W325E) or (W325F) or (W325G) or (W325I) or (W325K) or (W325L) or (W325M) or (W325Q) or (W325R) or (W325S) or (W325T) or (W325V) or (W325Y) or (W90D) or (W90Y) or (W90Y, V299G, G570D) or (Y51A) or (Y51C) or (Y51 E) or (Y51F) or (Y51G) or (Y51H) or (Y51I) or (Y51 L) or (Y51M) or (Y51N) or (Y51P) or (Y51Q) or (Y51S) or (Y51S, F77V) or (Y51T) or (Y51V) or (Y51W) or (A225C) or (A225G) or (A225M) or (A225N) or (A225Q) or (A225S) or (A225T) or (A225V) or (A225Y) or (A44G) or (A44Q) or (A44T) or (D182A) or (D182C) or (D182E) or (D182H) or (D182I) or (D182M) or (D182N) or (D182V) or (D182W) or (D80C) or (D80L) or (D80M) or (D80N) or (D80S) or (D80T) or (D80W) or (E140A) or (E140D) or (E140K) or (E140L) or (E140M) or (E140T) or (E183D) or (E183T) or (E337L) or (E337V) or (E409N) or (G114C) or (G114K) or (G114L) or (G114N) or (G114W) or (G227N) or (G227R) or (G240A) or (G240C) or (G240H) or (G240K) or (G240M) or (G240N) or (G240Q) or (G240R) or (G240S) or (G240V) or (G46T) or (H236A) or (H236C) or (H236D) or (H236F) or (H236G) or (H236I) or (H236K) or (H236M) or (H236P) or (H236R) or (H236S) or (H236Y) or (H92F) or (H92Y) or (I174T) or (I174V) or (I258C) or (I258L) or (I258M) or (I258V) or (K187A) or (K187G) or (K187N) or (K187R) or (K440C) or (K440M) or (K440N) or (K440T) or (K76C) or (K76E) or (K76F) or (K76H) or (K76I) or (K76L) or (K76N) or (K76V) or (M5C) or (M5E) or (M5P) or (M5Q) or (N186A) or (N186D) or (N186E) or (N186F) or (N186H) or (N186K) or (N186L) or (N186M) or (N186Q) or (N186T) or (N186V) or (N186Y) or (P172A) or (P172D) or (P172G) or (Q189E) or (Q189I) or (Q189K) or (Q189N) or (Q189V) or (Q397R) or (Q403K) or (Q403R) or (Q404F) or (Q404Y) or (Q73A) or (Q73E) or (Q73F) or (Q73L) or (Q73S) or (Q73T) or (Q73W) or (Q73Y) or (R179A) or (R179H) or (R179L) or (R179M) or (R179P) or (R179V) or (R190C) or (R190G) or (R190K) or (R190M) or (R190N) or (R190S) or (R190V) or (R190W) or (R79E) or (R79K) or (S270A) or (S270D) or (S270G) or (S53C) or (T175F) or (T175G) or (T175H) or (T175L) or (T175M) or (T175N) or (T175Q) or (T175R) or (T175Y) or (T269A) or (T269V) or (V299L) or (V299W) or (A225V, H236K, G240V, T269V) or (D80I, D182H, A225V, G240V, T269V) or(F173W, R179L, R190V, A225V) or (F173W, R179L, R190V, K440C) or (F173W, R179L, T269V, K440C) or (F173W, R190V, H236K, T269V) or (H236K, G240V, T269V, K440C) or (K76I, F173W, R179L, D182I, R190V, K440C) or (Q73W, D80I, N186V, G240V) or (R179L, A225S) or (R179L, A225V, G240V, K440C) or (R179L, R190V) or (R190V, A225V, T269V, K440C) or (T175M, H236D) or (T175M, R190V).
The variants according to the sixth embodiment were surprisingly found to show an improved product yield or titer or selectivity of the C19 hydroxylation, thereby further improving efficiency of the C19 hydroxylation process in comparison with the parent variant.
There is provided a first preferred subset comprising variants according to the sixth embodiment, wherein the P450-BM3 variants have an improved product yield or product titer for C19 hydroxylation in comparison with the parent variant. Comprised within this subset are variants comprising at least the mutation(s) (A184D) or (A184D, L188K) or (A184E) or (A184F) or (A184G) or (A184H) or (A184I) or (A184K) or (A184L) or (A184N) or (A184Q) or (A184R) or (A184W) or (A184W, L188F) or (A184Y) or (A184Y, L188K) or (A184Y, R50S) or (A221S) or (A264V) or (A321C) or (A321D) or (A321E) or (A321G) or (A321I) or (A321N) or (A321T) or (A321V) or (A330C) or (A330D) or (A330E) or (A330F) or (A330G) or (A330H) or (A330I) or (A330K) or (A330L) or (A330M) or (A330N) or (A330Q) or (A330R) or (A330S) or (A330T) or (A330T, E352A) or (A330V) or (A330W) or (A330Y) or (A330Y, W367C) or (A33V) or (A399C) or (A399E) or (A399G) or (A399I) or (A399L) or (A399M) or (A399N) or (A399Q) or (A399R) or (A399S) or (A399T) or (A399V) or (A74C) or (A74C, L75V) or (A74D) or (A74D, F81V) or (A74E) or(A74F) or(A74H) or(A74I) or(A74I, A184W, L188F) or (A74I, F77A, L181Y, A184W) or(A74I, L181Y, L188R) or (A74I, L188F) or (A74I, L188K) or (A74I, L188R) or (A74I, M177Y, A184R, L188F) or (A74I, M177Y, V178P, A184G) or (A74I, T146F) or (A74I, T146F, L181Y) or (A74I, T146F, L181Y, L188K) or (A74I, T146F, L181Y, L188R) or (A74I, T146F, L188F) or (A74I, T146F, L188K) or (A74I, T146F, V178P) or (A74I, T146F, V178P, L181Y, L188F) or (A74I, T146F, V178P, L181Y, L188K) or (A74I, T146F, V178P, L188K) or (A74I, T146F, V178P, L188R) or (A74I, T146F, V178W) or (A74I, T146F, V178W, L181Y, L188F) or (A74I, T146F, V178W, L181Y, L188R) or (A74I, T146F, V178W, L188F) or (A74I, T146F, V178W, L188K) or (A74I, T146F, V178W, L188R) or (A74I, V178P) or (A74I, V178P, L181Y) or (A74I, V178P, L181Y, L188R) or (A74I, V178P, L188F) or (A74I, V178P, L188K) or (A74I, V178P, L188R) or (A74I, V178W, L188F) or (A74I, V178W, L188R) or (A74K) or (A74L) or (A74M) or (A74N) or (A74P) or (A74Q) or (A74R) or (A74R, L75V) or (A74S) or (A74T) or (A74T, A184D) or (A74T, F77A, L181Y, A184D, L188K) or (A74T, F77A, V178W, A184Y) or (A74T, F77L, L181Y, A184D, L188K) or (A74T, L181Y) or (A74T, L181Y, A184D, L188K) or (A74T, L181Y, A184Y, L188K) or (A74T, L181Y, L188K) or (A74T, L181Y, L188R) or (A74T, L188K) or (A74T, M177Y, L181Y, A184D, L188K) or (A74T, T146F, L181Y, A184D, L188K) or (A74T, T146F, L181Y, L188K) or (A74T, T146F, L181Y, L188R) or (A74T, T146F, L188F) or (A74T, T146F, L188K) or (A74T, T146F, L188R) or (A74T, T146F, V178P) or (A74T, T146F, V178P, L181Y) or (A74T, T146F, V178P, L188K) or (A74T, T146F, V178P, L188R) or (A74T, T146F, V178W) or (A74T, T146F, V178W, L181Y) or (A74T, T146F, V178W, L181Y, L188K) or (A74T, T146F, V178W, L188R) or (A74T, V178P) or (A74T, V178P, L188F) or (A74T, V178P, L188K) or (A74T, V178P, L188R) or (A74T, V178W, L181Y, L188K) or (A74T, V178W, L188K) or (A74T, V178W, L188R) or (A74V) or (A74V, F77A, A184N, L188F) or (A74V, L181Y) or (A74V, L181Y, A184D, L188K) or (A74V, L181Y, A184W, L188F) or (A74V, L181Y, L188F) or (A74V, L188F) or (A74V, L188K) or (A74V, L75V) or (A74V, T146F, L181Y, L188K) or (A74V, T146F, L181Y, L188R) or (A74V, T146F, L188K) or (A74V, T146F, L188R) or (A74V, T146F, V178W, L181Y, L188F) or (A74V, V178P, A184W, L188K) or (A74V, V178P, L181Y, L188F) or (A74V, V178P, L181Y, L188K) or (A74V, V178P, L181Y, L188R) or (A74V, V178P, L188F) or (A74V, V178W, L188K) or (A74W) or (A74Y) or (D363G, T438C) or (E13D, R47L) or (E143A) or (E143C) or (E143D) or (E143F) or (E143G) or (E143I) or (E143K) or (E143L) or (E143M) or (E143N) or (E143P) or (E143Q) or (E143R) or (E143S) or (E143V) or (E143W) or (E143Y) or (E267D) or (E267R) or (E267T) or (E352A) or (E352D) or (E352F) or (E352G) or (E352I) or (E352L) or (E352M) or (E352N) or (E352P) or (E352R) or (E352S) or (E352T) or (E352V) or (E352W) or (E352Y) or (E64A) or (E64G) or (E64H) or (E64K) or (E64L) or (E64M) or (E64N) or (E64Q) or (E64V) or (E64Y) or (E82P) or (E93G) or (F173C) or (F173C, F205G) or (F173D) or (F173I) or (F173K) or (F173L) or (F173M) or (F173N) or (F173P) or (F173Q) or (F173R) or (F173S) or (F173S, F205P) or (F173V) or (F173W) or (F173Y) or (F173Y, I174F) or (F205A) or (F205C) or (F205D) or (F205D, D208N) or (F205E) or (F205G) or (F205H) or (F205I) or (F205K) or (F205L) or (F205M) or (F205N) or (F205P) or (F205R) or (F205S) or (F205T) or (F205V) or (F205W) or (F205Y) or (F261A) or (F261C) or (F261D) or (F261G) or (F261I) or (F261L) or (F261M) or (F261Q) or (F261S) or (F261T) or (F261V) or (F261Y) or (F331H) or (F331I) or (F331L) or (F331M) or (F331V) or (F331W) or (F331Y) or (F393M) or (F393W) or (F77A) or (F77A, A184W) or (F77A, L181P, A184Y, L188K) or (F77A, M 177Y) or (F77C) or (F77D) or (F77E) or (F77G) or (F77H) or (F77I) or (F77K) or (F77L) or (F77M) or (F77N) or (F77P) or (F77R) or (F77S) or (F77T) or (F77V) or (F77V, L86M) or (F77W) or (F77Y) or (F81D) or (F81I) or (F81L) or (F81R) or (F81S) or (F81V) or (F81W) or (F81Y) or (G271A) or (G271C) or (G271D) or (G271E) or (G271F) or (G271H) or (G271K) or (G271L) or (G271M) or (G271N) or (G271P) or (G271Q) or (G271S) or (G271T) or (G271V) or (G271W) or (G271Y) or (G402A) or (G415A) or (G415D) or (G415S) or (G415T) or (G415V) or (G85A) or (G85L) or (G85S) or (H266A) or (H266C) or (H266E) or (H266F) or (H266I) or (H266K) or (H266M) or (H266N) or (H266Q) or (H266S) or (H266V) or (H266W) or (H266Y) or (I153F, G271L) or (I153L) or (I153L, F173Y) or (I259A) or (I259C) or (I259D) or (I259F) or (I259H) or (I259K) or (I259L) or (I259M) or (I259N) or (I259Q) or (I259S) or (I259T) or (I259V) or (I263A) or (I263C) or (I263E) or (I263F) or (I263H, A264G) or (I263K) or (I263L) or (I263M) or (I263N) or (I263Q) or (I263S) or (I263V) or (I263Y) or (I401A) or (I401M) or (I401T) or (I401V) or (K210E) or (K210T, G271V) or (K224C) or (K224E) or (K224H) or (K224I) or (K224L) or (K224M) or (K224P) or (K224Q) or (K224W) or (K224Y) or (L150A) or (L150C) or (L150D) or (L150E) or (L150F) or (L150G) or (L150H) or (L150I) or (L150K) or (L150M) or (L150N) or (L150Q) or (L150R) or (L150R, F205R) or (L150S) or (L150S, F173L) or (L150T) or (L150V) or (L150W) or (L150Y) or (L181H) or (L181I) or (L181M) or (L181P) or (L181V) or (L181Y) or (L181Y, A184D) or (L181Y, L188F) or (L181Y, L188K) or (L181Y, L188K, H659R) or (L188A) or (L188D) or (L188E) or (L188F) or (L188H) or (L188I) or (L188K) or (L188M) or (L188N) or (L188Q) or (L188R) or (L188S) or (L188W) or (L20C) or (L20D) or (L20E) or (L20F) or (L20G) or (L20G, R47L) or (L20I) or (L20M) or (L20N) or (L20P) or (L20R) or (L20S) or (L20T) or (L20V) or (L20W) or (L20Y) or (L262I) or (L262V) or (L262W) or (L272A) or (L272C) or (L272E) or (L272F) or (L272G) or (L272I) or (L272K) or (L272M) or (L272N) or (L272Q) or (L272S) or (L272T) or (L272V) or (L272W) or (L272Y) or (L29A) or (L29C) or (L29D) or (L29F) or (L29H) or (L29I) or (L29M) or (L29M, R47G) or (L29Q) or (L29S) or (L29T) or (L29V) or (L29W) or (L29Y) or (L324F) or (L356C) or (L356F) or (L356I) or (L356M) or (L356V) or (L437I) or (L437M) or (L75I) or (L75I, F81C) or (L75I, F81I) or (L75I, F81L) or (L75I, F81S) or (L75I, F81V) or (L75V) or (L75V, F81C) or (L75V, F81L) or (L75V, F81V) or (L75V, F81Y) or (L78F) or (L78I) or (L78M) or (L78V) or (L86I) or (L86I, S89T) or (L86M) or (L86M, S89T) or (L86V) or (M118A) or (M118E) or (M118F) or (M118G) or (M118H) or (M118I) or (M118K) or (M118L) or (M118N) or (M118P) or (M118Q) or (M118S) or (M118T) or (M118V) or (M118W) or (M118Y) or (M177A) or (M177C) or (M177C, V178Y) or (M177D) or (M177E) or (M177F) or (M177G) or (M177H) or (M177I) or (M177L) or (M177N) or (M177P) or (M177Q) or (M177R) or (M177T) or (M177V) or (M177Y) or (M177Y, A184W) or (M177Y, A184Y) or (M177Y, M185V) or (M177Y, R50S) or (M177Y, V178P) or (M177Y, V178P, A184Y, L188F) or (M177Y, V178W) or (M177Y, V178W, A184Y) or (M177Y, V178W, A184Y, L188F) or (M185C) or (M185D) or (M185E) or (M185G) or (M185H) or (M185K) or (M185L) or (M185N) or (M185Q) or (M185V) or (M212A) or (M212C) or (M212D) or (M212E) or (M212F) or (M212G) or (M212H) or (M212K) or (M212L) or (M212L, I259F) or (M212Q) or (M212R) or (M212S) or (M212T) or (M212V) or (M212W) or (M212Y) or (M354A) or (M354C) or (M354D) or (M354E) or (M354G) or (M354I) or (M354K) or (M354K, D363Y) or (M354L) or (M354N) or (M354Q) or (M354R) or (M354S) or (M354T) or (M354V) or (M354W) or (M354Y) or (N70A) or (N70C) or (N70F) or (N70G) or (N70H) or (N70K) or (N70R) or (N70Y) or (P243T, H266L) or (P329A) or (P392C) or (P392G) or (P392Q) or (P392R) or (P392V) or (P9S) or (R147C, H266G) or (R161C, G271D) or (R190L, F261V) or (R203C) or (R255A) or (R255G) or (R255I) or (R255K) or (R255L) or (R255M) or (R255N) or (R255P) or (R255T) or (R255W) or (R47A) or (R47C) or (R47D) or (R47E) or (R47F) or (R47G) or (R47H) or (R47H, A74W) or (R47I) or (R47K) or (R47L) or (R47M) or (R47N) or (R47P) or (R47Q) or (R47S) or (R47T) or (R47V) or (R47W) or (R50A) or (R50C) or (R50D) or (R50E) or (R50F) or (R50G) or (R50I) or (R50L) or (R50M) or (R50N) or (R50P) or (R50Q) or (R50S) or (R50S, A184W, L188F) or (R50S, A74I, A184W, L188F) or (R50S, A74M, A184Y, L188K) or (R50S, A74T, L181Y, A184D, L188K, G240R) or (R50S, A74V) or (R50S, M177Y, V178P, A184Y) or (R50S, M177Y, V178W, A184W) or (R50S, M177Y, V178W, A184Y) or (R50S, M177Y, V178W, A184Y, A330R) or (R50S, M177Y, V178W, A184Y, A74V, G677D) or (R50S, M177Y, V178W, A184Y, L181P) or (R50S, M177Y, V178W, A184Y, L181Y) or (R50S, M177Y, V178W, A184Y, L188K) or (R50S, M177Y, V178W, A184Y, L188R) or (R50S, M177Y, V178W, A184Y, T146F) or (R50S, M177Y, V178W, A184Y, V26N) or (R50S, M177Y, V178W, L181Y, A184Y) or (R50S, S72G, A74V) or (R50S, S72G, A74V, M177Y, A184Y) or (R50S, S72G, A74V, M177Y, V178P, A184Y) or (R50S, S72G, A74V, M177Y, V178W, A184Y) or (R50S, S72G, A74V, M177Y, V178W, A184Y, L188R) or (R50S, T146F, M177Y, V178W, A184Y) or (R50V) or (R50Y) or (S176A, A184Y, L188K) or (S176C) or (S176D) or (S176E) or (S176F) or (S176G) or (S176H) or (S176K) or (S176L) or (S176M) or (S176N) or (S176P) or (S176Q) or (S72C, A74C) or (S72C, A74I) or (S72C, A74L) or (S72C, A74V) or (S72D, A74C) or (S72G) or (S72G, A74C) or (S72G, A74F) or (S72G, A74H) or (S72G, A74I) or (S72G, A74I, A184W, L188F) or (S72G, A74I, F77A, M177Y) or (S72G, A74I, F77L) or (S72G, A74I, F77L, M177Y) or (S72G, A74I, L181Y) or (S72G, A74I, L181Y, L188F) or (S72G, A74I, L181Y, L188K) or (S72G, A74I, L181Y, L188R) or (S72G, A74I, L188F) or (S72G, A74I, L188K) or (S72G, A74I, L188R) or (S72G, A74I, T146F) or (S72G, A74I, T146F, L181Y) or (S72G, A74I, T146F, L181Y, L188F) or (S72G, A74I, T146F, L181Y, L188K) or (S72G, A74I, T146F, L181Y, L188R) or (S72G, A74I, T146F, L188F) or (S72G, A74I, T146F, L188K) or (S72G, A74I, T146F, V178P) or (S72G, A74I, T146F, V178P, L181Y) or (S72G, A74I, T146F, V178P, L181Y, L188F) or (S72G, A74I, T146F, V178P, L181Y, L188K) or (S72G, A74I, T146F, V178P, L181Y, L188R) or (S72G, A74I, T146F, V178P, L188F) or (S72G, A74I, T146F, V178P, L188K) or (S72G, A74I, T146F, V178P, L188R) or (S72G, A74I, T146F, V178W) or (S72G, A74I, T146F, V178W, L181Y) or (S72G, A74I, T146F, V178W, L181Y, L188F) or (S72G, A74I, T146F, V178W, L181Y, L188K) or (S72G, A74I, T146F, V178W, L181Y, L188R) or (S72G, A74I, T146F, V178W, L188F) or (S72G, A74I, T146F, V178W, L188K) or (S72G, A74I, T146F, V178W, L188R) or (S72G, A74I, V178P) or (S72G, A74I, V178P, L181Y) or (S72G, A74I, V178P, L181Y, L188R) or (S72G, A74I, V178P, L188F) or (S72G, A74I, V178P, L188K) or (S72G, A74I, V178P, L188R) or (S72G, A74I, V178W) or (S72G, A74I, V178W, L181Y, L188F) or (S72G, A74I, V178W, L181Y, L188K) or (S72G, A74I, V178W, L181Y, L188R) or (S72G, A74I, V178W, L188F) or (S72G, A74I, V178W, L188R) or (S72G, A74L) or (S72G, A74M, A184Y, L188K) or (S72G, A74S) or (S72G, A74T) or (S72G, A74T, L181Y, A184D, L188K) or (S72G, A74T, L181Y, L188F) or (S72G, A74T, L181Y, L188K) or (S72G, A74T, L181Y, L188R) or (S72G, A74T, L188F) or (S72G, A74T, L188K) or (S72G, A74T, L188R) or (S72G, A74T, T146F) or (S72G, A74T, T146F, L181Y) or (S72G, A74T, T146F, L181Y, L188K) or (S72G, A74T, T146F, L181Y, L188R) or (S72G, A74T, T146F, L188F) or (S72G, A74T, T146F, L188K) or (S72G, A74T, T146F, L188R) or (S72G, A74T, T146F, V178P) or (S72G, A74T, T146F, V178P, L181Y) or (S72G, A74T, T146F, V178P, L188F) or (S72G, A74T, T146F, V178P, L188K) or (S72G, A74T, T146F, V178P, L188R) or (S72G, A74T, T146F, V178W) or (S72G, A74T, T146F, V178W, L181Y) or (S72G, A74T, T146F, V178W, L181Y, L188F) or (S72G, A74T, T146F, V178W, L181Y, L188K) or (S72G, A74T, T146F, V178W, L181Y, L188R) or (S72G, A74T, T146F, V178W, L188F) or (S72G, A74T, T146F, V178W, L188K) or (S72G, A74T, T146F, V178W, L188R) or (S72G, A74T, V178P) or (S72G, A74T, V178P, L181Y) or (S72G, A74T, V178P, L181Y, L188F) or (S72G, A74T, V178P, L181Y, L188K) or (S72G, A74T, V178P, L188F) or (S72G, A74T, V178P, L188K) or (S72G, A74T, V178P, L188R) or (S72G, A74T, V178W) or (S72G, A74T, V178W, L181Y, L188F) or (S72G, A74T, V178W, L181Y, L188K) or (S72G, A74T, V178W, L188F) or (S72G, A74T, V178W, L188K) or (S72G, A74T, V178W, L188R) or (S72G, A74V) or (S72G, A74V, A184W) or (S72G, A74V, A221V) or (S72G, A74V, F77A) or (S72G, A74V, F77A, M177Y) or (S72G, A74V, F77L, M177Y) or (S72G, A74V, F77S) or(S72G, A74V, L181Y) or (S72G, A74V, L181Y, L188F) or(S72G, A74V, L181Y, L188K) or (S72G, A74V, L188F) or (S72G, A74V, L188K) or (S72G, A74V, L188R) or (S72G, A74V, T146F) or (S72G, A74V, T146F, L181Y) or (S72G, A74V, T146F, L181Y, L188F) or (S72G, A74V, T146F, L181Y, L188K) or (S72G, A74V, T146F, L181Y, L188R) or (S72G, A74V, T146F, L188F) or (S72G, A74V, T146F, L188K) or (S72G, A74V, T146F, L188R) or (S72G, A74V, T146F, V178P) or (S72G, A74V, T146F, V178P, L181Y) or (S72G, A74V, T146F, V178P, L181Y, L188F) or (S72G, A74V, T146F, V178P, L181Y, L188K) or (S72G, A74V, T146F, V178P, L181Y, L188R) or (S72G, A74V, T146F, V178P, L188F) or (S72G, A74V, T146F, V178P, L188K) or (S72G, A74V, T146F, V178P, L188R) or (S72G, A74V, T146F, V178W) or (S72G, A74V, T146F, V178W, L181Y) or (S72G, A74V, T146F, V178W, L181Y, L188K) or (S72G, A74V, T146F, V178W, L181Y, L188R) or (S72G, A74V, T146F, V178W, L188F) or (S72G, A74V, T146F, V178W, L188K) or (S72G, A74V, T146F, V178W, L188R) or (S72G, A74V, V178P) or (S72G, A74V, V178P, L181Y) or (S72G, A74V, V178P, L181Y, L188F) or (S72G, A74V, V178P, L181Y, L188K) or (S72G, A74V, V178P, L181Y, L188R) or (S72G, A74V, V178P, L188F) or (S72G, A74V, V178P, L188K) or (S72G, A74V, V178P, L188R) or (S72G, A74V, V178W) or (S72G, A74V, V178W, L181Y) or (S72G, A74V, V178W, L181Y, L188F) or (S72G, A74V, V178W, L181Y, L188K) or (S72G, A74V, V178W, L181Y, L188R) or (S72G, A74V, V178W, L188F) or (S72G, A74V, V178W, L188K) or (S72G, A74V, V178W, L188R) or (S72G, A74Y) or (S72G, L181Y) or (S72G, L181Y, L188F) or (S72G, L181Y, L188K) or (S72G, L181Y, L188R) or (S72G, L188F) or (S72G, L188K) or (S72G, L188R) or (S72G, M177Y, V178W) or (S72G, M177Y, V178W, L188K) or (S72G, T146F) or (S72G, T146F, L181Y) or (S72G, T146F, L181Y, L188F) or (S72G, T146F, L181Y, L188K) or (S72G, T146F, L181Y, L188R) or (S72G, T146F, L188F) or (S72G, T146F, L188K) or (S72G, T146F, L188R) or (S72G, T146F, V178P) or (S72G, T146F, V178P, L181Y) or (S72G, T146F, V178P, L181Y, L188F) or (S72G, T146F, V178P, L181Y, L188K) or (S72G, T146F, V178P, L181Y, L188R) or (S72G, T146F, V178P, L188F) or (S72G, T146F, V178P, L188K) or (S72G, T146F, V178P, L188R) or (S72G, T146F, V178W) or (S72G, T146F, V178W, L181Y) or (S72G, T146F, V178W, L181Y, L188F) or (S72G, T146F, V178W, L181Y, L188K) or (S72G, T146F, V178W, L188F) or (S72G, T146F, V178W, L188K) or (S72G, T146F, V178W, L188R) or (S72G, V178P) or (S72G, V178P, L181Y) or (S72G, V178P, L181Y, L188F) or (S72G, V178P, L181Y, L188K) or (S72G, V178P, L181Y, L188R) or (S72G, V178P, L188F) or (S72G, V178P, L188K) or (S72G, V178P, L188R) or (S72G, V178W) or (S72G, V178W, L181Y) or (S72G, V178W, L181Y, L188F) or (S72G, V178W, L181Y, L188K) or (S72G, V178W, L181Y, L188R) or (S72G, V178W, L188F) or (S72G, V178W, L188K) or (S72G, V178W, L188R) or (S72H, A74C) or (S72N, A74C) or (S72N, A74I) or (S72N, A74V) or (S72W) or (S72W, A74V) or (S89C) or (S89E) or (S89G) or (S89I) or (S89L) or (S89N) or (S89Q) or (S89R) or (S89T) or (S89V) or (T146A) or (T146A, D222Y, I263M) or (T146A, F173D) or (T146A, F173V) or (T146A, F205G) or (T146A, F261C) or (T146A, F261S) or (T146A, F261V) or (T146A, G271S) or (T146A, G271T) or (T146A, H266V) or (T146A, I258T, I259S) or (T146A, I259H) or (T146A, I263F) or (T146A, I263L) or (T146A, I263N) or (T146A, I263V) or (T146A, L150A) or (T146A, L150G) or (T146A, L150K) or (T146A, L150R, F173I) or (T146A, L272H) or (T146A, L272S) or (T146A, L272W) or (T146A, M212A) or (T146C) or (T146D) or (T146E) or (T146F) or (T146F, L181Y) or (T146F, L181Y, L188F) or (T146F, L181Y, L188K) or (T146F, L188K) or (T146F, L188R) or (T146F, V178P) or (T146F, V178P, L181Y, L188R) or (T146F, V178P, L188K) or (T146F, V178P, L188R) or (T146F, V178W) or (T146F, V178W, L181Y, L188R) or (T146F, V178W, L188F) or (T146F, V178W, L188K) or (T146F, V178W, L188R) or (T146G) or (T146H) or (T146L) or (T146M) or (T146N) or (T146P) or (T146R) or (T146S) or (T146V) or (T146V, A191T) or (T146Y) or (T149P) or (T260W) or (T268A) or (T268S) or (T269K) or (T327A) or (T327A, A330W) or (T327C) or (T327D) or (T327E) or (T327I) or (T327L) or (T327M) or (T327N) or (T327P) or (T327Q) or (T327S) or (T327V) or (T365C) or (T365D) or (T365F) or (T365G) or (T365H) or (T365I) or (T365K) or (T365L) or (T365N) or (T365P) or (T365Q) or (T365V) or (T365W) or (T365Y) or (T436A) or (T436C) or (T436D) or (T436E) or (T436F) or (T436G) or (T436H) or (T436I) or (T436K) or (T436L) or (T436M) or (T436N) or (T436P) or (T436Q) or (T436R) or (T436S) or (T436V) or (T436W) or (T436Y) or (T438A) or (T438C) or (T438I) or (T438S) or (T438V) or (T49A) or (T49C) or (T49D) or (T49E) or (T49F) or (T49G) or (T49H) or (T49I) or (T49K) or (T49L) or (T49L, S54N) or (T49M) or (T49N) or (T49P) or (T49Q) or (T49R) or (T49R, R50C) or (T49R, R50S, M177Y, V178W, A184Y) or (T49S) or (T49V) or (T49W) or (T49Y) or (T88C) or (T88S) or (T88V) or (V178A) or (V178D) or (V178H) or (V178I) or (V178K) or (V178L) or (V178M) or (V178N) or (V178P) or (V178P, L181Y) or (V178P, L181Y, L188R) or (V178P, L188F) or (V178P, L188K) or (V178P, L188R) or (V178Q) or (V178R) or (V178S) or (V178T) or (V178W) or (V178W, A184Y) or (V178W, L181Y, L188K) or (V178W, L181Y, L188R) or (V178W, L188R) or (V178W, R50S) or (V178Y) or (V26A) or (V26C) or (V26F) or (V26G) or (V26H) or (V26I) or (V26K) or (V26L) or (V26M) or (V26N) or (V26N, R50S, M177Y, V178W, A184Y) or (V26Q) or (V26S) or (V26T) or (V26W) or (V26Y) or (V314A) or (V314E) or (V314F) or (V314H) or (V314P) or (V314Q) or (V314S) or (V314Y) or (V371P) or (V48A) or (V48D) or (V48E) or (V48F) or (V48G) or (V48H) or (V48I) or (V48L) or (V48M) or (V48P) or (V48Q) or (V48R) or (V48S) or (V48T) or (V48W) or (V48Y) or (W325A) or (W325C) or (W325D) or (W325E) or (W325F) or (W325G) or (W325I) or (W325K) or (W325L) or (W325M) or (W325Q) or (W325S) or (W325T) or (W325V) or (W325Y) or (W90Y) or (Y51A) or (Y51C) or (Y51F) or (Y51H) or (Y51I) or (Y51L) or (Y51M) or (Y51P) or (Y51S, F77V) or (Y51T) or (Y51V) or (Y51W) or (A225C) or (A225M) or (A225N) or (A225Q) or (A225S) or (A225T) or (A225Y) or (A44Q) or (A44T) or (D182A) or (D182C) or (D182E) or (D182H) or (D182I) or (D182M) or (D182N) or (D182V) or (D182W) or (D80C) or (D80L) or (D80M) or (D80N) or (D80S) or (D80T) or (D80W) or (E140A) or (E140D) or (E140K) or (E140L) or (E140M) or (E140T) or (E183D) or (E337L) or (E337V) or (G114C) or (G114K) or (G114L) or (G114N) or (G114W) or (G227N) or (G227R) or (G240A) or (G240C) or (G240H) or (G240K) or (G240N) or (G240R) or (G240S) or (H236A) or (H236C) or (H236D) or (H236F) or (H236I) or (H236K) or (H236M) or (H236P) or (H236R) or (H236S) or (H92F) or (H92Y) or (I174T) or (I174V) or (I258C) or (I258L) or (I258M) or (I258V) or (K187A) or (K187G) or (K187N) or (K187R) or (K440C) or (K440M) or (K440N) or (K440T) or (K76C) or (K76E) or (K76F) or (K76H) or (K76I) or (K76N) or (K76V) or (M5C) or (M5E) or (M5P) or (N186A) or (N186D) or (N186E) or (N186F) or (N186H) or (N186K) or (N186L) or (N186M) or (N186Q) or (N186T) or (N186V) or (N186Y) or (P172A) or (P172D) or (P172G) or (Q189E) or (Q189I) or (Q189K) or (Q189N) or (Q189V) or (Q397R) or (Q403K) or (Q403R) or (Q404F) or (Q404Y) or (Q73E) or (Q73F) or (Q73L) or (Q73S) or (Q73T) or (Q73W) or (Q73Y) or (R179A) or (R179H) or (R179L) or (R179M) or (R179P) or (R179V) or (R190C) or (R190G) or (R190K) or (R190M) or (R190N) or (R190S) or (R190V) or (R190W) or (R79K) or (S270A) or (S270D) or (S53C) or (T175F) or (T175G) or (T175H) or (T175L) or (T175M) or (T175N) or (T175Q) or (T175R) or (T175Y) or (T269V) or (V299L) or (V299W) or (A225V, H236K, G240V, T269V) or (D80I, D182H, A225V, G240V, T269V) or (F173W, R179L, T269V, K440C) or (F173W, R190V, H236K, T269V) or (H236K, G240V, T269V, K440C) or (Q73W, D80I, N186V, G240V) or (R179L, A225S) or (T175M, H236D) or (T175M, R190V).
There is provided a second preferred subset comprising variants according to the sixth embodiment, wherein the P450-BM3 variants have an improved selectivity (as determined by SF or TPR) for C19 hydroxylation in comparison with the parent variant (see
There is provided a third preferred subset comprising variants according to the sixth embodiment, wherein the P450-BM3 variants have an improved product yield or product titer and an improved selectivity (as determined by SF or TPR) for C19 hydroxylation in comparison with the parent variant. Comprised within this subset are variants comprising at least the mutation(s) (A184D) or(A184D, L188K) or (A184E) or (A184F) or (A184G) or (A184H) or (A184I) or (A184K) or (A184L) or (A184N) or (A184Q) or (A184R) or (A184W) or (A184W, L188F) or (A184Y) or (A184Y, L188K) or (A184Y, R50S) or (A221S) or (A264V) or (A321C) or (A321D) or (A321E) or (A321G) or (A321I) or (A321N) or (A321T) or (A321V) or (A330C) or (A330D) or (A330E) or (A330F) or (A330G) or (A330H) or (A330I) or (A330K) or (A330L) or (A330M) or (A330N) or (A330Q) or (A330R) or (A330S) or (A330T) or (A330T, E352A) or (A330V) or (A330W) or (A330Y) or (A330Y, W367C) or (A33V) or (A399C) or (A399E) or (A399G) or (A399I) or (A399L) or (A399M) or (A399N) or (A399Q) or (A399R) or (A399S) or (A399T) or (A399V) or (A74C) or (A74C, L75V) or (A74D) or (A74D, F81V) or (A74E) or (A74F) or (A74H) or (A74I) or (A74I, A184W, L188F) or (A74I, F77A, L181Y, A184W) or (A74I, L181Y, L188R) or (A74I, L188F) or (A74I, L188K) or (A74I, L188R) or (A74I, M177Y, A184R, L188F) or (A74I, M177Y, V178P, A184G) or (A74I, T146F) or (A74I, T146F, L181Y) or (A74I, T146F, L181Y, L188K) or (A74I, T146F, L181Y, L188R) or (A74I, T146F, L188F) or (A74I, T146F, L188K) or (A74I, T146F, V178P) or (A74I, T146F, V178P, L181Y, L188F) or (A74I, T146F, V178P, L181Y, L188K) or (A74I, T146F, V178P, L188K) or (A74I, T146F, V178P, L188R) or (A74I, T146F, V178W) or (A74I, T146F, V178W, L181Y, L188F) or (A74I, T146F, V178W, L181Y, L188R) or (A74I, T146F, V178W, L188F) or (A74I, T146F, V178W, L188K) or (A74I, T146F, V178W, L188R) or (A74I, V178P) or (A74I, V178P, L181Y) or (A74I, V178P, L181Y, L188R) or (A74I, V178P, L188F) or (A74I, V178P, L188K) or (A74I, V178P, L188R) or (A74I, V178W, L188F) or (A74I, V178W, L188R) or (A74K) or (A74L) or (A74M) or (A74N) or (A74P) or (A74Q) or (A74R) or (A74R, L75V) or (A74S) or (A74T) or (A74T, A184D) or (A74T, F77A, L181Y, A184D, L188K) or (A74T, F77A, V178W, A184Y) or (A74T, F77L, L181Y, A184D, L188K) or (A74T, L181Y) or (A74T, L181Y, A184D, L188K) or (A74T, L181Y, A184Y, L188K) or (A74T, L181Y, L188K) or (A74T, L181Y, L188R) or (A74T, L188K) or (A74T, M177Y, L181Y, A184D, L188K) or (A74T, T146F, L181Y, A184D, L188K) or (A74T, T146F, L181Y, L188K) or (A74T, T146F, L181Y, L188R) or (A74T, T146F, L188F) or (A74T, T146F, L188K) or (A74T, T146F, L188R) or (A74T, T146F, V178P) or (A74T, T146F, V178P, L181Y) or (A74T, T146F, V178P, L188K) or (A74T, T146F, V178P, L188R) or (A74T, T146F, V178W) or (A74T, T146F, V178W, L181Y) or (A74T, T146F, V178W, L181Y, L188K) or (A74T, T146F, V178W, L188R) or (A74T, V178P) or (A74T, V178P, L188F) or (A74T, V178P, L188K) or (A74T, V178P, L188R) or (A74T, V178W, L181Y, L188K) or (A74T, V178W, L188K) or (A74T, V178W, L188R) or (A74V) or (A74V, F77A, A184N, L188F) or (A74V, L181Y) or (A74V, L181Y, A184D, L188K) or (A74V, L181Y, A184W, L188F) or (A74V, L181Y, L188F) or (A74V, L188F) or (A74V, L188K) or (A74V, L75V) or (A74V, T146F, L181Y, L188K) or (A74V, T146F, L181Y, L188R) or (A74V, T146F, L188K) or (A74V, T146F, L188R) or (A74V, T146F, V178W, L181Y, L188F) or (A74V, V178P, A184W, L188K) or (A74V, V178P, L181Y, L188F) or (A74V, V178P, L181Y, L188K) or (A74V, V178P, L181Y, L188R) or (A74V, V178P, L188F) or (A74V, V178W, L188K) or (A74W) or (A74Y) or (D363G, T438C) or (E13D, R47L) or (E143A) or (E143C) or (E143D) or (E143F) or (E143G) or (E143I) or (E143K) or (E143L) or (E143M) or (E143N) or (E143P) or (E143Q) or (E143R) or (E143S) or (E143V) or (E143W) or (E143Y) or (E267D) or (E267R) or (E267T) or (E352A) or (E352D) or (E352F) or (E352G) or (E352I) or (E352L) or (E352M) or (E352N) or (E352P) or (E352R) or (E352S) or (E352T) or (E352V) or (E352W) or (E352Y) or (E64A) or (E64G) or (E64H) or (E64K) or (E64L) or (E64M) or (E64N) or (E64Q) or (E64V) or (E64Y) or (E82P) or (E93G) or (F173C) or (F173C, F205G) or (F173D) or (F173I) or (F173K) or (F173L) or (F173M) or (F173N) or (F173P) or (F173Q) or (F173R) or (F173S) or (F173S, F205P) or (F173V) or (F173W) or (F173Y) or (F173Y, I174F) or (F205A) or (F205C) or (F205D) or (F205D, D208N) or (F205E) or (F205G) or (F205H) or (F205I) or (F205K) or (F205L) or (F205M) or (F205N) or (F205P) or (F205R) or (F205S) or (F205T) or (F205V) or (F205W) or (F205Y) or (F261A) or (F261C) or (F261D) or (F261G) or (F261I) or (F261L) or (F261M) or (F261Q) or (F261S) or (F261T) or (F261V) or (F261Y) or (F331H) or (F331I) or (F331L) or (F331M) or (F331V) or (F331W) or (F331Y) or (F393M) or (F393W) or (F77A) or (F77A, A184W) or (F77A, L181P, A184Y, L188K) or (F77A, M177Y) or (F77C) or (F77D) or (F77E) or (F77G) or (F77H) or (F77I) or (F77K) or (F77L) or (F77M) or (F77N) or (F77P) or (F77R) or (F77S) or (F77T) or (F77V) or (F77V, L86M) or (F77W) or (F77Y) or (F81D) or (F81I) or (F81L) or (F81R) or (F81S) or (F81V) or (F81W) or (F81Y) or (G271A) or (G271C) or (G271D) or (G271E) or (G271F) or (G271H) or (G271K) or (G271L) or (G271M) or (G271N) or (G271P) or (G271Q) or (G271S) or (G271T) or (G271V) or (G271W) or (G271Y) or (G402A) or (G415A) or (G415D) or (G415S) or (G415T) or (G415V) or (G85A) or (G85L) or (G85S) or (H266A) or (H266C) or (H266E) or (H266F) or (H266I) or (H266K) or (H266M) or (H266N) or (H266Q) or (H266S) or (H266V) or (H266W) or (H266Y) or (I153F, G271L) or (I153L) or (I153L, F173Y) or (I259A) or (I259C) or (I259D) or (I259F) or (I259H) or (I259K) or (I259L) or (I259M) or (I259N) or (I259Q) or (I259S) or (I259T) or (I259V) or (I263A) or (I263C) or (I263E) or (I263F) or (I263H, A264G) or (I263K) or (I263L) or (I263M) or (I263N) or (I263Q) or (I263S) or (I263V) or (I263Y) or (I401A) or (I401M) or (I401T) or (I401V) or (K210E) or (K210T, G271V) or (K224C) or (K224E) or (K224H) or (K224I) or (K224L) or (K224M) or (K224P) or (K224Q) or (K224W) or (K224Y) or (L150A) or (L150C) or (L150D) or (L150E) or (L150F) or (L150G) or (L150H) or (L150I) or (L150K) or (L150M) or (L150N) or (L150Q) or (L150R) or (L150R, F205R) or (L150S) or (L150S, F173L) or (L150T) or (L150V) or (L150W) or (L150Y) or (L181H) or (L181I) or (L181M) or (L181P) or (L181V) or (L181Y) or (L181Y, A184D) or (L181Y, L188F) or (L181Y, L188K) or (L181Y, L188K, H659R) or (L188A) or (L188D) or (L188E) or (L188F) or (L188H) or (L188I) or (L188K) or (L188M) or (L188N) or (L188Q) or (L188R) or (L188S) or (L188W) or (L20C) or (L20D) or (L20E) or (L20F) or (L20G) or (L20G, R47L) or (L20I) or (L20M) or (L20N) or (L20P) or (L20R) or (L20S) or (L20T) or (L20V) or (L20W) or (L20Y) or (L262I) or (L262V) or (L262W) or (L272A) or (L272C) or (L272E) or (L272F) or (L272G) or (L272I) or (L272K) or (L272M) or (L272N) or (L272Q) or (L272S) or (L272T) or (L272V) or (L272W) or (L272Y) or (L29A) or (L29C) or (L29D) or (L29F) or (L29H) or (L29I) or (L29M) or (L29M, R47G) or (L29Q) or (L29S) or (L29T) or (L29V) or (L29W) or (L29Y) or (L324F) or (L356C) or (L356F) or (L356I) or (L356M) or (L356V) or (L437I) or (L437M) or (L75I) or (L75I, F81C) or (L75I, F81I) or (L75I, F81L) or (L75I, F81S) or (L75I, F81V) or (L75V) or (L75V, F81C) or (L75V, F81L) or (L75V, F81V) or (L75V, F81Y) or (L78F) or (L78I) or (L78M) or (L78V) or (L86I) or (L86I, S89T) or (L86M) or (L86M, S89T) or (L86V) or (M118A) or (M118E) or (M118F) or (M118G) or (M118H) or (M118I) or (M118K) or (M118L) or (M118N) or (M118P) or (M118Q) or (M118S) or (M118T) or (M118V) or (M118W) or (M118Y) or (M177A) or (M177C) or (M177C, V178Y) or (M177D) or (M177E) or (M177F) or (M177H) or (M177I) or (M177L) or (M177N) or (M177P) or (M177Q) or (M177R) or (M177T) or (M177V) or (M177Y) or (M177Y, A184W) or (M177Y, A184Y) or (M177Y, M185V) or (M177Y, R50S) or (M177Y, V178P) or (M177Y, V178P, A184Y, L188F) or (M177Y, V178W) or (M177Y, V178W, A184Y) or (M177Y, V178W, A184Y, L188F) or (M185C) or (M185D) or (M185E) or (M185G) or (M185H) or (M185K) or (M185L) or (M185N) or (M185Q) or (M185V) or (M212A) or (M212C) or (M212D) or (M212E) or (M212F) or (M212G) or (M212H) or (M212K) or (M212L) or (M212L, I259F) or (M212Q) or (M212R) or (M212S) or (M212T) or (M212V) or (M212W) or (M212Y) or (M354A) or (M354C) or (M354D) or (M354E) or (M354G) or (M354I) or (M354K) or (M354K, D363Y) or (M354L) or (M354N) or (M354Q) or (M354R) or (M354S) or (M354T) or (M354V) or (M354W) or (M354Y) or (N70A) or (N70C) or (N70F) or (N70G) or (N70H) or (N70K) or (N70R) or (N70Y) or (P243T, H266L) or (P329A) or (P392C) or (P392G) or (P392Q) or (P392R) or (P392V) or (P9S) or (R147C, H266G) or (R161C, G271D) or (R190L, F261V) or (R203C) or (R255A) or (R255G) or (R255I) or (R255K) or (R255L) or (R255M) or (R255N) or (R255P) or (R255T) or (R255W) or (R47A) or (R47C) or (R47D) or (R47E) or (R47F) or (R47G) or (R47H) or (R47H, A74W) or (R47I) or (R47K) or (R47L) or (R47M) or (R47N) or (R47P) or (R47Q) or (R47S) or (R47T) or (R47V) or (R47W) or (R50A) or (R50C) or (R50D) or (R50E) or (R50F) or (R50G) or (R50I) or (R50L) or (R50M) or (R50N) or (R50P) or (R50Q) or (R50S) or (R50S, A184W, L188F) or (R50S, A74I, A184W, L188F) or (R50S, A74M, A184Y, L188K) or (R50S, A74T, L181Y, A184D, L188K, G240R) or (R50S, A74V) or (R50S, M177Y, V178P, A184Y) or (R50S, M177Y, V178W, A184W) or (R50S, M177Y, V178W, A184Y) or (R50S, M177Y, V178W, A184Y, A330R) or (R50S, M177Y, V178W, A184Y, A74V, G677D) or (R50S, M177Y, V178W, A184Y, L181P) or (R50S, M177Y, V178W, A184Y, L181Y) or (R50S, M177Y, V178W, A184Y, L188K) or (R50S, M177Y, V178W, A184Y, L188R) or (R50S, M177Y, V178W, A184Y, T146F) or (R50S, M177Y, V178W, A184Y, V26N) or (R50S, M177Y, V178W, L181Y, A184Y) or (R50S, S72G, A74V) or (R50S, S72G, A74V, M177Y, A184Y) or (R50S, S72G, A74V, M177Y, V178P, A184Y) or (R50S, S72G, A74V, M177Y, V178W, A184Y) or (R50S, S72G, A74V, M177Y, V178W, A184Y, L188R) or (R50S, T146F, M177Y, V178W, A184Y) or (R50V) or (R50Y) or (S176A, A184Y, L188K) or (S176C) or (S176D) or (S176E) or (S176F) or (S176G) or (S176H) or (S176K) or (S176L) or (S176M) or (S176N) or (S176P) or (S176Q) or (S72C, A74C) or (S72C, A74I) or (S72C, A74L) or (S72C, A74V) or (S72D, A74C) or (S72G) or (S72G, A74C) or (S72G, A74F) or (S72G, A74H) or (S72G, A74I) or (S72G, A74I, A184W, L188F) or (S72G, A74I, F77A, M177Y) or (S72G, A74I, F77L) or (S72G, A74I, F77L, M177Y) or (S72G, A74I, L181Y) or (S72G, A74I, L181Y, L188F) or (S72G, A74I, L181Y, L188K) or (S72G, A74I, L181Y, L188R) or (S72G, A74I, L188F) or (S72G, A74I, L188K) or (S72G, A74I, L188R) or (S72G, A74I, T146F) or (S72G, A74I, T146F, L181Y) or (S72G, A74I, T146F, L181Y, L188F) or (S72G, A74I, T146F, L181Y, L188K) or (S72G, A74I, T146F, L181Y, L188R) or (S72G, A74I, T146F, L188F) or (S72G, A74I, T146F, L188K) or (S72G, A74I, T146F, V178P) or (S72G, A74I, T146F, V178P, L181Y) or (S72G, A74I, T146F, V178P, L181Y, L188F) or (S72G, A74I, T146F, V178P, L181Y, L188K) or (S72G, A74I, T146F, V178P, L181Y, L188R) or (S72G, A74I, T146F, V178P, L188F) or (S72G, A74I, T146F, V178P, L188K) or (S72G, A74I, T146F, V178P, L188R) or (S72G, A74I, T146F, V178W) or (S72G, A74I, T146F, V178W, L181Y) or (S72G, A74I, T146F, V178W, L181Y, L188F) or (S72G, A74I, T146F, V178W, L181Y, L188K) or (S72G, A74I, T146F, V178W, L181Y, L188R) or (S72G, A74I, T146F, V178W, L188F) or (S72G, A74I, T146F, V178W, L188K) or (S72G, A74I, T146F, V178W, L188R) or (S72G, A74I, V178P) or (S72G, A74I, V178P, L181Y) or (S72G, A74I, V178P, L181Y, L188R) or (S72G, A74I, V178P, L188F) or (S72G, A74I, V178P, L188K) or (S72G, A74I, V178P, L188R) or (S72G, A74I, V178W) or (S72G, A74I, V178W, L181Y, L188F) or (S72G, A74I, V178W, L181Y, L188R) or (S72G, A74I, V178W, L188F) or (S72G, A74I, V178W, L188R) or (S72G, A74L) or (S72G, A74M, A184Y, L188K) or (S72G, A74S) or (S72G, A74T) or (S72G, A74T, L181Y, A184D, L188K) or (S72G, A74T, L181Y, L188F) or (S72G, A74T, L181Y, L188K) or (S72G, A74T, L181Y, L188R) or (S72G, A74T, L188F) or (S72G, A74T, L188K) or (S72G, A74T, L188R) or (S72G, A74T, T146F) or (S72G, A74T, T146F, L181Y) or (S72G, A74T, T146F, L181Y, L188K) or (S72G, A74T, T146F, L181Y, L188R) or (S72G, A74T, T146F, L188F) or (S72G, A74T, T146F, L188K) or (S72G, A74T, T146F, L188R) or (S72G, A74T, T146F, V178P) or (S72G, A74T, T146F, V178P, L181Y) or (S72G, A74T, T146F, V178P, L188F) or (S72G, A74T, T146F, V178P, L188K) or (S72G, A74T, T146F, V178P, L188R) or (S72G, A74T, T146F, V178W) or (S72G, A74T, T146F, V178W, L181Y) or (S72G, A74T, T146F, V178W, L181Y, L188F) or (S72G, A74T, T146F, V178W, L181Y, L188K) or (S72G, A74T, T146F, V178W, L181Y, L188R) or (S72G, A74T, T146F, V178W, L188F) or (S72G, A74T, T146F, V178W, L188K) or (S72G, A74T, T146F, V178W, L188R) or (S72G, A74T, V178P) or (S72G, A74T, V178P, L181Y) or (S72G, A74T, V178P, L181Y, L188F) or (S72G, A74T, V178P, L181Y, L188K) or (S72G, A74T, V178P, L188F) or (S72G, A74T, V178P, L188K) or (S72G, A74T, V178P, L188R) or (S72G, A74T, V178W) or (S72G, A74T, V178W, L181Y, L188F) or (S72G, A74T, V178W, L181Y, L188K) or (S72G, A74T, V178W, L188F) or (S72G, A74T, V178W, L188K) or (S72G, A74T, V178W, L188R) or (S72G, A74V) or (S72G, A74V, A184W) or (S72G, A74V, A221V) or (S72G, A74V, F77A) or (S72G, A74V, F77A, M177Y) or (S72G, A74V, F77L, M177Y) or (S72G, A74V, F77S) or(S72G, A74V, L181Y) or (S72G, A74V, L181Y, L188F) or (S72G, A74V, L181Y, L188K) or (S72G, A74V, L188F) or (S72G, A74V, L188K) or (S72G, A74V, L188R) or (S72G, A74V, T146F) or (S72G, A74V, T146F, L181Y) or (S72G, A74V, T146F, L181Y, L188F) or (S72G, A74V, T146F, L181Y, L188K) or (S72G, A74V, T146F, L181Y, L188R) or (S72G, A74V, T146F, L188F) or (S72G, A74V, T146F, L188K) or (S72G, A74V, T146F, L188R) or (S72G, A74V, T146F, V178P) or (S72G, A74V, T146F, V178P, L181Y) or (S72G, A74V, T146F, V178P, L181Y, L188F) or (S72G, A74V, T146F, V178P, L181Y, L188K) or (S72G, A74V, T146F, V178P, L181Y, L188R) or (S72G, A74V, T146F, V178P, L188F) or (S72G, A74V, T146F, V178P, L188K) or (S72G, A74V, T146F, V178P, L188R) or (S72G, A74V, T146F, V178W) or (S72G, A74V, T146F, V178W, L181Y) or (S72G, A74V, T146F, V178W, L181Y, L188K) or (S72G, A74V, T146F, V178W, L181Y, L188R) or (S72G, A74V, T146F, V178W, L188F) or (S72G, A74V, T146F, V178W, L188K) or (S72G, A74V, T146F, V178W, L188R) or (S72G, A74V, V178P) or (S72G, A74V, V178P, L181Y) or (S72G, A74V, V178P, L181Y, L188F) or (S72G, A74V, V178P, L181Y, L188K) or (S72G, A74V, V178P, L181Y, L188R) or (S72G, A74V, V178P, L188F) or (S72G, A74V, V178P, L188K) or (S72G, A74V, V178P, L188R) or (S72G, A74V, V178W) or (S72G, A74V, V178W, L181Y) or (S72G, A74V, V178W, L181Y, L188F) or (S72G, A74V, V178W, L181Y, L188K) or (S72G, A74V, V178W, L181Y, L188R) or (S72G, A74V, V178W, L188F) or (S72G, A74V, V178W, L188K) or (S72G, A74V, V178W, L188R) or (S72G, A74Y) or (S72G, L181Y) or (S72G, L181Y, L188F) or (S72G, L181Y, L188K) or (S72G, L181Y, L188R) or (S72G, L188F) or (S72G, L188K) or (S72G, L188R) or (S72G, M177Y, V178W) or (S72G, M177Y, V178W, L188K) or (S72G, T146F) or (S72G, T146F, L181Y) or (S72G, T146F, L181Y, L188F) or (S72G, T146F, L181Y, L188K) or (S72G, T146F, L181Y, L188R) or (S72G, T146F, L188F) or (S72G, T146F, L188K) or (S72G, T146F, L188R) or (S72G, T146F, V178P) or (S72G, T146F, V178P, L181Y) or (S72G, T146F, V178P, L181Y, L188F) or (S72G, T146F, V178P, L181Y, L188K) or (S72G, T146F, V178P, L181Y, L188R) or (S72G, T146F, V178P, L188F) or (S72G, T146F, V178P, L188K) or (S72G, T146F, V178P, L188R) or (S72G, T146F, V178W) or (S72G, T146F, V178W, L181Y) or (S72G, T146F, V178W, L181Y, L188F) or (S72G, T146F, V178W, L181Y, L188K) or (S72G, T146F, V178W, L188F) or (S72G, T146F, V178W, L188K) or (S72G, T146F, V178W, L188R) or (S72G, V178P) or (S72G, V178P, L181Y) or (S72G, V178P, L181Y, L188F) or (S72G, V178P, L181Y, L188K) or (S72G, V178P, L181Y, L188R) or (S72G, V178P, L188F) or (S72G, V178P, L188K) or (S72G, V178P, L188R) or (S72G, V178W) or (S72G, V178W, L181Y) or (S72G, V178W, L181Y, L188F) or (S72G, V178W, L181Y, L188K) or (S72G, V178W, L181Y, L188R) or (S72G, V178W, L188F) or (S72G, V178W, L188K) or (S72G, V178W, L188R) or (S72H, A74C) or (S72N, A74C) or (S72N, A74I) or (S72N, A74V) or (S72W) or (S72W, A74V) or (S89C) or (S89E) or (S89G) or (S89I) or (S89L) or (S89N) or (S89Q) or (S89R) or (S89T) or (S89V) or (T146A) or (T146A, D222Y, I263M) or (T146A, F173D) or (T146A, F173V) or (T146A, F205G) or (T146A, F261C) or (T146A, F261S) or (T146A, F261V) or (T146A, G271S) or (T146A, G271T) or (T146A, H266V) or (T146A, I258T, I259S) or (T146A, I259H) or (T146A, I263F) or (T146A, I263L) or (T146A, I263N) or (T146A, I263V) or (T146A, L150A) or (T146A, L150G) or (T146A, L150K) or (T146A, L150R, F173I) or (T146A, L272H) or (T146A, L272S) or (T146A, L272W) or (T146A, M212A) or (T146C) or (T146D) or (T146E) or (T146F) or (T146F, L181Y) or (T146F, L181Y, L188F) or (T146F, L181Y, L188K) or (T146F, L188K) or (T146F, L188R) or (T146F, V178P) or (T146F, V178P, L181Y, L188R) or (T146F, V178P, L188K) or (T146F, V178P, L188R) or (T146F, V178W) or (T146F, V178W, L181Y, L188R) or (T146F, V178W, L188F) or (T146F, V178W, L188K) or (T146F, V178W, L188R) or (T146G) or (T146H) or (T146L) or (T146M) or (T146N) or (T146P) or (T146R) or (T146S) or (T146V) or (T146V, A191T) or (T146Y) or (T149P) or (T268A) or (T268S) or (T269K) or (T327A) or (T327A, A330W) or (T327C) or (T327D) or (T327E) or (T327I) or (T327L) or (T327M) or (T327N) or (T327P) or (T327Q) or (T327S) or (T327V) or (T365C) or (T365D) or (T365F) or (T365G) or (T365H) or (T365I) or (T365K) or (T365L) or (T365N) or (T365P) or (T365Q) or (T365V) or (T365W) or (T365Y) or (T436A) or (T436C) or (T436D) or (T436E) or (T436F) or (T436G) or (T436H) or (T436I) or (T436K) or (T436L) or (T436M) or (T436N) or (T436P) or (T436Q) or (T436R) or (T436S) or (T436V) or (T436W) or (T436Y) or (T438A) or (T438C) or (T438I) or (T438S) or (T438V) or (T49A) or (T49C) or (T49D) or (T49E) or (T49F) or (T49G) or (T49H) or (T49I) or (T49K) or (T49L) or (T49L, S54N) or (T49M) or (T49N) or (T49P) or (T49Q) or (T49R) or (T49R, R50C) or (T49R, R50S, M177Y, V178W, A184Y) or (T49S) or (T49V) or (T49W) or (T49Y) or (T88C) or (T88S) or (T88V) or (V178D) or (V178H) or (V178I) or (V178K) or (V178L) or (V178M) or (V178N) or (V178P) or (V178P, L181Y) or (V178P, L181Y, L188R) or (V178P, L188F) or (V178P, L188K) or (V178P, L188R) or (V178Q) or (V178R) or (V178S) or (V178T) or (V178W) or (V178W, A184Y) or (V178W, L181Y, L188K) or (V178W, L181Y, L188R) or (V178W, L188R) or (V178W, R50S) or (V178Y) or (V26A) or (V26C) or (V26F) or (V26G) or (V26H) or (V26I) or (V26K) or (V26L) or (V26M) or (V26N) or (V26N, R50S, M177Y, V178W, A184Y) or (V26Q) or (V26S) or (V26T) or (V26W) or (V26Y) or (V314A) or (V314E) or (V314F) or (V314H) or (V314P) or (V314Q) or (V314S) or (V314Y) or (V48A) or (V48D) or (V48E) or (V48F) or (V48G) or (V48H) or (V48I) or (V48L) or (V48M) or (V48P) or (V48Q) or (V48R) or (V48S) or (V48T) or (V48W) or (V48Y) or (W325A) or (W325C) or (W325D) or (W325E) or (W325F) or (W325G) or (W325I) or (W325K) or (W325L) or (W325M) or (W325Q) or (W325S) or (W325T) or (W325V) or (W325Y) or (W90Y) or (Y51A) or (Y51C) or (Y51F) or (Y51H) or (Y51I) or (Y51L) or (Y51M) or (Y51P) or (Y51S, F77V) or (Y51T) or (Y51V) or (Y51W) or (A225C) or (A225N) or (A225Q) or (A225S) or (A225T) or (A225Y) or (A44Q) or (D182A) or (D182C) or (D182E) or (D182H) or (D182I) or (D182M) or (D182N) or (D182V) or (D182W) or (E140A) or (E140D) or (E140K) or (E140M) or (E140T) or (E337L) or (G114C) or (G114K) or (G114L) or (G114N) or (G114W) or (G240A) or (G240C) or (G240H) or (G240K) or (G240N) or (G240R) or (G240S) or (H236A) or (H236C) or (H236D) or (H236F) or (H236I) or (H236K) or (H236P) or (H236R) or (H236S) or (H92F) or (H92Y) or (I174T) or (I174V) or (I258L) or (I258M) or (I258V) or (K187A) or (K187G) or (K187N) or (K187R) or (K440C) or (K440N) or (K440T) or (K76C) or (K76E) or (K76F) or (K76H) or (K76I) or (K76N) or (K76V) or (M5C) or (M5E) or (M5P) or (N186A) or (N186D) or (N186E) or (N186F) or (N186H) or (N186K) or (N186L) or (N186M) or (N186Q) or (N186T) or (N186V) or (N186Y) or (P172A) or (P172D) or (P172G) or (Q189I) or (Q189K) or (Q189V) or (Q403K) or (Q403R) or (Q404F) or (Q404Y) or (Q73E) or (Q73F) or (Q73L) or (Q73S) or (Q73T) or (Q73W) or (R179A) or (R179H) or (R179L) or (R179M) or (R179P) or (R179V) or (R190C) or (R190G) or (R190K) or (R190M) or (R190N) or (R190S) or (R190V) or (R190W) or (R79K) or (S270A) or (S270D) or (S53C) or (T175F) or (T175G) or (T175H) or (T175L) or (T175M) or (T175N) or (T175Q) or (T175R) or (T175Y) or (T269V) or (V299L) or (V299W) or (F173W, R179L, T269V, K440C) or (H236K, G240V, T269V, K440C).
According to one embodiment there is provided a BM3 variant wherein said variant has an improved selectivity and/or product yield or product titer for C19 hydroxylation compared with at least one BM3 variant according to SEQ ID No. 2 to 28, or 117 to 119, preferably with at least one BM3 variant selected from BM3-254, BM3-261, BM3-263, or BM3-268. This embodiment can be and is suggested to be combined with all previous embodiments, in particular with the first, second, third, fourth, fifth and/or sixth embodiment or the described subsets.
According to a highly preferred embodiment of the current invention, there is provided a BM3 variant for catalyzing the C19 hydroxylation of a steroid or steroid derivative, wherein an amino acid sequence (insert) has been introduced between amino acid positions 458 and 477, and wherein the insert has at least 90%, 95%, 99% or 100% sequence identity with a sequence according to any of SEQ ID No. 123 to 173. Most preferably this embodiment is a BM3 variant according to any of the previously described embodiments of the first aspect.
It was surprisingly found that BM3 variants for C19 hydroxylation of steroids comprising these amino acid sequence inserts had improved product titer or TPR, for example if the insert was introduced into a BM3 variant with Seq ID No. 118 or 119.
According to a separate aspect 1B there is provided a BM3 variant characterized by an improved BM3 protein expression relative to BM3 wildtype (WT), wherein the BM3 variant comprises mutations at at least one, two, three, four, five, six, seven, eight, nine, ten or all amino acid positions of (C62), (S106), (Q110), (A117), (Q128), (T152), (G157), (G227), (P243), (H285), (Q288), (A295), (N319), (G368), (V371), (K391), (N395) and (H408). These BM3 variants may also be in accordance with some embodiments of the first aspect.
In particular provided herein is a BM3 variant characterized by an improved BM3 protein expression relative to BM3 wildtype (WT), wherein the BM3 variant comprises at least one, two, three, four, five, six, seven, eight, nine, ten or all mutations of (C62L), (S106A), (Q110E), (A117P), (Q128E), (T152I), (G157L), (G227P), (P243E), (H285E), (Q288E), (A295D), (N319E), (G368E), (V371P), (K391L), (N395F) and (H408L).
In some more specific embodiments, there is provided the BM3 variant according to the previous embodiments, wherein the BM3 variant comprises the mutations (S106A, Q128E) or (S106A, H285E, Q288E) or (S106A, Q128E, H285E) or (C62L, A117P, G157L, N319E) or (C62L, G227P, N319E, K391L) or (Q110E, Q128E, Q288E, N319E) or (Q128E, H285E, Q288E, N319E) or (C62L, S106A, A117P, A295D, V371P) or (C62L, S106A, A117P, G227P, P243E) or (C62L, S106A, Q128E, G227P, K391L) or (S106A, Q110E, G227P, H285E, K391L) or (C62L, S106A, Q128E, G227P, Q288E, K391L) or (S106A, G227P, H285E, V371P, K391L, H408L) or (S106A, Q110E, G227P, N319E, K391L, N395F) or (C62L, S106A, Q128E, T152I, G227P, P243E, H408L) or (S106A, Q110E, Q128E, G227P, Q288E, N319E, K391L) or (C62L, S106A, Q128E, G157L, P243E, A295D, G368E, K391L) or (S106A, A117P, Q128E, T152I, G227P, N319E, V371P, N395F).
As shown in Table E7E and Example 7E, introduction of these mutations resulted in an improved protein expression of the BM3 protein. Protein expression can be measured as known in the art, e.g. by use of SDS PAGE, Western Blot, Mass Spectrometry or any other suitable method. These mutations and BM3 variants can thus be used to solve the same problem, i.e. to improve the C19 hydroxylation.
As understood by the skilled person, the BM3 embodiments for the improved protein production can be combined and are suggested to be combined with each of the previously described embodiments, which are suitable to improve selectivity or yield for C19 hydroxylation.
In addition, the BM3 embodiments for the improved protein production are however not restricted to C19 hydroxylation and can also be used more broadly, i.e to improve the protein production for any BM3 protein, independent of its suitability for steroid hydroxylation.
According to a second aspect of the current invention, there is provided a nucleic acid encoding for a Cytochrome P450 BM3 monooxygenase variant as described herein.
A defined protein or amino acid sequence may be encoded by various nucleic acid sequences. Where an amino acid sequence has been defined herein, each of the nucleic acids encoding this amino acid sequence shall also be deemed disclosed herein. It is therefore well understood that the nucleic acid sequence as provided e.g. in SEQ ID No. 59 for the wildtype sequence has to be read in an exemplary fashion.
Preferably, the nucleic acid encodes for a variant according to the first aspect. Even more preferably, the nucleic acid encodes for a variant according to the first aspect, first embodiment or the nucleic acid encodes for a variant according to the first aspect, second embodiment, e.g. variants according to SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 117, 118 or 119 or functional variants derived thereof. Furthermore, the nucleic acid can be a nucleic acid encoding for a variant according to the third, fourth, fifth, or sixth embodiment of the first aspect.
Nucleic acids according to the second aspect can be generated de novo as known in the art or can be generated by mutating available BM3 nucleic acid sequences. The nucleic acid encoding the P450-BM3 variant according to the current invention can then be introduced into a suitable expression system or host cell for expression of the BM3 variant. Sequences of the nucleic acids can be verified as known in the art. Exemplary methods for sequence verification are disclosed for example in (Green and Sambrook 2012).
According to a third aspect of the current invention, there is provided a host cell for the production of a Cytochrome P450 BM3 monooxygenase variant. In a preferred embodiment, the host cell comprises a nucleic acid encoding for a BM3 variant according to any of the aspects dercribed herein.
Suitable expression systems are well known in the art and example systems comprise E. coli strains such as DH5alpha, BL21 (DE3), or Rosetta (DE3), but also various other bacterial and non bacterial systems. In some preferred embodiments, the host cell is a procaryotic cell (e.g. an E. coli cell) or a eukaryotic cell (e.g. a CHO cell). In a preferred embodiment, the host cell is an E. coli cell, e.g. DH5alpha, BL21 (DE3), or Rosetta (DE3). In another preferred embodiment, the host cell is from E. coli strain W3110 (NC_007779.1), JM101 or MG1655 (NC_000913.3). Introduction of nucleic acids encoding for a BM3 variant, e.g. according to the second aspect, into the host cell may occur as known in the art, i.e. using suitable vectors or transfer systems. For example, where bacterial expression systems such as DH5alpha, BL21 (DE3), or Rosetta (DE3) are used, suitable vectors are known in the art, such as pSE420 (Invitrogen), pET21a (EMD Biosciences), pET22b (EMD Millipore), pLys (EMD Biosciences) or pETM11 (EMBL Vector Collection, Germany). For a detailed decription see e.g. section 4.2 “Cloning of cyp102A1 and pETM11-BM3 construction” of Supporting Information of (Acevedo-Rocha 2018) and the examples provided herein.
According to a fourth aspect of the current invention, there is provided the use of a Cytochrome P450 BM3 monooxygenase (BM3) variant for the production of a compound according to formula I, wherein R1 and R2 form a six-membered ring as part of a steroid.
In a preferred embodiment according to the fourth aspect, formula I is formula (II), wherein R3 is a beta-hydroxy group (-OH) or an oxo group (=O).
As described also elsewhere herein, it was surprisingly found, that BM3 variants can be used for the production of a compound according to formula I and II in a commercially relevant setup, i.e. for an industrial application. To this end, a BM3 variant suitable for catalyzing C19 hydroxylation is incubated with a suitable substrate, e.g. as described in examples 2, 8, 10, 13, 17, 20, 21, 24, 25.
In some highly preferred embodiments according to the fourth aspect, the substrate for the BM3 variant is a steroid or steroid derivative.
According to a fifth aspect of the current invention, there is provided the use of a BM3 variant for the C19-hydroxylation of a steroid or steroid derivative.
As described also elsewhere herein, it was surprisingly found, that BM3 variants can be used for the C19-hydroxylation of a steroid or steroid derivative in a commercially relevant setup, i.e. for an industrial application. To this end, a BM3 variant suitable for catalyzing C19 hydroxylation is incubated with a suitable substrate, e.g. as described in examples 2, 8, 10, 13, 17, 20, 21, 24, 25.
According to some highly preferred embodiments of the fourth or fifth aspect, the BM3 variant is a variant as described herein. Preferably, the BM3 variant can be a variant according to the first aspect. Even more preferably, the BM3 variant can be a variant according to the first aspect, first embodiment or the BM3 variant can be a variant according to the first aspect, second embodiment, e.g. a variant according to SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 117, 118 or 119, or a variant derived from any of these. Furthermore, the BM3 variant can be a variant according to the third, fourth, fifth, or sixth embodiment of the first aspect.
In some embodiments according to the fourth or fifth aspect, the BM3 variant is purified prior to incubation with the substrate using a purification technique known in the art, such as but not restricted to immobilized metal affinity chromatography, ion exchange chromatography or size exclusion chromatography. In some other embodiments according to the fourth aspect, the BM3 variant remains in a cell suspension when incubated with the substrate, e.g. no separation of BM3 variant and expression system occurs prior to the biotransformation, e.g. whole-cell biotransformation.
According to some preferred embodiments of the fourth or fifth aspect said steroid or steroid derivatives comprise a 1,4-dien-3-one-A-ring or a 4-en-3-one-A-ring. According to some even more preferred embodiments according to the fourth or fifth aspect said steroid or steroid derivative is androsta-1,4-dien-3,17-dione (ADD) or (17beta)-17-hydroxyandrosta-1,4-dien-3-one (delta1 testosterone).
In some embodiments of the fourth or fifth aspect the substrate steroid or steroid derivative is added to the biotransformation reaction as a purified compound. In some embodiments the substrate steroid or steroid derivative is dissolved in a suitable solvent and can be added in solution to the biotransformation mixture. In other embodiments the substrate steroid or steroid derivative is formed in the course of a reaction preceding the C19 hydroxylation. The reaction preceding the C19 hydroxylation reaction may be another biotransformation reaction or a chemical reaction, e.g. a reduction of delta1 testosterone to testosterone or an oxidation of delta1 testosterone to ADD.
In some embodiments of the fourth or fifth aspect the formed C19 hydroxylated steroid or steroid derivative is extracted from the biotransformation reaction mixture using a method known in the art, e.g. with 4-methyl-2-pentanone (see example 10). In some other embodiments according to the fourth or fifth aspect the formed C19 hydroxylated steroid or steroid derivative is converted into a secondary product. This conversion may occur e.g. by means of biotransformation or chemical reaction. A secondary product may be for example estrone or a derivative thereof.
Extraction of the C19 hydroxylated steroid or steroid derivative or a secondary product thereof may occur as known in the art, e.g. by stirring the reaction mixture with 4-methyl-2-pentanone, separation of the organic phase and concentration by evaporation of the organic solvents. Further purification of the C19 hydroxylated steroid or steroid derivative or a secondary product thereof may be performed using chromatographic techniques, e.g. flash chromatography using silica gel or preparative HPLC, or crystallization.
Analysis of the production of the C19 hydroxylated steroid, steroid derivative or secondary product can occur as known in the art, e.g. by using MS- or NMR-methods.
Suitable biotransformation conditions are described in the examples provided herein. However, it is routine for the skilled person to optimize the reaction conditions for a BM3 variant to obtain optimal yields for the reaction product. For example, incubation may occur in a KH2PO4/K2HPO4 buffer comprising an aqueous EDTA solution in a pH range of 6.6 to 7.4. Suitable incubation times may be for example between 12 and 48 hours.
According to a sixth aspect of the current invention, there is provided a process for C19-hydroxylation of a steroid or derivative thereof comprising
The recombinant BM3 variant producing microorganism is a microorganism comprising a nucleic acid encoding a BM3 variant. The recombinant BM3 variant producing microorganism can be derived from any suitable expression system. Preferably, the recombinant BM3 variant producing microorganism is a microorganism according to the third aspect. For example, the BM3 variant may be a variant according to the first aspect.
In some embodiments of the sixth aspect, the process comprises culturing the recombinant BM3 variant producing microorganism in a culture medium. This can occur as known in the art, e.g. as described in the examples. However, the skilled person is aware of a variety of setups for the cultivation of microorganisms. Suitable culture media are described in the art for the respective expression system and can be easily adapted.
In some embodiments of the sixth aspect, the BM3 variant is purified prior to incubation with the substrate using a purification technique known in the art, such as immobilized metal affinity chromatography, ion exchange chromatography or size exclusion chromatography. In some other embodiments of the sixth aspect, the BM3 variant remains in a cell suspension when incubated with the substrate, e.g. no separation of BM3 variant and expression system occurs prior to the biotransformation, e.g. whole-cell biotransformation.
In some embodiments, the process comprises incubating a substrate-containing reaction medium with a BM3 variant (e.g. purified or crude mixture with cells of the expression system). Suitable media and conditions are described in the examples provided herein. However, it is routine for the skilled person to optimize the reaction conditions for a BM3 variant to optimize yields for the reaction product.
For example, the substrate can be an exogenous substrate. Such an exogenous substrate is added to the biotransformation reaction, e.g. as a purified compound, solution or mixture. The substrate can also be an intermediately formed substrate, i.e. the substrate can be formed in the course of a reaction preceding the C19 hydroxylation. The reaction preceding the C19 hydroxylation reaction may be another biotransformation reaction or a chemical reaction.
Suitable substrates are steroids or steroid derivatives, in particular those comprising a 1,4-dien-3-one-A-ring or a 4-en-3-one-A-ring. For example ADD or d1-testosterone are suitable substrates for the described BM3 variants. In some preferred embodiments according to the sixth aspect, said steroid or derivative thereof comprises a 1,4-dien-3-one-A-ring and a 19-methyl group. In some preferred embodiments according to the sixth aspect, said steroid or derivative thereof comprises a 4-en-3-one-A-ring and a 19-methyl group.
Following the biotransformation, the C19 hydroxylation product can be isolated from the reaction medium as described in the art. In the alternative, a secondary product may be isolated from the reaction medium. The isolation may be performed for example by chromatographic techniques such as preparative or analytical HPLC, or by extraction. Extraction of the C19 hydroxylated steroid or steroid derivative or a secondary product thereof may occur as known in the art, e.g. with 4-methyl-2-pentanone (see example 10). Further purification of the C19 hydroxylated steroid or steroid derivative or a secondary product thereof may be performed using chromatographic techniques, e.g. flash chromatography using silica gel or preparative HPLC, or crystallization.
Analysis of the production of the C19 hydroxylated steroid, steroid derivative or secondary product can occur as known in the art, e.g. by MS- or NMR-methods.
According to a seventh aspect there is provided a method for obtaining optimized BM3 variants for the C19 hydroxylation of steroids, said method comprising
The description provided for the process according to the fifth and sixth aspect applies mutatis mutandis, in particular with regard to step (i) a and b.
In a preferred embodiment, the steroid or steroid derivative comprises an 1,4-dien-3-one-A-ring or a 4-en-3-one-A-ring. In a highly preferred embodiment the steroid or steroid derivative is ADD or d1-testosterone. In some preferred embodiments of the seventh aspect, the steroid or derivative thereof comprises a 1,4-dien-3-one-A-ring and a 19-methyl group. In some preferred embodiments of the seventh aspect, said steroid or derivative thereof comprises a 4-en-3-one-A-ring and a 19-methyl group.
Product yield and selectivity (factor) are calculated as disclosed elsewhere herein. The product yield can be the product yield for the C19 hydroxylated steroid or steroid derivative, or a product derived thereof. Preferably the product yield is the product yield for estradiol or estrone. The selectivity factor can be the selectivity factor for the C19 hydroxylated steroid or steroid derivative.
Preferably, the daughter variant has the sequence of its parent variant and at least one further mutation. Preferably, the parent variant is a variant according to the first aspect.
If not stated otherwise, culture media were prepared in demineralized water and sterilized at 121° C. for 20 minutes or sterile filtered. All solutions were prepared with MilliQ water and sterile filtered, unless otherwise described. All preparations were performed under sterile conditions.
Oxford trace element solution contained FeCl3 × 6 H2O (27 g/L), ZnCl2 (1.31 g/L), CoCl2 × 6 H2O (2.87 g/L), CuCl2 × 2 H2O (1.27 g/L), Boric acid (0.5 g/L), CaCl2 × 2 H2O (1.32 g/L), Na2MoO4 × 2 H2O (2.35 g/L), Hydrochloric acid (37%) (100 mL/L).
List of abbreviations: Δ...deletion from amino acid; DMSO...Dimethyl sulfoxide; DMF...Dimethylformamide; EDTA...Ethylendiamintetraacetate; × g...times gravity; IPTG... isopropyl β-D-thiogalactopyranoside; LB... Luria-Bertani broth; OD550... optical density at 550 nm; rpm... revolutions per minute; SOC Outgrowth Medium...#B9020S-from BioLabs: 2 % Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCI, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose. Pluronic® PE 8100... Pluronic PE types are low-foaming, nonionic surfactants. They are block copolymers in which the central polypropylene glycol group is flanked by two polyethylene glycol groups. PE 8100 conforms to the following structural formula: HO(CH2CH2O)x(CH2C(CH3)HO)y(CH2CH2O)zH.
PE 8100 is a polypropylene glycol block copolymer with a molar mass of 2300 g/mol and 10 % polypropylene glycol in the molecule.
Nucleotide sequences encoding P450-BM3 variants as described herein can be synthesized as known in the art, e.g. as offered by respective service providers such as Eurofins Genomics GmbH (Eurofins Genomics GmbH, Anzinger Str. 7a, 85560 Ebersberg, Germany). In brief, nucleic acid sequences encoding P450-BM3 WT (e.g. SEQ ID No. 59) or other P450-BM3 variants as described herein were cloned into an expression vector based on the vector pETM-11 (EMBL Vector Collection, Germany) or another suitable vector. For a detailed description see section 4.2 “Cloning of cyp102A1 and pETM11-BM3 construction” of Supporting Information of (Acevedo-Rocha 2018). Genetic elements were introduced into the modified pETM-11 vector by means of commonly known methods. For expression of BM3 variants, the expression vector was introduced into Escherichia coli strain BL21 Star™ (DE3) (Invitrogen™, Lifetechnologies) cells, E. coli BL 21 (DE3) cells or E. coli BL 21 Gold (DE3) ΔdkgA :: FRT T7 gdh (the construction is described in (Agudo R 2012)) cells.
Nucleotide substitutions (replacements) were introduced into the nucleic acid parent sequences, e.g. to obtain a T88 exchange into other amino acids on SEQ ID No. 5 as parent. Several Molecular Biological methods can be used to achieve these replacements. A useful method for preparing a mutated nucleic acid according to the invention and the corresponding protein comprises carrying out site-directed mutagenesis on codons encoding one or more amino acids, which are selected in advance. The methods for obtaining these site-directed mutations are well known to the skilled person and widely described in the literature (in particular: Directed Mutagenesis: A Practical Approach, 1991, Edited by M.J. McPHERSON, IRL PRESS), or are methods for which it is possible to employ commercial kits (for example the QUIKCHANGE™ lightening mutagenesis kit from Qiagen or Stratagene). After site-directed mutagenesis, nucleic acids were transformed into the Escherichia coli strain BL21 Star™ (DE3) (Invitrogen™, Lifetechnologies). Replacements within the BM3 gene were generated by first introducing Bsal-sites into a vector backbone with harboring SEQ ID No. 122, yielding an entry vector, and subsequent ligation of insert sequences from a library with different length and properties inframe into the BM3 gene.
Transformed cells were tested in appropriate biotransformation reactions, in order to determine product yield and selectivity. Appropriate biotransformation reactions are described below, see e.g. Example 2. Sequence verification was performed as known in the art.
Glycerol stocks of the E. coli cultures transformed with the respective expression plasmids were prepared by adding one volume of 40% glycerol solution to one volume of E. coli culture.
For isolation of single bacterial colonies, appropriate dilutions of E.coli cultures were plated onto LB-Agar plates containing suitable concentrations of kanamycin and incubated at 37° C. until single colonied were obtained.
For screening purposes E. coli strain BL21 Star™ (DE3) (Invitrogen™, Lifetechnologies) was used as host for expression plasmids (Example 1). For pre-cultures sterilized 2.5-mL 96-well deep-well plates (850301, HJ-Bioanalytik, Erkelenz, Germany) were filled with 490 µL LB-medium (LB Broth Miller, Fisher Bioreagents, BP1426-500) supplemented with kanamycin (Sigma-Aldrich, 50 µg/mL), inoculated with 10 µL of glycerol stocks of the respective variants (that were prepared in 96-well microtiter plates) or alternatively 500 µL LB-medium were inoculated with cell material from Agar plate colonies. Pre-cultures were incubated for 17 hours at 37° C. and 250 rpm in a climo shaker ISF1-X (Kühner AG, Birsfelden, Switzerland).
Medium for expression cultures contained Tryptone (12 g/L), Yeast extract (24 g/L), Meat Peptone, tryptically digested (2 g/L), KH2PO4 (2.2 g/L), K2HPO4 (9.4 g/L), glycerol (0.4% [v/v]), Riboflavin (0.001 g/L), Thiamine hydrochloride (0.337 g/L) and Oxford trace element solution (250 µL/L). Tryptone, Yeast extract and Meat peptone were prepared as 50 g/L stock solutions, and K-PO4 was prepared as a 50x stock solution. These stock solutions were sterilized at 121° C. for 20 minutes. Riboflavin and Thiamine hydrochloride were freshly prepared as 0.06 g/L and 10 g/L stock solutions and sterile-filtered, respectively. The medium was prepared from the stock solutions and Kanamycin (Kanamycin solution, K0254, Sigma-Aldrich, St. Louis, MO, USA) was added for a final concentration of 50 mg/L.
For expression cultures sterilized 2.5-mL 96-well deep-well plates (850301, HJ-Bioanalytik, Erkelenz, Germany) were filled with 440 µL expression medium supplemented with kanamycin (50 mg/L); expression cultures were inoculated with 50 µL of pre-cultures. The expression culture plates were incubated at 37° C. and 250 rpm in a climo shaker. After 4 h incubation 10 µL of 30 mM IPTG diluted in expression medium (supplemented with 50 mg/L kanamycin) was added per well to induce enzyme expression (resulting in a final concentration of 0.6 mM IPTG). The expression culture plates were subsequently incubated for 20 hours at 27° C. in a shaking incubator.
Cells were harvested by centrifugation of the expression cultures for 15 minutes at 4° C. and 2500 × g. The culture supernatant was discarded, and the remaining cell pellets were resuspended in 495 µL of P450 assay buffer (100 mM KPO4-buffer at pH 7.4, 50 g/L glucose, 35 g/L glycerol, 1 mM EDTA) or 250 µL of 2x P450 assay buffer (200 mM KPO4-buffer at pH 7.0, 100 g/L glucose, 70 g/L glycerol, 2 mM EDTA). The deep-well plates were stored at -80° C. until use.
The deep-well plates containing the cell suspensions were thawed at room temperature. The biotransformation reaction was started (i) by addition of 5 µL of Δ1-testosterone or androsta-1,4-diene-3,17-dione (ADD) stock solution (at 5 mg/mL or 10 mg/mL in N,N-Dimethylformamide) to cells resuspended in 495 µL of P450 assay buffer, or (ii) by addition of 250 µL of androsta-1,4-diene-3,17-dione (ADD) stock (at 4000 mg/mL) to 250 µL cell suspension in 2x P450 assay buffer, and incubation occured for 22 h at 27° C. in a shaking incubator. Reactions were stopped by adding 500 µL of Stop solution (containing Acetonitrile, Methanol and Dimethylsulfoxide in a 1:1:1 ratio), and for extraction of steroides incubated on a shaker for 30 min at 20° C. Afterwards the plates were centrifuged for 15 min at 4° C. and 2500× g. From each well 120 µL of the supernatant was transferred to a 96-well filter plate (Corning, 3504) that was subsequently centrifuged for 5 min at 4° C. and 650× g. The filtered samples were stored at -20° C. or directly submitted to HPLC analysis.
Analytical HPLC was performed with the following setup: Instrument: Agilent Technologies 1290 Infinity; Column: Ascentis Express C18, 50 x 2.1 mm, 2.7 µm; Eluent A: Water (+0.025% or 0.005% Trifluoroacetic acid); Eluent B: Acetonitrile; Flow 1.5 mL/min; Temperature: 45° C.; injection volume: 4 µL, detection at 280 nm. Gradient: 0 - 0.18 min 5% solvent B, 0.18 - 0.4 min 5 - 10% solvent B, 0.4 - 4.6 min 10 - 27% solvent B, 4.6 - 7.0 min 27 - 95% solvent B, 7.01 - 7.5 min 5% solvent B.
Androsta-1,4-diene-3,17-dione (ADD), Δ1-testosterone, estradiol and estrone (dissolved in N,N-Dimethylformamide) were used as reference substances and as a standard for quantification. Appropriate dilutions were prepared covering the range up to the maximum concentration used in the biotransformation.
Observed retention times were 4.01 min for Δ1-testosterone, 4.22 min for ADD, 4.37 min for estradiol and 4.86 min for estrone. The concentration of the respective compounds in samples was calculated by comparing the obtained peak areas with those of the standards.
Variants of BM3-268 (Seq ID No. 5) with mutations in one or two positions were prepared as described in Example 1 and screened for Δ1-testosterone to estradiol conversion. Cultivation, biotransformation and HPLC analysis was performed as described in Example 2. Glycerol stocks were used as inoculum for cultivation, and the initial substrate (Δ1-testosterone) concentration was 50 mg/L.
Results for estradiol product yield and the selectivity factor for estradiol are shown in
Variants of the BM3 derivative BM3-268; M177Y, A184Y (Seq ID No. 15) with further mutations in up to six positions (Example 1) were screened for Δ1-testosterone to estradiol conversion. Cultivation, biotransformation and HPLC analysis was performed as described in Example 2. Glycerol stocks were used as inoculum for cultivation, and the initial Δ1-testosterone concentration used in the experiment was 100 mg/L.
Resulting estradiol product yields and estradiol selectivity factors are shown in
Variants of BM3-268 (Seq ID No. 5) with up to seven mutations in different positions (Example 1) were screened for Δ1-testosterone to estradiol conversion. Cultivation, biotransformation and HPLC analysis was performed as described in Example 2. Agar plate colonies were used as inoculum for cultivation, and the initial Δ1-testosterone concentration used in the experiment was 50 mg/L. Results for estradiol yield and the selectivity factor for estradiol are shown in
Variants of the BM3-268; M177Y, A184Y (Seq ID No. 15) with further mutations in up to six positions (Example 1) were screened for androsta-1,4-diene-3,17-dione (ADD) to estrone conversion. Cultivation, biotransformation and HPLC analysis was performed as described in Example 2. Glycerol stocks were used as inoculum for cultivation, and the initial ADD concentration used in the experiment was 100 mg/L.
Results for estrone yield and selectivity factor for estrone are shown in
Variants of BM3-268 (Seq ID No. 5) with up to six mutations in different positions (Example 1) were screened for androsta-1,4-diene-3,17-dione (ADD) to estrone conversion. Cultivation, biotransformation and HPLC analysis was performed as described in Example 2. Agar plate colonies were used as inoculum for cultivation, and the initial ADD concentration used in the experiment was 100 mg/L. Results for estrone yield and the selectivity factor for estrone are shown in
Variants of the BM3-268; S72G, V78L, A82E, F87A, M177Y, V178P, L181Y, A184Y, L188F (SEQ ID No. 117) with further mutations in one or two positions (Example 1) were screened for androsta-1,4-diene-3,17-dione (ADD) to estrone conversion. Cultivation, biotransformation and HPLC analysis was performed as described in Example 2. Glycerol stocks were used as inoculum for cultivation, and the initial ADD concentration used in the experiment was 2000 mg/L.
Results for obtained estrone titers and target product ratio for estrone are shown in
Variants of BM3-268; S72G, V78L, A82E, F87A, M177Y, V178P, L181Y, A184Y, L188F (SEQ ID No. 117) with up to five mutations in different positions (Example 1) were screened for androsta-1,4-diene-3,17-dione (ADD) to estrone conversion. Cultivation, biotransformation and HPLC analysis was performed as described in Example 2. Agar plate colonies were used as inoculum for cultivation, and the initial ADD concentration used in the experiment was 2000 mg/L. Results for estrone titer and the target product ratio for estrone are shown in
Variant G457S, I458G, A477N of BM3-268; S72G, V78L, A82E, F87A, T146F, M177Y, V178W, L181Y, A184Y (SEQ ID No. 118) (see Example 1) was screened for androsta-1,4-diene-3,17-dione (ADD) to estrone conversion. Cultivation, biotransformation and HPLC analysis was performed as described in Example 2. Glycerol stocks were used as inoculum for cultivation, and the initial ADD concentration used in the experiment was 2000 mg/L. Results for the obtained estrone titer and target product ratio (TPR) are listed in Table E7C.
Table E7C: Obtained estrone titers and target product ratio (TPR) with BM3 variants Seq ID No. 118 and 119.
Variants of BM3-268; S72G, T146F, M177Y, V178W, L181Y, A184Y, G457S, I458G, A477N (SEQ ID No. 119) with amino acid sequence exchanges between positions 458 and 477 (Example 1) were screened for androsta-1,4-diene-3,17-dione (ADD) to estrone conversion. Cultivation, biotransformation and HPLC analysis was performed as described in Example 2. Glycerol stocks were used as inoculum for cultivation, and the initial ADD concentration used in the experiment was 2000 mg/L.
Results for the obtained estrone titers and target product ratios (TPR) are listed in Table E7D. Only those variants are listed where - relative to the parent type - the insert (“linker”) improved (i) product yield as determined by titer and/or (ii) selectivity as determined by TPR.
Table E7D: Obtained estrone titer and target product ratio (TPR) with BM3 variants with amino acid sequence exchanges between positions 458 and 477 in BM3-268; S72G, T146F, M177Y, V178W, L181Y, A184Y, G457S, I458G, A477N (SEQ ID No. 119)
Variants of wild type BM3 (SEQ ID No. 1) with up to eight mutations (Example 1) in different positions were screened for BM3 protein level. Cultivation of cells was carried out as described in Example 2. Cellular protein levels were analyzed on PAGE gel (NuPAGE™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 15-well; Thermo Fisher Scientific). Expression cultures were adjusted to an OD600nm of 0.5, and one volume of a 2-fold blend of NuPAGE® LDS Sample Buffer (4X) and NuPAGE® Sample Reducing Agent (10X) (both Thermo Fisher Scientific) were added. Samples were denatured for 5 minutes at 99° C. in a Thermocycler. Subsequently, 10 µL of denatured samples were loaded on the gel, and electrophoresis was carried out at for 60 minutes at 180 Volts in NuPAGE® MOPS SDS Running Buffer (Thermo Fisher Scientific). After electrophoresis the gels were stained using SimplyBlue™ SafeStain (Thermo Fisher Scientific), and a digital image was taken. Quantification of protein bands was carried out using the software package ImageJ (ImageJ version 1.53e, National Institutes of Health, Bethesda, MD, USA). The gel image was converted to 8-bit grey scale and a background subtraction was performed using the setting ‘light background’ at a rolling ball radius of 50. Areas in the gel profile were calculated for each lane for the BM3 protein peak and for the sum of all peaks (total protein). Based on the area values of BM3 protein and total protein the ratio of BM3 to total protein was calculated for each sample, and those values were compared to the wild type control samples ((area BM3/ area total protein for variant)/ (area BM3/ area total protein for wild type)). The results for the BM3 protein level relative to the BM3 wild type are listed in Table E7E.
Table E7E: BM3 protein levels of BM3 variants with up to eight mutations relative to the wild type (SEQ ID No. 1)
Tryptone (10 g/L), sodium chloride (10 g/L) and yeast extract (5 g/L) in demineralized water. The medium was sterilized at 121° C. for 20 minutes. Afterwards kanamycin (50 mg/L) was added.
Each well of a 96 deep well plate contained 0.25 ml of the medium which was inoculated with the E. coli strains (2 µL) containing the P450-BM3 variants from glycerol stocks. The plate was shaken at 37° C. and 800 rpm for 17 hours.
Each well of a 96 deep well plate contained 1.5 ml of the medium which was inoculated with 50 µL from the preculture. The cultures were shaken at 37° C. and 800 rpm for 3 hours. Then the temperature was decreased to 27° C. within 1 hour and IPTG (1 mM) in demineralized water was added to start the protein expression. After 4 hours of expression, the plates were harvested by centrifugation, the supernatant was discarded, the pellets were resuspended in 500 µL reaction buffer (KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at-80° C.
The plates were thawed and the reaction was started by addition of 5 µL of steroid stock solution of (17beta)-17-hydroxyandrosta-1,4-dien-3-one (12.5 µg dissolved in 5 µL of DMF) and plates were shaken for 22 h at 700 rpm and 27° C. while covered with lids. The plates were frozen in liquid nitrogen and stored at -80° C. The thawed samples were prepared for HPLC screening by adding 500 µL mixture of acetonitrile / methanol / DMSO 1:1:1 (HPLC grade). The plates were shaken and centrifuged and 200 µl of the supernatant was transferred to an analytics plate. The plate was covered with a silicone sealing mat and submitted to HPLC analysis.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Thermo Accucore C30 2.6 µm, 50x2.1 mm; Eluent A: water + 0.05 vol % formic acid, eluent B: acetonitrile + 0.05 vol % formic acid; Gradient: 0.0-2.0 min 5-30% B, 2.0-5.0 min 85% B; injection volume 4 µL; flow 0.9 mL/min; temperature: 45° C.; DAD scan: 248-280 nm.
After testing hundreds of P450-BM3 variants, only few mutants allowed for the C19-hydroxylation of delta-1-testosterone leading to estradiol as final product:
In a subsequent screening experiment of four BM3 variants the following screening yields were found for the following (side) products:
Media, preculture and main culture were prepared as decribed for delta-1-testosterone (Example 8).
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Ascentis Express C18 2.7 µm, 50×2.1 mm; Eluent A: water + 0.05 vol % formic acid, eluent B: acetonitrile + 0.05 vol % formic acid; Gradient: 0.0-3.0 min 5-30% B, 3.0-5.0 95% B, 5.0-5.5 5% B; injection volume 4 µL; flow 0.9 mL/min; temperature: 45° C.; DAD scan: 244 nm.
After testing hundreds of P450-BM3 variants, only few variants allowed for the C19-hydroxylation of testosterone:
(17beta)-estra-1 (10),2,4-triene-3,17-diol
Expression system E. coli BL 21(DE3) pETM11 with BM3-268 was provided by M. T. Reetz and is described in (Kille Sabrina 2011) (DOI:10.1038/NCHEM.1113). The respective protein and gene sequence such as pETM11-BM3-268 is given in SEQ ID No. 5 and 63, respectively.
Preculture medium comprised tryptone (10 g/L), sodium chloride (10 g/L) and yeast extract (5 g/L) in demineralized water. The medium was sterilized before kanamycin (50 mg/L) was added. One preculture (100 mL) was inoculated with the strain E. coli BL 21 (DE3) pETM11-BM3-268 (SEQ ID No.5) (50 µL) containing the desired plasmids and was shaken at 37° C. and 165 rpm for 16 hours. This preculture (100 mL) was used to inoculate one 5 L steel fermenter. The cultivation medium was prepared in the fermenter. Tryptone (20 g/L), sodium chloride (20 g/L), yeast extract (10 g/L) and Pluronic® PE 8100 (0.5 mL) were dissolved in demineralized water (4.0 L) and sterilized for 30 minutes at 121° C. in the fermenter. Afterwards kanamycin (0.25 g) in demineralized water (10 mL) was added. After the inoculation of the fermenter (16 hours old preculture), the inoculated culture was stirred at 500 rpm at 37° C. with an aeration rate of 2.5 L/min and an oxygen partial pressure of 30% regulated by the stirring rate of up to 1500 rpm. After 3 hours an aqueous glucose solution (25 %, 40 g/h) was added. After 4 hours and 25 minutes an OD550 of 16.16 was reached, the temperature was decreased to 28° C. within 10 minutes and IPTG (1.19 g) in demineralized water (50 mL) was added to start the protein expression. After additional 8 hours and 35 minutes the cells (124.6 g) were harvested by centrifugation, suspended in buffer (125 mL; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 4 × 45 mL, 1 × 35 mL und 10 × 1 mL.
To a 5 L steel fermenter which contained KH2PO4 (15 g), K2HPO4 (46.15 g), Pluronic® PE 8100 (0.5 mL), demineralized water (3.8 L), aqueous glucose solution (50%, 375 mL) and aqueous EDTA solution (0.5 M, 3.75 mL) (17beta)-17-hydroxyandrosta-1,4-dien-3-one (500 mg, 1.75 mmol) dissolved in DMF (10 mL) was added and the mixture was stirred at 140 rpm at 27° C. with an aeration rate of 2.0 L/min. Cells in buffer (215 mL) were added and the biotransformation was stirred at an oxygen partial pressure of 50 % regulated by the stirring rate of up to 800 rpm. The pH value was maintained at pH 7.4 by addition of an aqueous solution of sodium hydroxide (16%). After 26 hours and 5 minutes the biotransformation was extracted with 4-methyl-2-pentanone. The organic layer was concentrated to give a crude oil (4.98 g) which was slurried with methanol, filtered and concentrated to give an oil (4 g). The crude product was further purified by flash chromatography using silica gel (dichloromethane/acetone gradient) and by preparative HPLC to give the title compound (4.3 mg, 0.8% yield).
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 100×30 mm; Eluent A: water + 0.2 vol-% aqueous ammonia (32%), Eluent B: acetonitrile; gradient: 0.00-0.50 min 17% B (40-70 mL/min), 0.51-5.50 min 34-44 % B (70 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; eluent A: water + 0.2 vol % aqueous ammonia (32%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 mL/min; temperature: 60° C.; DAD scan: 210-400 nm.
LC-MS: Rt = 1.01 min; MS (ESIneg): m/z = 271 [M-H]-1H-NMR (400 MHz, CHLOROFORM-d) δ [ppm]: 0.000 (1.83), 0.710 (4.29), 1.102 (0.18), 1.113 (0.30), 1.132 (0.31), 1.141 (0.29), 1.160 (0.32), 1.185 (0.85), 1.213 (0.54), 1.224 (0.54), 1.243 (0.60), 1.255 (0.59), 1.273 (0.57), 1.286 (0.46), 1.303 (0.52), 1.317 (0.44), 1.329 (0.32), 1.348 (0.47), 1.355 (0.45), 1.375 (0.54), 1.398 (0.55), 1.407 (0.56), 1.429 (0.57), 1.438 (0.51), 1.460 (0.41), 1.471 (0.41), 1.509 (0.63), 1.590 (0.24), 1.599 (0.21), 1.616 (0.31), 1.621 (0.29), 1.633 (0.23), 1.640 (0.26), 1.775 (0.23), 1.783 (0.23), 1.790 (0.24), 1.807 (0.22), 1.814 (0.24), 1.821 (0.20), 1.851 (0.23), 1.859 (0.34), 1.867 (0.24), 1.882 (0.22), 1.890 (0.31), 1.898 (0.20), 2.030 (0.22), 2.043 (0.24), 2.053 (0.23), 2.063 (0.26), 2.077 (0.30), 2.100 (0.32), 2.211 (0.25), 2.218 (0.25), 2.244 (0.24), 2.252 (0.22), 2.554 (16.00), 2.736 (0.53), 2.749 (0.54), 2.776 (0.22), 3.642 (0.29), 3.664 (0.50), 3.685 (0.27), 6.498 (0.70), 6.546 (0.36), 6.552 (0.31), 6.567 (0.39), 6.574 (0.34), 7.076 (0.58), 7.097 (0.53).
(1alpha,2alpha,17beta)-17-hydroxy-1,2-epoxyandrost-4-en-3-one
For the preparation of the title compound see preparation of Example 10. The crude product was further purified by flash chromatography using silica gel (dichloromethane/acetone gradient) and by preparative HPLC to give the title compound (13 mg).
Instrument: Waters Autopurification system; Column: Waters XBrigde C18 5 µ 100×30 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99 %), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 16% B (25-70 mL/min), 0.51-5.50 min 33-48% B (70 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99 %), Eluent B: Acetonitrile; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; Flow: 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.
Analytical Chiral HPLC: Rt = 2.04 min.
1H-NMR (600 MHz, Pyridine-d5): δ [ppm]: 0.77 - 0.91 (m, 2H), 0.98 - 1.05 (m, 1H), 0.99 (s, 3H), 1.11 (td, 1H), 1.23 - 1.32 (m, 1H), 1.34 (s, 3H), 1.43 - 1.53 (m, 2H), 1.55 - 1.67 (m, 2H), 1.73 -1.81 (m, 2H), 2.01 - 2.14 (m, 3H), 2.20 (td, 1H), 3.63 (dd, 1H), 3.74 (d, 1H), 3.89 (td, 1H), 5.91 -5.93 (m, 1H), 6.18 (d, 1H).
(15beta,17beta)-15,17-dihydroxyandrosta-1,4-dien-3-one
For the preparation of the title compound see preparation of Example 10. The crude product was further purified by flash chromatography using silica gel (dichloromethane/acetone gradient) and by preparative HPLC to give the title compound (78 mg).
Analytical HPLC: Rt = 0.74 min; MS (ESIpos): m/z = 303 [M+H]+
1H-NMR (600 MHz, CDCl3) δ [ppm]: 0.82 (dd, 1H), 1.05 - 1.16 (m, 3H), 1.11 (s, 3H), 1.28 (s, 3H), 1.58 - 1.65 (m, 1H), 1.71 (qd, 1H), 1.75 - 1.80 (m, 1H), 1.86 (dt, 1H), 2.09 (qd, 1H), 2.20 -2.29 (m, 1H), 2.37 - 2.42 (m, 1H), 2.55 (tdd, 1H), 2.63 (ddd, 1H), 3.58 (t, 1H), 4.21 (ddd, 1H), 6.09 (s, 1H), 6.24 (dd, 1H), 7.08 (d, 1H).
(15beta,17beta)-estra-1(10),2,4-triene-3,15,17-triol
Medium of the preculture with tryptone (16 g/L), sodium chloride (10 g/L) and yeast extract (10 g/L) in demineralized water was adjusted to pH 7.3 with sodium hydroxide solution (16% in water), and sterilized at 121° C. for 20 minutes. Afterwards kanamycin (50 mg/L) was added.
One preculture (100 mL each) was inoculated with the Escherichia coli strain BL 21 Gold (DE3) ΔdkgA :: FRT T7 gdh (50 µL) expressing BM3-268; F77L and was shaken at 37° C. and 165 rpm for 16 hours. These precultures (100 mL) were used to inoculate one 10 L steel fermenter. The cultivation media was prepared in the fermenters. Tryptone (12 g/L), yeast extract (24 g/L), predigested beef extract (2 g/L), KH2PO4 (2.2 g/L), K2HPO4 (9.4 g/L) and glycerol (87%, 4.6 g/L) were dissolved in demineralized water (9.2 L) and sterilized for 20 minutes at 121° C. in the fermenter. Afterwards kanamycin (0.5 g) in water (20 mL), riboflavin (10 mg) in water (20 mL), thiamine hydrochloride (3.37 g) in water (10 mL) and Oxford trace metal solution (2.5 mL) were added. After the inoculation with the 17 hours old preculture, the inoculated culture was stirred at 315 rpm at 37° C. with an aeration rate of 3.3 L/min partial pressure at pH 6.6 which was regulated by addition of aqueous sodium hydroxide solution (16%) or aqueous phosphoric acid solution (16%). After 2 hours an OD550 of 0.92 was reached, the temperature was decreased to 27° C. within 15 minutes and IPTG (2.38 g) in demineralized water (40 mL) and aminolevulinic acid (838 mg) in demineralized water (40 mL) were added to start the protein expression. After 12 hours the aqueous phosphoric acid solution (16%) was substituted for an aqueous glucose solution (50%) for pH regulation. After additional 24 hours the cells were harvested by centrifugation (96.86 g), suspended in buffer (97 mL; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 2 ×40 mL, 1 ×80 mL und 5 × 1 mL.
To one 1 L Biostat Q fermenter which contained KH2PO4 (4 g), K2HPO4 (12.3 g), Pluronic® PE 8100 (0.1 mL), demineralized water (930 mL), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 M, 1 mL) (17beta)-17-hydroxyandrosta-1,4-dien-3-one (50 mg, 174.5 µmol) dissolved in DMF (4 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. The pH value was maintained at pH 7.4. Cells in buffer (20 mL) were added and the biotransformation was maintained at an oxygen partial pressure of 50 % by varying the stirring rate to up to 1200 rpm. After 24 hours and 40 minutes the biotransformation was frozen and stored at -20° C.
To one 1 L Biostat Q fermenter which contained KH2PO4 (4 g), K2HPO4 (12.3 g), Pluronic® PE 8100 (0.1 mL), demineralized water (930 mL), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 M, 1 mL) (17beta)-17-hydroxyandrosta-1,4-dien-3-one (50 mg, 174,5 µmol) dissolved in DMF (4 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. The pH value was maintained at pH 7.4. Cells in buffer (40 mL) were added and the biotransformation was maintained at an oxygen partial pressure of 50 % by varying the stirring rate to up to 1200 rpm. After 24 hours and 40 minutes the biotransformation was harvested, frozen and stored at -20° C.
To two 1 L Biostat Q fermenter which contained KH2PO4 (4 g), K2HPO4 (12.3 g), Pluronic® PE 8100 (0.1 mL), demineralized water (930 mL), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 M, 1 mL) (17beta)-17-hydroxyandrosta-1,4-dien-3-one (50 mg, 174,5 µmol) dissolved in DMF (4 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. The pH value was maintained at pH 7.4. Cells in buffer (40 mL) were added and the biotransformation was maintained at an oxygen partial pressure of 50% by varying the stirring rate to up to 1200 rpm. After 5 hours and 20 minutes the culture was harvested, frozen and stored at -20° C.
The harvested four biotransformations were thawed, combined and extracted with 4-methyl-2-pentanone.
The organic phases were concentrated to give a crude oil (1.69 g) which was slurried with methanol and was filtered and concentrated to give an oil (1.04 g). The crude product was further purified by flash chromatography using silica gel (dichloromethane/ethanol gradient) and by preparative HPLC to give the title compound (13.0 mg).
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 50×50 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 5% B (50-100 mL/min), 0.51-8.00 min 5 - 35% B (100 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; Flow 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.
Analytical Chiral HPLC: Rt = 0.78 min.
1H-NMR (600 MHz, Pyridine-d5): δ [ppm]: 1.08 (dd, 1H), 1.37 (td, 1H), 1.49 (qd, 1H), 1.61 (s, 3H), 1.65 - 1.74 (m, 1H), 2.17 - 2.24 (m, 2H), 2.28 (ddd, 1H), 2.32 - 2.39 (m, 2H), 2.49 - 2.56 (m, 1H), 2.81 - 2.89 (m, 2H), 2.94 - 3.01 (m, 1H), 3.96 (td, 1H), 4.55 (s, 1H), 5.85 (d, 1H), 6.25 (d, 1H), 7.02 (d, 1H), 7.12 (dd, 1H), 7.38 (d, 1H), 11.16 (s, 1H).
(1alpha,2alpha,17beta)-17,19-dihydroxy-1,2-epoxyandrost-4-en-3-one
For the preparation of the title compound see Example 13. The crude product (1.04 g) was further purified by flash chromatography using silica gel (dichloromethane/ethanol gradient) and by preparative HPLC to give the title compound (7.4 mg).
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 50×50 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 5% B (50-100 mL/min), 0.51-8.00 min 5 - 35% B (100 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; Flow 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.
Analytical Chiral HPLC: Rt = 0.80 min.
1H-NMR (600 MHz, CDCl3) δ [ppm]: -0.006 (0.17), 0.000 (4.06), 0.005 (0.19), 0.772 (0.44), 0.816 (16.00), 0.880 (0.19), 0.969 (0.54), 0.981 (0.62), 0.987 (0.73), 0.989 (0.80), 0.999 (0.79), 1.001 (0.80), 1.007 (0.70), 1.015 (0.41), 1.020 (0.77), 1.036 (0.77), 1.044 (0.88), 1.057 (0.93), 1.064 (0.94), 1.069 (1.08), 1.076 (1.00), 1.086 (0.47), 1.090 (0.59), 1.096 (0.51), 1.253 (0.81), 1.260 (0.82), 1.266 (0.73), 1.278 (0.76), 1.282 (1.02), 1.287 (0.83), 1.299 (0.81), 1.305 (0.86), 1.313 (0.39), 1.324 (0.90), 1.334 (0.94), 1.344 (1.03), 1.354 (1.03), 1.365 (0.45), 1.374 (0.43), 1.432 (0.59), 1.437 (0.64), 1.445 (0.72), 1.451 (0.96), 1.454 (0.88), 1.457 (0.79), 1.460 (0.87), 1.465 (0.68), 1.468 (0.78), 1.471 (0.71), 1.474 (0.96), 1.480 (0.52), 1.488 (0.46), 1.494 (0.47), 1.506 (0.43), 1.512 (0.49), 1.528 (0.85), 1.534 (1.04), 1.550 (0.85), 1.556 (0.84), 1.572 (0.55), 1.574 (0.61), 1.579 (0.75), 1.586 (0.60), 1.590 (0.81), 1.595 (0.92), 1.602 (0.83), 1.607 (1.01), 1.611 (0.91), 1.616 (0.83), 1.623 (1.01), 1.628 (1.22), 1.640 (1.51), 1.761 (0.34), 1.767 (0.37), 1.779 (0.68), 1.785 (0.77), 1.799 (0.70), 1.806 (0.68), 1.816 (0.29), 1.823 (0.30), 1.862 (0.36), 1.868 (0.80), 1.873 (1.01), 1.882 (1.30), 1.884 (1.47), 1.889 (1.08), 1.898 (1.30), 1.905 (1.12), 1.910 (0.58), 1.992 (0.36), 2.000 (0.71), 2.004 (0.72), 2.014 (0.49), 2.022 (0.68), 2.026 (0.67), 2.033 (0.35), 2.052 (0.38), 2.062 (0.43), 2.067 (0.72), 2.074 (0.56), 2.077 (0.75), 2.083 (0.62), 2.090 (0.70), 2.092 (0.54), 2.100 (0.62), 2.105 (0.40), 2.115 (0.33), 2.312 (0.50), 2.319 (0.68), 2.324 (0.56), 2.334 (0.85), 2.339 (1.05), 2.346 (0.78), 2.372 (0.43), 2.396 (0.84), 2.403 (0.73), 2.405 (0.74), 2.418 (0.98), 2.426 (0.94), 2.439 (0.42), 2.441 (0.42), 2.447 (0.39), 3.358 (2.18), 3.361 (2.22), 3.364 (2.35), 3.367 (2.14), 3.633 (0.88), 3.647 (1.53), 3.662 (0.85), 3.944 (2.87), 3.950 (2.82), 4.173 (0.59), 4.263 (2.71), 4.281 (1.88), 5.900 (2.52), 7.267 (12.12).
(1alpha,2alpha,15beta,17beta)-15,17-dihydroxy-1,2-epoxyandrost-4-en-3-one
For the preparation of the title compound see Example 13. The crude product (1.04 g) was further purified by flash chromatography using silica gel (dichloromethane/ethanol gradient) and by preparative HPLC to give the title compound (2.8 mg).
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 50x50 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 5% B (50-100 mL/min), 0.51-8.00 min 5 - 35% B (100 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99 %), Eluent B: Acetonitrile; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99 % B; Flow 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.
Analytical Chiral HPLC: Rt = 0.73 min.
1H-NMR (600 MHz, CHLOROFORM-d) δ [ppm]: -0.006 (0.48), 0.000 (11.79), 0.005 (0.44), 0.825 (1.12), 0.835 (1.15), 0.844 (1.18), 0.854 (1.23), 0.857 (1.22), 0.881 (0.35), 1.070 (0.62), 1.076 (0.66), 1.091 (1.49), 1.104 (13.48), 1.112 (1.31), 1.119 (1.11), 1.134 (0.39), 1.141 (0.37), 1.227 (0.58), 1.234 (0.66), 1.254 (2.09), 1.266 (0.93), 1.272 (0.78), 1.398 (0.70), 1.440 (15.22), 1.457 (1.29), 1.513 (0.45), 1.546 (0.44), 1.571 (2.00), 1.576 (2.63), 1.590 (16.00), 1.610 (1.91), 1.614 (2.15), 1.619 (1.27), 1.635 (0.91), 1.641 (0.91), 1.657 (0.40), 1.663 (0.37), 1.786 (0.38), 1.792 (0.80), 1.797 (0.85), 1.804 (0.43), 1.808 (0.34), 1.815 (0.61), 1.820 (0.62), 1.872 (0.61), 1.877 (0.96), 1.883 (0.58), 1.893 (0.64), 1.898 (0.85), 1.905 (0.51), 2.108 (0.34), 2.114 (0.32), 2.126 (0.65), 2.133 (0.70), 2.145 (0.62), 2.152 (0.65), 2.215 (0.47), 2.219 (0.62), 2.226 (0.54), 2.236 (0.60), 2.240 (0.78), 2.247 (0.68), 2.262 (0.38), 2.269 (0.60), 2.274 (0.52), 2.283 (0.45), 2.291 (0.57), 2.294 (0.51), 2.405 (0.51), 2.414 (0.47), 2.428 (0.86), 2.435 (0.79), 2.449 (0.41), 2.457 (0.36), 2.603 (0.67), 2.616 (0.88), 2.617 (0.84), 2.627 (0.79), 2.630 (0.83), 2.640 (0.83), 2.642 (0.82), 2.654 (0.65), 3.385 (1.56), 3.388 (1.69), 3.391 (1.66), 3.395 (1.55), 3.571 (0.58), 3.585 (1.12), 3.600 (0.56), 3.678 (2.43), 3.684 (2.34), 4.182 (0.51), 4.191 (0.87), 4.199 (0.51), 5.746 (2.23).
(6beta, 17beta)-6, 17-dihydroxyandrosta-1,4-dien-3-one
For the preparation of the title compound see Example 13. The crude product (1.04 g) was further purified by flash chromatography using silica gel (dichloromethane/ethanol gradient) and by preparative HPLC to give the title compound (2.4 mg).
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 50×50 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99 %), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 5% B (50-100 mL/min), 0.51-8.00 min 5 - 35% B (100 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; Flow 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.
Analytical Chiral HPLC: Rt = 0.75 min.
1H-NMR (600 MHz, CDCl3) δ [ppm]: 0.000 (7.05), 0.850 (0.34), 0.857 (10.48), 0.868 (0.34), 0.960 (0.40), 0.972 (0.39), 0.979 (0.55), 0.990 (0.55), 0.998 (0.48), 1.010 (0.46), 1.056 (0.37), 1.065 (0.43), 1.075 (0.61), 1.082 (0.65), 1.089 (0.52), 1.094 (0.57), 1.096 (0.56), 1.103 (1.02), 1.110 (0.72), 1.118 (0.75), 1.131 (0.41), 1.139 (0.42), 1.215 (0.47), 1.220 (0.44), 1.235 (0.62), 1.238 (0.70), 1.241 (0.73), 1.243 (0.71), 1.253 (1.01), 1.258 (1.09), 1.264 (0.79), 1.377 (0.64), 1.387 (0.69), 1.398 (0.76), 1.407 (0.80), 1.418 (0.40), 1.427 (0.45), 1.440 (1.03), 1.456 (12.77), 1.464 (0.72), 1.470 (0.66), 1.473 (0.63), 1.476 (0.51), 1.478 (0.58), 1.487 (0.52), 1.490 (0.47), 1.492 (0.64), 1.499 (0.39), 1.506 (0.37), 1.512 (0.39), 1.562 (0.37), 1.571 (0.42), 1.602 (1.00), 1.608 (1.08), 1.614 (1.25), 1.619 (1.41), 1.623 (1.48), 1.628 (1.35), 1.631 (1.37), 1.635 (1.48), 1.638 (1.35), 1.644 (1.17), 1.651 (1.05), 1.656 (0.96), 1.705 (0.40), 1.712 (0.39), 1.727 (0.56), 1.734 (0.56), 1.748 (0.66), 1.754 (0.70), 1.768 (0.66), 1.773 (0.86), 1.777 (0.87), 1.780 (0.57), 1.785 (0.56), 1.792 (0.36), 1.795 (0.39), 1.799 (0.37), 1.883 (0.57), 1.889 (0.73), 1.895 (0.53), 1.905 (0.56), 1.910 (0.66), 1.916 (0.46), 2.052 (0.49), 2.057 (0.81), 2.062 (0.63), 2.069 (0.34), 2.074 (0.85), 2.081 (1.05), 2.085 (1.41), 2.090 (0.63), 2.093 (0.33), 2.097 (0.54), 2.102 (0.65), 2.107 (0.74), 2.112 (0.39), 2.122 (0.72), 2.127 (0.45), 3.646 (0.92), 3.660 (1.30), 3.674 (0.85), 4.543 (0.81), 4.548 (1.50), 4.553 (0.85), 6.162 (2.12), 6.165 (2.42), 6.210 (1.34), 6.213 (1.21), 6.226 (1.36), 6.229 (1.27), 7.052 (1.95), 7.069 (1.96), 7.264 (16.00).
(17beta)-17,19-dihydroxyandrost-4-en-3-one
The strain E. coli BL 21 (DE3) pETM11-BM3-254 was provided by M. T. Reetz and is described in (Kille Sabrina 2011) (DOI:10.1038/NCHEM.1113). The protein and gene sequence are listed under SEQ ID No. 2 and 60, respectively.
Medium of the preculture: tryptone (10 g/L), sodium chloride (10 g/L) and yeast extract (5 g/L) in demineralized water. The media was sterilized at 121° C. for 20 minutes. Afterwards kanamycin (50 mg/L) was added.
One preculture (100 mL) was inoculated with the strain E. coli BL 21 (DE3) pETM11-BM3-254 (50 µL) containing the desired plasmids and was shaken at 37° C. and 165 rpm for 16 hours. This preculture (10 mL) was used to inoculate one 1 L Biostat Q fermenter. The cultivation medium was prepared in the fermenter. Tryptone (20 g/L), sodium chloride (20 g/L), yeast extract (10 g/L) and Pluronic® PE 8100 (0.1 mL) were dissolved in demineralized water (1.0 L) and sterilized for 30 minutes at 121° C. in the fermenter. Afterwards kanamycin (50 mg) in demineralized water (5 mL) was added. After the inoculation of the fermenter the inoculated culture was stirred at 500 rpm at 37° C. with an aeration rate of 0.4 L/min and an oxygen partial pressure of 30 % was maintained by the stirring at a rate of up to 1500 rpm. After 3 hours an aqueous glucose solution (50%, 4 g/h) was added. After 4 hours and 50 minutes an OD550 of 16.1 was reached, the temperature was decreased to 28° C. within 5 minutes and IPTG (238 mg) in demineralized water (10 mL) was added to start the protein expression. After additional 9 hours the cells (39.1 g) were harvested by centrifugation, suspended in buffer (39.1 mL; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 1 × 75 mL, 3 × 1 mL.
To two 1 L Biostat Q fermenter each containing KH2PO4 (3 g), K2HPO4 (9.23 g), Pluronic® PE 8100 (0.1 mL), demineralized water (0.75 L), aqueous glucose solution (50 %, 15 mL) and aqueous EDTA solution (0.5 M, 0.75 mL) (17beta)-17-hydroxyandrost-4-en-3-one (75.0 mg, 260 µmol) dissolved in DMF (5 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. Cells in buffer (each 37 mL) were added and the biotransformation was maintained at an oxygen partial pressure of 50% by varying the stirring rate to up to 800 rpm. The pH value was maintained at pH7.4. After 26 hours aqueous glucose solution was added (50%, 60 mL). After 102.5 hours the biotransformation was extracted with 4-methyl-2-pentanone. The organic layer was concentrated to give an oil (1.09 g) which was dissolved in methanol and was concentrated to give an oil (0.91 g). The crude product was further purified by flash chromatography using silica gel (dichloromethane/ methanol gradient) and by preparative HPLC to give the title compound (4.0 mg, 2,52 % yield).
Instrument: Labomatic HD5000, Labocord-5000; Gilson GX-241, Labcol Vario 4000, Column: Chiralpak IA 5 µ 250×30 mm; Eluent A: Acetonitrile + 0.1 Vol-% Diethylamine (99%); Eluent B: Ethanol; Isocratic: 90% A + 10% B; Flow 50.0 mL/min; UV 254 nm.
Instrument: Agilent HPLC 1260; Column: Chiralpak IA 3 µ 100×4.6 mm; Eluent A: Acetonitrile + 0.1 Vol-% Diethylamine (99%); Eluent B: Ethanol; Isocratic: 90% A + 10% B; Flow 1.4 mL/min; Temperature: 25° C.; DAD 254 nm.
1H-NMR (600 MHz, CHLOROFORM-d) δ [ppm]: 0.79 (s, 3H), 0.92 - 0.99 (m, 1H), 1.02 - 1.09 (m, 3H), 1.31 (qd, 2H), 2.05 - 2.13 (m, 1H), 2.32 - 2.44 (m, 4H), 2.75 (ddd, 1H), 3.65 (t, 1H), 3.92 (d, 1H), 4.08 (dd, 1H), 5.96 (s, 1H).
In a second preparation the title compound was synthesized with the strain E. coli BL 21 Gold (DE3) ΔdkgA :: FRT T7 gdh and plasmid pETM11-BM3-254. The construction of the E. coli BL 21 Gold (DE3) ΔdkgA :: FRT T7 gdh strain is described in (Agudo R 2012). The strain was rendered competent and transformed with pETM11-BM3-254.
Required solutions: TFB1 buffer containing KCH3COO (2.95 g/L), KCI (7.46 g/L), CaCl2 (1.11 g/L), glycerol (130 mL). TFB2 buffer containing KCI (0.75 g/L), CaCl2(11.1 g/L), MOPS buffer (2.1 g/L), glycerol (130 mL). The buffers were autoclaved. A solution of MgCl2x6H2O (95.2 g/L) was prepared including sterile filtration.
A culture (5 mL) of Luria-Bertani broth medium (10 g/L bacto tryptone, 5 g/L bacto yeast extract, 10 g/L sodium chloride) was inoculated with a glycerol culture (2 µl) of the strain E. coli BL 21 Gold (DE3) ΔdkgA :: FRT T7 gdh. This culture was shaken at 37° C. and 165 rpm overnight. The overnight culture (1 mL) was used to inoculate a culture (100 mL) of Luria-Bertani broth medium. The culture was incubated at 37° C. and 165 rpm until an OD600 of 0.4-0.5 was reached, whereupon the culture was subjected to centrifugation at 4° C. and 4000 rpm for 15 minutes. All following substances were pre-cooled in ice. The supernatant of the culture was discarded and the cell pellet was resuspended in TFB1 buffer (30 mL) and 3.2 ml MgCl2 solution (3.2 mL) and incubated on ice for 15 min. The mixture was subjected to centrifugation at 4° C. and 4000 rpm for 10 minutes. The supernatant was discarded and the cell pellet was resuspended in TFB2 buffer (4 mL) and was incubated for 15 min on ice. The cells were aliquoted à 50 µL, were snap frozen in liquid nitrogen and stored at -80° C.
For transformation 50 µL of the competent cells were thawed on ice for 15 min, 1-2 µL of plasmid (pETM11-BM3-254) were added, and the mixture was incubated on ice for 30 min. A heat shock was carried out for 45 sec at 42° C., followed by incubation on ice for 5 min. After the addition of SOC Outgrowth Medium (350 µL), the mixture was incubated for 1 h at 37° C. and 1000 rpm shaking. The cells (100 µL) were placed on Luria-Bertani broth agar plates with kanamycin (50 mg/L) and incubated overnight at 37° C. Single colonies were picked and sequenced.
Media of the preculture: tryptone (10 g/L), sodium chloride (10 g/L) and yeast extract (5 g/L) in demineralized water. The media was sterilized at 121° C. for 20 minutes. Afterwards kanamycin (50 mg/L) was added.
One preculture (100 mL) was inoculated with the strain E. coli BL 21 Gold (DE3) ΔdkgA :: FRT T7 gdh pETM11-BM3-254 (50 µL) containing the desired plasmid and was shaken at 37° C. and 165 rpm for 16 hours. This preculture (10 mL) was used to inoculate one 1 L Biostat Q fermenter. The cultivation media was prepared in the fermenters. Tryptone (20 g/L), sodium chloride (20 g/L), yeast extract (10 g/L) and Pluronic® PE 8100 (0.1 mL) were dissolved in demineralized water (1.0 L) and sterilized for 30 minutes at 121° C. in the fermenter. Afterwards kanamycin (50 mg) in demineralized water (5 mL) and Oxford trace metal solution (0.25 mL) was added. After the inoculation of the fermenter, the inoculated culture was stirred at 500 rpm at 37° C. with an aeration rate of 0.4 L/min and an oxygen partial pressure of 30 % maintained by the stirring up to 1500 rpm. After 3 hours an aqueous glucose solution was added (25 %, 8 g/h). After 5 hours and 30 minutes OD550 of 15.72 was reached, the temperature was decreased to 28° C. within 10 minutes and IPTG (238 mg) in demineralized water (10 mL) was added to start the protein expression. After additional 9 hours and 50 minutes the cells (34.88 g) were harvested by centrifugation, suspended in buffer (34.88 mL; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 2 × 25 mL, 3 × 1 mL and 1 × 13 ml.
One preculture (100 mL) was inoculated with the strain E. coli BL 21 Gold (DE3) ΔdkgA :: FRT T7 gdh pETM11-BM3-254 (50 µL) containing the desired plasmid and was shaken at 37° C. and 165 rpm for 16 hours. This preculture (10 mL) was used to inoculate one 1 L Biostat Q fermenter. The cultivation media was prepared in the fermenters. Tryptone (20 g/L), sodium chloride (20 g/L), yeast extract (10 g/L) and Pluronic® PE 8100 (0.1 mL) was dissolved in demineralized water (4.0 L) and sterilized for 30 minutes at 121° C. in the fermenter. Afterwards kanamycin (50 mg) in demineralized water (5 mL) and Oxford trace metal solution (0.25 mL) was added. After inoculation of the fermenter, the inoculated culture was stirred at 500 rpm at 37° C. with an aeration rate of 0.4 L/min, and an oxygen partial pressure of 30% was maintained by the stirring up to 1500 rpm. After 3 hours an aqueous glucose solution (25%, 8 g/h) was added. After 5 hours and 5 minutes an OD550 of 16.72 was reached, the temperature was decreased to 28° C. within 5 minutes and IPTG (238 mg) in demineralized water (10 mL) and aminolevulinic acid (42 mg) in demineralized water (5 mL) were added to start the protein expression. After additional 9 hours and 10 minutes the cells (38 g) were harvested by centrifugation, suspended in buffer (38 g; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 2 × 25 mL, 3 × 1 mL and 1 × 19 ml.
One preculture (100 mL) was inoculated with the strain E. coli BL 21 Gold (DE3) ΔdkgA :: FRT T7 gdh pETM11-BM3-254 (50 µL) containing the desired plasmid and was shaken at 37° C. and 165 rpm for 16 hours. This preculture (100 mL) was used to inoculate one 10 L steel fermenter. The cultivation media was prepared in the fermenter. Tryptone (20 g/L), sodium chloride (20 g/L), yeast extract (10 g/L) and Pluronic® PE 8100 (0.1 mL) were dissolved in demineralized water (8.8 L) and sterilized for 30 minutes at 121° C. in the fermenter. Afterwards kanamycin (500 mg) in demineralized water (10 mL) and Oxford trace metal solution (2.5 mL) was added. After inoculation of the fermenter (16 hours old preculture), the inoculated culture was stirred at 400 rpm at 37° C. with an aeration rate of 4.0 L/min and an oxygen partial pressure of 30% maintained by stirring up to 1200 rpm. After 3 hours an aqueous glucose solution (25%, 80 g/h) was added. After 5 hours and 20 minutes an OD550 of 16.69 was reached, the temperature was decreased to 28° C. within 5 minutes and IPTG (2.38 g) in demineralized water (20 mL) and aminolevulinic acid (420 mg) in demineralized water (20 mL) was added to start the protein expression. After additional 9 hours and 25 minutes the cells (298.42 g) were harvested by centrifugation, suspended in buffer (298.42 g; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 7 × 25 mL, 3 × 1 mL and 7 × 50 ml.
To one 1 L Biostat Q fermenter which contained KH2PO4 (4 g), K2HPO4 (12.3 g), Pluronic® PE 8100 (0.1 mL), demineralized water (930 mL), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 M, 1 mL) (17beta)-17-hydroxyandrost-4-en-3-one (100 mg, 347 µmol) dissolved in DMF (5 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. The pH value was maintained at pH 7.4. Cells in buffer (50 mL of cell cultivation 1) were added, and the biotransformation was maintained at an oxygen partial pressure of 50% by varying the stirring rate to up to 800 rpm. After 26.5 hours the biotransformation was extracted with 4-methyl-2-pentanone.
To one 1 L Biostat Q fermenter which contained KH2PO4 (4 g), K2HPO4 (12.3 g), Pluronic® PE 8100 (0.1 mL), demineralized water (930 mL), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 M, 1 mL) (17beta)-17-hydroxyandrost-4-en-3-one (100 mg, 347 µmol) dissolved in DMF (5 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. The pH value was maintained at pH 7.4. Cells in buffer (50 mL of cell cultivation 2) were added, and the biotransformation was maintained at an oxygen partial pressure of 50% by varying the stirring rate to up to 800 rpm. After 26.5 hours the biotransformation was extracted with 4-methyl-2-pentanone.
To one 1 L Biostat Q fermenter which contained KH2PO4 (4 g), K2HPO4 (12.3 g), Pluronic® PE 8100 (0.1 mL), demineralized water (930 mL), aqueous glucose solution (50 %, 20 mL) and aqueous EDTA solution (0.5 M, 1 mL) (17beta)-17-hydroxyandrost-4-en-3-one (100 mg, 347 µmol) dissolved in DMF (5 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. The pH value was maintained at pH 7.4. Cells in buffer (50 mL of cell cultivation 3) were added, and the biotransformation was maintained at an oxygen partial pressure of 50 % by varying the stirring rate to up to 1200 rpm. After 4 hours additional cells in buffer (25 mL of cell cultivation 3) were added. After 26 hours and 25 minutes the biotransformation was extracted with 4-methyl-2-pentanone.
To two 1 L Biostat Q fermenter each containing KH2PO4 (4 g), K2HPO4 (12.3 g), Pluronic® PE 8100 (0.1 mL), demineralized water (930 mL), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 M, 1 mL) (17beta)-17-hydroxyandrost-4-en-3-one (25 mg, 87 µmol) dissolved in DMF (2.5 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. The pH value was maintained at pH 7.4. Cells in buffer (50 mL of cell cultivation 3) were added, and the biotransformation was maintained at an oxygen partial pressure of 50 % by varying the stirring rate to up to 800 rpm. After 26 hours and 25 minutes the biotransformation was extracted with 4-methyl-2-pentanone.
The organic phases of the five biotransformations were combined and concentrated to give an oil (4.45 g) which was slurried with methanol and was filtered and concentrated to give an oil (1.23 g). The crude product was further purified by flash chromatography using silica gel (dichloromethane/ethanol gradient) and by preparative HPLC to give the title compound (13.0 mg).
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; Eluent A: Water + 0.1 Vol-% formic acid (99%), Eluent B: Acetonitrile; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; Flow 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 150×50 mm; Eluent A: Water + 0.1 Vol-% formic acid (99 %), Eluent B: Acetonitril; Gradient: 0.00-0.50 min 9% B (40-100 mL/min), 0.51-8.50 min 19-41% B (100 mL/min), DAD scan: 210-400 nm.
(15beta,17beta)-15,17,19-trihydroxyandrost-4-en-3-one
For the preparation of the title compound see second preparation of Example 17. The crude product (1.23 g) was further purified by flash chromatography using silica gel (dichloromethane/ethanol gradient) and by preparative HPLC to give the title compound (2.2 mg).
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 150×50 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 5 % B (40-100 mL/min), 0.51-8.50 min 5-30% B (100 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; Flow: 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.
Analytical Chiral HPLC: Rt = 0.63 min.
1H-NMR (600 MHz, DMSO-d6) δ [ppm]: 0.61 (dd, 1H), 0.82 - 0.97 (m, 6H), 1.37 - 1.47 (m, 2H), 1.51 - 1.57 (m, 1H), 1.61 (td, 1H), 1.64 - 1.68 (m, 1H), 1.89 - 1.97 (m, 1H), 2.07 - 2.17 (m, 2H), 2.22 - 2.32 (m, 3H), 2.37 - 2.45 (m, 2H), 2.58 - 2.67 (m, 2H), 3.26 - 3.32 (m, 1H), 3.73 (dd, 1H), 3.85 (dd, 1H), 3.91 - 3.98 (m, 1H), 4.35 (d, 1H), 4.46 (d, 1H), 4.72 (t, 1H), 5.74 (s, 1H).
1H-NMR (600 MHz, DMSO-d6) δ [ppm]: -0.006 (0.85), 0.005 (0.77), 0.593 (1.06), 0.603 (1.08), 0.612 (1.10), 0.621 (1.08), 0.832 (0.48), 0.847 (0.83), 0.853 (0.89), 0.869 (0.54), 0.875 (0.56), 0.889 (16.00), 0.900 (0.68), 0.907 (0.64), 0.924 (1.06), 0.943 (1.10), 0.957 (0.56), 0.964 (0.54), 1.383 (0.68), 1.387 (0.70), 1.398 (0.85), 1.402 (0.95), 1.406 (0.83), 1.410 (0.77), 1.421 (1.12), 1.425 (1.31), 1.440 (0.68), 1.446 (0.66), 1.529 (0.77), 1.535 (0.83), 1.552 (0.58), 1.557 (0.54), 1.588 (0.46), 1.602 (0.83), 1.611 (0.85), 1.625 (0.48), 1.634 (0.46), 1.651 (1.02), 1.656 (0.60), 1.666 (0.64), 1.671 (0.95), 1.918 (0.73), 1.924 (0.79), 1.937 (0.73), 1.942 (0.75), 2.076 (0.44), 2.087 (0.60), 2.092 (0.79), 2.099 (0.56), 2.113 (0.73), 2.120 (1.37), 2.126 (1.14), 2.134 (`0.54), 2.141 (0.58), 2.145 (0.64), 2.230 (0.77), 2.234 (0.56), 2.248 (0.79), 2.255 (1.31), 2.270 (1.35), 2.275 (0.93), 2.280 (1.24), 2.283 (1.22), 2.294 (1.43), 2.306 (0.77), 2.382 (0.81), 2.385 (1.16), 2.388 (1.64), 2.391 (1.31), 2.405 (0.75), 2.411 (0.70), 2.519 (3.59), 2.522 (3.63), 2.525 (3.05), 2.600 (0.58), 2.610 (1.08), 2.613 (1.04), 2.616 (1.33), 2.619 (1.02), 2.624 (0.79), 2.628 (0.75), 2.633 (0.70), 2.637 (0.62), 2.652 (0.62), 2.661 (0.48), 3.265 (0.54), 3.273 (0.58), 3.279 (1.14), 3.287 (1.16), 3.293 (0.58), 3.302 (0.56), 3.713 (0.99), 3.723 (1.04), 3.732 (1.31), 3.741 (1.26), 3.837 (1.02), 3.846 (1.08), 3.855 (0.81), 3.863 (0.77), 3.943 (0.87), 3.949 (0.87), 4.348 (2.88), 4.355 (2.82), 4.454 (2.47), 4.462 (2.38), 4.711 (1.18), 4.719 (2.47), 4.728 (1.16), 5.740 (3.34), 6.552 (0.56), 8.318 (1.16).
(2beta, 15beta, 17beta)-2,15,17-trihydroxyandrost-4-en-3-one
For the preparation of the title compound see second preparation of Example 17. The crude product (1.23 g) was further purified by flash chromatography using silica gel (dichloromethane/ethanol gradient) and by preparative HPLC to give the title compound (1 mg).
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 150×50 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99 %), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 5% B (40-100 mL/min), 0.51-8.50 min 5-30% B (100 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; Flow: 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.
Analytical Chiral HPLC: Rt = 0.64 min.
1H-NMR (400 MHz, CHLOROFORM-d) δ [ppm]: 0.774 (1.91), 0.788 (1.77), 0.802 (1.30), 0.816 (1.29), 0.997 (0.71), 1.010 (14.33), 1.027 (0.71), 1.031 (0.70), 1.040 (0.91), 1.051 (1.46), 1.061 (0.86), 1.072 (0.57), 1.082 (0.82), 1.092 (0.42), 1.129 (0.40), 1.136 (0.42), 1.148 (0.61), 1.160 (16.00), 1.170 (0.89), 1.174 (0.86), 1.184 (3.29), 1.191 (1.47), 1.210 (0.65), 1.349 (0.55), 1.358 (0.57), 1.381 (0.95), 1.389 (0.89), 1.407 (0.92), 1.415 (0.98), 1.434 (0.69), 1.439 (0.56), 1.443 (0.76), 1.456 (0.60), 1.464 (2.08), 1.474 (1.47), 1.498 (3.80), 1.507 (1.42), 1.517 (1.52), 1.523 (1.55), 1.533 (2.86), 1.538 (1.94), 1.554 (1.66), 1.559 (1.68), 1.640 (0.47), 1.708 (0.49), 1.716 (0.89), 1.724 (0.89), 1.733 (0.51), 1.740 (0.42), 1.748 (0.63), 1.757 (0.62), 1.779 (0.74), 1.788 (1.01), 1.796 (0.61), 1.811 (0.61), 1.819 (0.93), 1.828 (0.54), 1.943 (0.50), 1.979 (0.98), 2.026 (1.06), 2.036 (0.61), 2.057 (0.70), 2.063 (0.57), 2.106 (0.64), 2.191 (0.71), 2.198 (1.06), 2.207 (1.84), 2.215 (0.62), 2.227 (1.79), 2.234 (1.28), 2.240 (0.81), 2.400 (1.05), 2.414 (1.07), 2.434 (0.99), 2.448 (0.98), 2.512 (0.43), 2.516 (0.45), 2.521 (0.76), 2.532 (0.66), 2.535 (0.71), 2.540 (1.23), 2.543 (1.28), 2.558 (0.89), 2.562 (1.02), 2.568 (0.44), 2.577 (1.01), 2.580 (1.05), 2.598 (0.68), 3.504 (1.26), 3.526 (2.58), 3.547 (1.18), 4.093 (1.59), 4.101 (0.86), 4.107 (1.71), 4.114 (1.31), 4.119 (0.79), 4.127 (1.90), 4.133 (0.80), 4.142 (1.13), 5.234 (4.88), 5.755 (3.02), 5.757 (2.97).
(2beta, 17beta)-2,17,19-trihydroxyandrost-4-en-3-one
Media of the preculture: tryptone (10 g/L), sodium chloride (10 g/L) and yeast extract (5 g/L) in demineralized water. The media was sterilized at 121° C. for 20 minutes. Afterwards kanamycin (50 mg/L) was added.
One preculture (100 mL) was inoculated with the strain E. coli BL 21 Gold (DE3) ΔdkgA :: FRT T7 gdh pETM11-BM3-254 (50 µL) containing the desired plasmids and was shaken at 37° C. and 165 rpm for 16 hours. This preculture (10 mL) was used to inoculate one 1 L Biostat Q fermenter. The cultivation media was prepared in the fermenter. Tryptone (20 g/L), sodium chloride (20 g/L), yeast extract (10 g/L) and Pluronic® PE 8100 (0.5 mL) were dissolved in demineralized water (1.0 L) and sterilized for 30 minutes at 121° C. in the fermenter. Afterwards kanamycin (50 mg) in demineralized water (5 mL) was added. After the inoculation of the fermenter, the inoculated culture was stirred at 500 rpm at 37° C. with an aeration rate of 0.4 L/min and an oxygen partial pressure of 30% was maintained by stirring of up to 1500 rpm. After 3 hours an aqueous glucose solution (25%, 8 g/h) was added. After 5 hours and 30 minutes an OD550 of 16.2 was reached, the temperature was decreased to 28° C. within 15 minutes and IPTG (0.24 g) in demineralized water (10 mL) was added to start the protein expression. After additional 10 hours and 5 minutes the cells were harvested by centrifugation (38.8 g), suspended in buffer (38.8 mL; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 3 × 1 mL and 7 × 10 mL.
One preculture (100 mL) was inoculated with the strain E. coli BL 21 Gold (DE3) ΔdkgA :: FRT T7 gdh pETM11-BM3-254 (50 µL) containing the desired plasmids and was shaken at 37° C. and 165 rpm for 16 hours. This preculture (10 mL) was used to inoculate one 1 L Biostat Q fermenter. The cultivation media was prepared in the fermenter. Tryptone (20 g/L), sodium chloride (20 g/L), yeast extract (10 g/L) and Pluronic® PE 8100 (0.5 mL) were dissolved in demineralized water (1.0 L) and sterilized for 30 minutes at 121° C. in the fermenter. Afterwards kanamycin (50 mg) in demineralized water (5 mL) was added. After the inoculation of the fermenter, the inoculated culture was stirred at 500 rpm at 37° C. with an aeration rate of 0.4 L/min and an oxygen partial pressure of 30% was maintained by stirring of up to 1500 rpm. After 3 hours an aqueous glucose solution (25%, 8 g/h) was added. After 5 hours an OD550 of 16.6 was reached, the temperature was decreased to 28° C. within 15 minutes and IPTG (0.24 g) in demineralized water (10 mL) was added to start the protein expression. After additional 9 hours and 15 minutes the cells were harvested by centrifugation (42 g), suspended in buffer (42 mL; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 3 × 1 mL and 7 × 10 mL.
One preculture (100 mL) was inoculated with the strain E. coli BL 21 Gold (DE3) ΔdkgA :: FRT T7 gdh pETM11-BM3-254 (50 µL) containing the desired plasmids and was shaken at 37° C. and 165 rpm for 16 hours. This preculture (10 mL) was used to inoculate one 1 L Biostat Q fermenter. The cultivation media was prepared in the fermenter. Tryptone (20 g/L), sodium chloride (20 g/L), yeast extract (10 g/L) and Pluronic® PE 8100 (0.5 mL) were dissolved in demineralized water (1.0 L) and sterilized for 30 minutes at 121° C. in the fermenter. Afterwards kanamycin (50 mg) in demineralized water (5 mL) was added. After the inoculation of the fermenter, the inoculated culture was stirred at 500 rpm at 37° C. with an aeration rate of 0.4 L/min and an oxygen partial pressure of 30% was maintained by stirring of up to 1500 rpm. After 3 hours an aqueous glucose solution (25%, 8 g/h) was added. After 5 hours and 5 minutes an OD550 of 16.72 was reached, the temperature was decreased to 28° C. within 15 minutes and IPTG (0.238 g) in demineralized water (10 mL) and 5-aminolevulinic acid hydrochloride (0.042 g) in demineralized water (5 mL) was added to start the protein expression. After additional 9 hours and 40 minutes the cells were harvested by centrifugation (43 g), suspended in buffer (43 mL; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 3 × 1 mL and 5 × 15 mL.
To a 1 L Biostat Q fermenter which contained KH2PO4 (4 g), K2HPO4 (12.3 g), Pluronic® PE 8100 (0.1 mL), demineralized water (0.93 L), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 M, 1 mL) (17beta)-17-hydroxyandrosta-4-en-3-one (100 mg, 0.35 mmol) dissolved in DMF (5 mL) was added, and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. Cells in buffer (50 mL of preculture 2) were added, and the biotransformation was maintained at an oxygen partial pressure of 50% by varying the stirring rate to up to 800 rpm. The pH value was maintained at pH 7.4 by addition of an aqueous solution of sodium hydroxide (16%). After 4 hours, cells (20 mL) in buffer were added again. After 28 hours and 10 minutes the biotransformation was stopped.
To a 1 L Biostat Q fermenter which contained KH2PO4 (4 g), K2HPO4 (12.3 g), Pluronic® PE 8100 (0.1 mL), demineralized water (0.93 L), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 M, 1 mL) (17beta)-17-hydroxyandrosta-4-en-3-one (100 mg, 0.35 mmol) dissolved in DMF (5 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. Cells in buffer (50 mL of cell cultivation 3) were added, and the biotransformation was maintained at an oxygen partial pressure of 50% by varying the stirring rate to up to 800 rpm. The pH value was maintained at pH 7.4 by addition of an aqueous solution of sodium hydroxide (16%). After 27 hours and 45 minutes the biotransformation was stopped.
To a 1 L Biostat Q fermenter which contained KH2PO4 (4 g), K2HPO4 (12.3 g), Pluronic® PE 8100 (0.1 mL), demineralized water (0.93 L), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 M, 1 mL). (17beta)-17-hydroxyandrosta-4-en-3-one (4 × 25 mg, 0.35 mmol) dissolved in DMF (4 × 2.5 mL) was added in intervals after 0 h, 2 h, 4 h and 6 h and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. Cells in buffer (50 mL of cell cultivation 1) were added at 0 h, and the biotransformation was maintained at an oxygen partial pressure of 50% by varying the stirring rate between 800 and 1200 rpm. The pH value was maintained at pH 7.4 by addition of an aqueous solution of sodium hydroxide (16%). After 28 hours the biotransformation.
The three biotransformations were combined and extracted with 4-methyl-2-pentanone. The organic layer was concentrated to give an oil (2.2 g) which was slurried with methanol and was filtered and concentrated to give an oil (1.06 g). The crude product was further purified by flash chromatography using silica gel (dichloromethane/methanol gradient) and by preparative HPLC to give the title compound (7.8 mg, 2% yield).
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 100×30 mm; Eluent A: water + 0.1 vol-% aqueous ammonia (99%), Eluent B: acetonitrile; gradient: 0.00-0.50 min 10% B (40-70 mL/min), 0.51-5.50 min 10-40% B (70 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; eluent A: water + 0.1 vol % aqueous ammonia (99%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 mL/min; temperature: 60° C.; DAD scan: 210-400 nm.
Analytical Chiral HPLC: Rt = 0.73 min.
1H-NMR (600 MHz, CDCl3) δ [ppm]: 0.79 (s, 3H), 0.95 - 1.11 (m, 3H), 1.24 - 1.36 (m, 3H), 1.38 - 1.50 (m, 3H), 1.57 - 1.63 (m, 1H), 1.69 - 1.77 (m, 1H), 1.81 (dq, 1H), 1.87 (dt, 1H), 1.95 - 2.02 (m, 1H), 2.03 - 2.14 (m, 1H), 2.20 - 2.27 (m, 2H), 2.30 (s, 1H), 2.35 - 2.41 (m, 1H), 2.44 - 2.54 (m, 1H), 3.60 - 3.73 (m, 3H), 4.09 - 4.17 (m, 2H).
3-hydroxyestra-1 (10),2,4-trien-17-one
E. coli BL21 Star™ (DE3) pETM11-BM3-268; S72G, A74V, F77A
Media of the preculture: tryptone (16 g/L), sodium chloride (10 g/L) and yeast extract (10 g/L) in demineralized water. The pH value was maintained at pH 7.3. The medium was sterilized at 121° C. for 20 minutes. Afterwards kanamycin (50 mg/L) was added.
One preculture (100 mL) was inoculated with the strain E. coli BL21 Star™ (DE3) pETM11-BM3-268; S72G, A74V, F77A (50 µL) containing the desired plasmids and was shaken at 37° C. and 165 rpm for 17 hours. This preculture (100 mL) was used to inoculate one 10 L steel fermenter. The cultivation media were prepared in the fermenter. Tryptone (12 g/L), yeast extract (24 g/L), predigested beef extract (2 g/L), KH2PO4 (2.2 g/L), K2HPO4 (9.4 g/L) and glycerol (87%, 4.6 g/L) were dissolved in demineralized water (9.2 L) and sterilized for 20 minutes at 121° C. in the fermenter. Afterwards kanamycin (0.5 g) in water (20 mL), riboflavin (10 mg) in water (20 mL), thiamine hydrochloride (3.37 g) in water (10 mL) and Oxford trace metal solution (2.5 mL) were added. After the inoculation of the fermenter, the culture was stirred at 315 rpm at 37° C. with an aeration rate of 3.3 L/min at pH 6.6 which was regulated by addition of aqueous sodium hydroxide solution (16%) or aqueous phosphoric acid solution (16%). After 2.55 hours, an OD550 of 0.94 was reached, the temperature was decreased to 27° C. within 10 minutes and IPTG (1.43 g) in water (40 mL) were added to start the protein expression. After 9.15 hours the aqueous phosphoric acid solution (16%) was substituted for an aqueous glucose solution (50%) for pH regulation. After additional 23.5 hours an OD550 of 23.92 was reached, the cells were harvested by centrifugation (215.6 g), suspended in buffer (215.6 mL; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 1 × 150 mL, 1 × 130 mL, 2 × 40 mL und 5 × 1 mL.
To a 10 L steel fermenter which contained KH2PO4 (40 g), K2HPO4 (123 g), Pluronic® PE 8100 (1 mL), demineralized water (9.0 L), aqueous glucose solution (50%, 200 mL) and aqueous EDTA solution (0.5 M, 10 mL) androsta-1,4-diene-3,17-dione (500 mg, 1.75 mmol) dissolved in DMF (10 mL) was added and the mixture was stirred at 315 rpm at 27° C. with an aeration rate of 3.3 L/min. Cells in buffer (400 mL) were added, and the biotransformation was maintained at an oxygen partial pressure of 50% by varying the stirring rate to up to 1200 rpm. The pH value was maintained at pH 7.4 by addition of an aqueous solution of sodium hydroxide (16%). After 26 hours the biotransformation was extracted with 4-methyl-2-pentanone. The organic layer was concentrated to give an oil (2.37 g) which was slurried with methanol and was filtered and concentrated to give an oil (2.05 g). The crude product was further purified by flash chromatography using silica gel (dichloromethane/methanol gradient) and twice by preparative HPLC to give the title compound (14 mg).
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 50×50 mm; Eluent A: Water + 0.2 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 23% B (50-100 mL/min), 0.51-13.50 min 23 - 43% B (100 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0-2.6 min 1-99% B, 2.6-3.0 min 99% B; Flow 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.
Analytical HPLC: Rt = 1.10 min.
Instrument: Sepiatec: Prep SFC100; Column: Chiralpak IG 5 µ 250×30 mm; eluent A: CO2; eluent B: methanol; isocratic: 20% B; flow: 100 mL/min; temperature: 40° C.; BPR: 150 bar; UV: 280 nm.
Instrument: Agilent: 1260, Aurora SFC-Modul; Column: Chiralpak IG 5 µ 100×4.6 mm; eluent A: CO2; eluent B: methanol; isocratic: 20% B; flow: 4 mL/min; temperature: 37.5° C.; BPR: 100 bar; UV: 280 nm.
Analytical HPLC: Rt = 3.66 min.
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 0.820 (16.00), 1.293 (0.48), 1.322 (0.95), 1.329 (0.75), 1.348 (2.38), 1.371 (1.82), 1.401 (0.24), 1.450 (0.87), 1.461 (0.83), 1.476 (1.50), 1.492 (0.75), 1.523 (0.55), 1.529 (0.44), 1.545 (0.36), 1.552 (0.59), 1.575 (0.36), 1.583 (0.32), 1.726 (0.99), 1.733 (0.91), 1.748 (0.79), 1.754 (0.67), 1.882 (0.51), 1.890 (0.55), 1.896 (0.59), 1.915 (0.75), 1.921 (0.83), 1.935 (0.63), 1.943 (0.67), 1.964 (0.48), 1.976 (0.44), 2.011 (0.59), 2.033 (0.91), 2.058 (0.83), 2.071 (0.20), 2.080 (1.19), 2.102 (0.51), 2.136 (0.55), 2.284 (0.40), 2.301 (0.71), 2.314 (0.71), 2.326 (0.51), 2.331 (0.24), 2.394 (0.95), 2.414 (1.07), 2.440 (0.75), 2.461 (0.95), 2.518 (0.71), 2.522 (0.48), 2.539 (0.44), 2.664 (0.20), 2.669 (0.24), 2.673 (0.20), 2.710 (0.20), 2.735 (1.35), 2.747 (1.43), 2.775 (0.51), 3.298 (0.20), 6.444 (2.06), 6.450 (2.42), 6.496 (1.35), 6.502 (1.07), 6.517 (1.39), 6.523 (1.15), 7.036 (1.82), 7.056 (1.66), 9.033 (4.63).
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 0.82 (s, 3H), 1.22 - 1.41 (m, 3H), 1.42 - 1.61 (m, 3H), 1.69 - 1.79 (m, 1H), 1.86 - 1.99 (m, 2H), 2.00 - 2.19 (m, 2H), 2.25 - 2.35 (m, 1H), 2.43 (dd, 1H), 2.68 - 2.83 (m, 2H), 6.45 (d, 1H), 6.51 (dd, 1H), 7.05 (d, 1H), 9.03 (s, 1H).
For another preparation of the title compound see Example 25. The crude product was further purified by flash chromatography using silica gel (dichloromethane/methanol gradient) and by preparative HPLC to give the title compound (91 mg).
Furthermore the title compound was synthesized using androsta-1,4-diene-3,17-dione (ADD) as a substrate by the following procedures with the screening yields mentioned below.
A: E. coli BL21 Star™ (DE3) pETM11-BM3-268; M177Y, A184Y (Prot Seq ID No. 15; DNA Seq ID No. 73) + S72G, V178P, L181Y, L188F
B: E. coli BL21 Star™ (DE3) pETM11-BM3-268; M177Y, A184Y (Prot Seq ID No. 15; DNA Seq ID No. 73) + S72G, T146F, V178W, L181Y
C: E. coli BL21 Star™ (DE3) pETM11-BM3-268; S72G, M177Y, V178P, L181Y, A184Y, L188F (Prot SEQ ID No. 117; DNA Seq ID No. 120) + R179L
D: E. coli BL21 Star™ (DE3) pETM11-BM3-268; S72G, M177Y, V178P, L181Y, A184Y, L188F (Prot SEQ ID No. 117; DNA Seq ID No. 120)
E: E. coli BL21 Star™ (DE3) pETM11-BM3-268; S72G, T146F, M177Y, V178W, L181Y, A184Y, G457S, 1458G, A477N (Prot SEQ ID No. 119; DNA-Seq ID No. 122)
F: E. coli BL21 Star™ (DE3) pETM11-BM3-268; S72G, T146F, M177Y, V178W, L181Y, A184Y, G457S, 1458G, A477N (Prot SEQ ID No. 119; DNA-Seq ID No. 122) + insert with SEQ ID No. 137
G: E. coli BL21 Star™ (DE3) pETM11-BM3-268; S72G, T146F, M177Y, V178W, L181Y, A184Y, G457S, 1458G, A477N (Prot SEQ ID No. 119; DNA-Seq ID No. 122) + insert with SEQ ID No. 126
H: E. coli BL21 Star™ (DE3) pETM11-BM3-268; S72G, T146F, M177Y, V178W, L181Y, A184Y, G457S, 1458G, A477N (Prot SEQ ID No. 119; DNA-Seq ID No. 122) + insert with Seq ID No. 139
I: E. coli BL21 Star™ (DE3) pETM11-BM3-268; S72G, T146F, M177Y, V178W, L181Y, A184Y, G457S, 1458G, A477N (Prot SEQ ID No. 119; DNA-Seq ID No. 122) + insert with SEQ ID No. 145
Media of the preculture: tryptone (16 g/L), sodium chloride (10 g/L) and yeast extract (10 g/L) in demineralized water. The pH value was maintained at pH 7.3. The medium was sterilized at 121° C. for 20 minutes. Afterwards kanamycin (50 mg/L) was added.
One preculture (100 mL) was inoculated with the strain (50 µL) containing the desired plasmids and was shaken at 37° C. and 165 rpm for 17 hours. This preculture (100 mL) was used to inoculate one 10 L steel fermenter. The cultivation media were prepared in the fermenter. Tryptone (12 g/L), yeast extract (24 g/L), predigested beef extract (2 g/L), KH2PO4 (2.2 g/L), K2HPO4 (9.4 g/L) and glycerol (87%, 4.6 g/L) were dissolved in demineralized water (9.2 L) and sterilized for 20 minutes at 121° C. in the fermenter. Afterwards kanamycin (0.5 g) in water (20 mL), riboflavin (10 mg) in water (20 mL), thiamine hydrochloride (3.37 g) in water (10 mL) and Oxford trace metal solution (2.5 mL) were added. After the inoculation of the fermenter, the culture was stirred at 315 rpm at 37° C. with an aeration rate of 3.3 L/min at pH 6.6 which was regulated by addition of aqueous sodium hydroxide solution (16%) or aqueous phosphoric acid solution (16%). After an OD550 of 0.9-1.0 was reached, the temperature was decreased to 27° C. within 10 minutes and IPTG (1.43 g) in water (40 mL) was added to start the protein expression. The aqueous phosphoric acid solution (16%) was substituted for an aqueous glucose solution (50%) for pH regulation after the time described in the table below. After the culturing time mentioned in the table below the cells were harvested by centrifugation and resuspended in buffer, 1 g cell pellet in 1 mL buffer (KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 80 mL.
To a 1 L glass vessel fermenter which contained KH2PO4 (9.54 g), K2HPO4 (5.19 g), 0.1 mL of Pluronic® PE 8100, demineralized water (0.9 L), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 mM, 1 mL), androsta-1,4-diene-3,17-dione (400 mg, 1.41 mmol/L) dissolved in DMF (8 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. Cells in buffer (80 mL) were added, and the biotransformation was maintained at a dissolved oxygen partial pressure of 50% by varying the stirring rate up to 2000 rpm. After Glucose concentration dropped below 5 g/L an aqueous glucose solution (50%) feeding started. The pH value was maintained at pH 6.6 by addition of an aqueous solution of sodium hydroxide (16%). After 0 h, 1 h, 2 h, 3 h, 5 h, 7 h, 23 h and 47 h an analytical sample (2 mL) was taken and the titer for Example 21 was measured with the methods mentioned below.
To a 1 L glass vessel fermenter which contained KH2PO4 (6.8 g), K2HPO4 (8.7 g), 0.1 mL of Pluronic® PE 8100, demineralized water (0.9 L), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 mM, 1 mL), androsta-1,4-diene-3,17-dione (400 mg, 1.41 mmol/L) dissolved in DMF (8 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. Cells in buffer (80 mL) were added, and the biotransformation was maintained at a dissolved oxygen partial pressure of 50% by varying the stirring rate up to 2000 rpm. After the glucose concentration dropped below 5 g/L an aqueous glucose solution (50%) feeding started. The pH value was maintained at pH 7.0 by addition of an aqueous solution of sodium hydroxide (16 %). After 0 h, 1 h, 2 h, 3 h, 5h, 7 h and 23 h an analytical sample (2 mL) was taken and the titer for Example 21 was measured with methods mentioned below.
To a 1 L glass vessel fermenter which contained KH2PO4 (9.54 g), K2HPO4 (5.19 g), 0.1 mL of Pluronic® PE 8100, demineralized water (0.5 L), aqueous glucose solution (50%, 20 mL), 20 mL of DMF and aqueous EDTA solution (0.5 mM, 1 mL), androsta-1,4-diene-3,17-dione (1500 mg, 5.27 mmol/L) as aqueous suspension was added (15.4 mL of an 125 g/L stock solution; 78%) and the mixture was stirred at 500 rpm at 27° C. with an aeration rate of 0.5 L/min. Cells in buffer (400 mL) were added, and the biotransformation was maintained at an dissolved oxygen partial pressure of 50% by varying the stirring rate up to 2000 rpm and addition of pure oxygen to aeration within the range of 0.4 L/min and reducing air aeration by the same volume per minute to keep aeration rate of 0.5 L/min. After 3 hours an aqueous glucose solution (50%) feeding started. The amount of glucose measured should not drop below 5 g/L. The total amount of added glucose was 97.9 g. The pH value was maintained at pH 6.6 by addition of an aqueous solution of sodium hydroxide (16 %). After 0 h, 1 h, 2 h, 3 h, 5 h, 7 h and 23 h an analytical sample (2 mL) was taken and the titer for Example 21 was measured with methods mentioned below.
To a 1 L glass vessel fermenter which contained KH2PO4 (9.54 g), K2HPO4 (5.19 g), 0.1 mL of Pluronic® PE 8100, demineralized water (0.9 L), aqueous glucose solution (50%, 20 mL) and aqueous EDTA solution (0.5 mM, 1 mL), androsta-1,4-diene-3,17-dione (800 mg, 2.81 mmol/L) dissolved in DMF (16 mL) was added and the mixture was stirred at 300 rpm at 27° C. with an aeration rate of 0.5 L/min. Cells in buffer (80 mL) were added, and the biotransformation was maintained at a dissolved oxygen partial pressure of 50% by varying the stirring rate up to 2000 rpm. After 23 hours an aqueous glucose solution (50%) feeding started. The amount of glucose measured should not drop below 5 g/L. The total amount of added glucose was 67.5 g. The pH value was maintained at pH 6.6 by addition of an aqueous solution of sodium hydroxide (16%). After 0 h, 1 h, 2 h, 3 h, 5 h, 7 h and 23 h an analytical sample (2 mL) was taken and the titer for Example 21 was measured with methods mentioned below.
Instrument: Waters Acquity UPLC (I-Class, H-Class) or Thermo Scientific ISQ EM SingleQuad; Column: Ascentis Express C18 2.7 µm, 150×2.1 mm; Eluent A: Water + 0.05 % Formic acid (99%); Eluent B: Acetonitrile + 0.05% Formic acid; Gradient: 0-9 min 5-30% B, 9-15 min 30-95% B; Flow 0.7 mL/min; Temperature: 45° C.; DAD: 243 nm + 280 nm.
15alpha-hydroxyandrosta-1,4-diene-3, 17 -dione
For the preparation of the title compound see Example 21. The crude product was further purified by flash chromatography using silica gel (dichloromethane/methanol gradient) and twice by preparative HPLC to give the title compound (14 mg).
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 50×50 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 17% B (50-100 mL/min), 0.51-8.50 min 17 - 37% B (100 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99 %), Eluent B: Acetonitrile; Gradient: 0-2.6 min 1-99% B, 2.6-3.0 min 99% B; Flow 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.
Analytical HPLC: Rt = 0.82 min.
Instrument: PrepCon Labomatic HPLC; Column: YMC Cellulose SC 5 µ, 250×30 mm; eluent A: hexane + 0.1 vol % diethylamine; eluent B: 2-propanol; isocratic: 70 % A + 30 % B; flow: 50 mL/min; temperature: 25° C.; UV: 254 nm.
Instrument: Waters Alliance 2695; Column: YMC Cellulose SC 3 µ, 100×4.6 mm; eluent A: hexane + 0.1 vol % diethylamine; eluent B: 2-propanol; isocratic: 70% A + 30% B; flow: 1.4 mL/min; temperature: 25° C.; UV: 254 nm.
Analytical HPLC: Rt = 9.28 min.
1H-NMR (600 MHz, CDCl3) δ [ppm]: 0.000 (2.45), 0.736 (0.49), 0.839 (0.64), 0.848 (0.52), 0.856 (0.53), 0.862 (0.46), 0.869 (0.88), 0.881 (1.19), 0.892 (0.62), 1.071 (0.16), 1.139 (0.57), 1.146 (0.65), 1.157 (0.67), 1.159 (0.78), 1.163 (0.80), 1.166 (0.69), 1.177 (0.65), 1.184 (0.62), 1.189 (0.46), 1.197 (0.55), 1.210 (0.78), 1.217 (0.88), 1.233 (1.06), 1.239 (1.17), 1.254 (5.41), 1.262 (3.03), 1.270 (14.17), 1.281 (2.20), 1.288(1.62), 1.297 (1.04), 1.306 (16.00), 1.333 (0.30), 1.348 (0.42), 1.356 (0.21), 1.376 (0.22), 1.399 (0.16), 1.412 (0.17), 1.601 (0.17), 1.642 (0.51), 1.700 (0.19), 1.724 (0.23), 1.731 (0.33), 1.747 (0.53), 1.753 (0.50), 1.768 (0.70), 1.774 (0.62), 1.789 (0.32), 1.796 (0.33), 1.840 (0.70), 1.844 (1.02), 1.851 (1.63), 1.858 (0.95), 1.861 (0.97), 1.866 (1.73), 1.868 (1.14), 1.873 (1.06), 1.877 (0.57), 1.884 (0.28), 2.163 (0.24), 2.169 (0.28), 2.181 (0.55), 2.187 (0.64), 2.201 (0.54), 2.206 (0.60), 2.219 (0.21), 2.225 (0.26), 2.267 (0.30), 2.271 (0.41), 2.276 (0.50), 2.280 (0.46), 2.288 (0.41), 2.292 (0.42), 2.297 (0.46), 2.301 (0.44), 2.307 (0.28), 2.434 (0.41), 2.438 (0.48), 2.441 (0.51), 2.445 (0.42), 2.456 (0.59), 2.461 (0.67), 2.463 (0.68), 2.468 (0.52), 2.503 (0.69), 2.513 (0.64), 2.535 (1.79), 2.545 (1.79), 2.567 (0.41), 2.569 (0.53), 2.575 (2.58), 2.590 (0.64), 2.592 (0.66), 2.598 (0.63), 2.600 (0.64), 2.607 (0.88), 2.612 (0.34), 2.614 (0.31), 2.621 (0.27), 2.623 (0.37), 4.576 (0.66), 4.585 (1.06), 4.593 (0.65), 6.107 (1.41), 6.109 (2.45), 6.112 (1.50), 6.245 (1.70), 6.248 (1.64), 6.262 (1.64), 6.266 (1.59), 7.060 (2.35), 7.078 (2.17), 7.267 (8.94).
6beta-hydroxyandrosta-1 ,4-diene-3, 17 -dione
For the preparation of the title compound see Example 21. The crude product was further purified by flash chromatography using silica gel (dichloromethane/methanol gradient) and twice by preparative HPLC to give the title compound (2 mg).
Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5 µ 50x50 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 17 % B (50-100 mL/min), 0.51-8.50 min 17 - 37% B (100 mL/min), DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µm, 50×2.1 mm; Eluent A: Water + 0.1 Vol-% Formic acid (99%), Eluent B: Acetonitrile; Gradient: 0-2.6 min 1-99% B, 2.6-3.0 min 99% B; Flow 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.
Analytical HPLC: Rt = 0.82 min.
Instrument: PrepCon Labomatic HPLC; Column: YMC Cellulose SC 5 µ, 250×30 mm; eluent A: hexane + 0.1 vol % diethylamine; eluent B: 2-propanol; isocratic: 70 % A + 30 % B; flow: 50 mL/min; temperature: 25° C.; UV: 254 nm.
Instrument: Waters Alliance 2695; Column: YMC Cellulose SC 3 µ, 100×4.6 mm; eluent A: hexane + 0.1 vol % diethylamine; eluent B: 2-propanol; isocratic: 70% A + 30% B; flow: 1.4 mL/min; temperature: 25° C.; UV: 254 nm.
Analytical HPLC: Rt = 6.58 min.
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 0.697 (1.62), 0.717 (0.16), 0.833 (0.65), 0.851 (1.06), 0.879 (16.00), 0.986 (0.57), 0.996 (0.57), 1.014 (0.89), 1.023 (0.97), 1.042 (0.73), 1.052 (0.65), 1.083 (0.32), 1.147 (0.65), 1.164 (1.06), 1.172 (0.89), 1.184 (1.38), 1.195 (1.87), 1.232 (5.28), 1.256 (1.38), 1.272 (1.06), 1.289 (1.38), 1.293 (1.46), 1.300 (1.22), 1.317 (0.97), 1.330 (0.97), 1.347 (0.65), 1.351 (0.65), 1.373 (16.00), 1.472 (0.41), 1.495 (0.81), 1.502 (0.65), 1.517 (0.57), 1.526 (1.14), 1.534 (0.57), 1.548 (0.73), 1.557 (0.65), 1.579 (0.32), 1.598 (0.32), 1.609 (0.41), 1.631 (0.65), 1.641 (0.81), 1.668 (2.60), 1.692 (0.89), 1.698 (1.22), 1.708 (0.65), 1.784 (0.73), 1.795 (0.81), 1.805 (0.57), 1.822 (0.65), 1.833 (0.81), 1.847 (0.65), 1.853 (0.73), 1.867 (0.73), 1.883 (0.57), 1.898 (0.49), 1.959 (0.81), 1.967 (0.81), 1.975 (1.22), 1.981 (1.71), 2.006 (1.46), 2.017 (0.73), 2.028 (1.38), 2.051 (0.49), 2.080 (0.32), 2.089 (0.32), 2.109 (0.73), 2.116 (0.65), 2.138 (0.73), 2.165 (0.32), 2.173 (0.24), 2.331 (0.97), 2.336 (0.49), 2.380 (1.14), 2.401 (1.14), 2.428 (1.30), 2.450 (1.79), 2.518 (3.65), 2.522 (2.44), 2.539 (0.49), 2.673 (0.89), 2.678 (0.41), 4.398 (1.71), 4.405 (1.71), 5.331 (2.27), 5.336 (2.19), 6.054 (3.49), 6.058 (4.30), 6.084 (2.60), 6.089 (1.79), 6.109 (2.36), 6.114 (1.95), 7.168 (3.17), 7.193 (3.09).
16beta-hydroxyandrosta-1 ,4-diene-3, 17 -dione
E. coli BL21 Star™ (DE3) pETM11-BM3-268; F77A, M177Y
Media of the preculture: tryptone (10 g/L), sodium chloride (10 g/L) and yeast extract (5 g/L) in demineralized water. The media was sterilized at 121° C. for 20 minutes. Afterwards kanamycin (50 mg/L) was added.
One preculture (100 mL) was inoculated with the strain E. coli BL21 Star™ (DE3) pETM11-BM3-268; F77A, M177Y (50 µL) containing the desired plasmids and was shaken at 37° C. and 165 rpm for 17 hours. This preculture (100 mL) was used to inoculate one 10 L steel fermenter. The cultivation media were prepared in the fermenter. Tryptone (12 g/L), yeast extract (24 g/L), predigested beef extract (2 g/L), KH2PO4 (2.2 g/L), K2HPO4 (9.4 g/L) and glycerol (87%, 4.6 g/L) were dissolved in demineralized water (9.2 L) and sterilized for 20 minutes at 121° C. in the fermenter. Afterwards kanamycin (0.5 g) in water (20 mL), riboflavin (10 mg) in water (20 mL), thiamine hydrochloride (3.37 g) in water (10 mL) and Oxford trace metal solution (2.5 mL) were added. After the inoculation of the fermenter, the culture was stirred at 315 rpm at 37° C. with an aeration rate of 3.3 L/min at pH 6.6 which was regulated by addition of aqueous sodium hydroxide solution (16%) or aqueous phosphoric acid solution (16%). After 3.15 hours, an OD550 of 1.0 was reached, the temperature was decreased to 27° C. within 10 minutes and IPTG (1.43 g) in water (40 mL) were added to start the protein expression. After 8 hours the aqueous phosphoric acid solution (16%) was substituted for an aqueous glucose solution (50%) for pH regulation. After additional 24 hours an OD550 of 25.2 was reached, the cells were harvested by centrifugation (284.2 g), suspended in buffer (284.2 mL; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 2 × 150 mL, 1 × 140 mL, 2 × 40 mL und 5 × 1 mL.
To a 10 L steel fermenter which contained KH2PO4 (40 g), K2HPO4 (123 g), Pluronic® PE 8100 (1 mL), demineralized water (9.0 L), aqueous glucose solution (50 %, 200 mL) and aqueous EDTA solution (0.5 M, 10 mL) androsta-1,4-diene-3,17-dione (500 mg, 1.75 mmol) dissolved in DMF (10 mL) was added and the mixture was stirred at 315 rpm at 27° C. with an aeration rate of 3.3 L/min. Cells in buffer (400 mL) were added, and the biotransformation was maintained at an oxygen partial pressure of 50% by varying the stirring rate to up to 1200 rpm. The pH value was maintained at pH7.4 by addition of an aqueous solution of sodium hydroxide (16%). After 26 hours the biotransformation was extracted with 4-methyl-2-pentanone. The organic layer was concentrated to give an oil (1.71 g) which was slurried with methanol and was filtered and concentrated to give an oil (1.55 g). The crude product was further purified by flash chromatography using silica gel (dichloromethane/methanol gradient) and by preparative HPLC to give the title compound (11 mg).
Instrument: Sepiatec: Prep SFC100; Column: Reprosil Chiral NR 8µ 250x30 mm; eluent A: CO2; eluent B: 2-propanol; isocratic: 25% B; flow: 100 mL/min; temperature: 40° C.; BPR: 150 bar; UV: 254 nm.
Instrument: Agilent: 1260, Aurora SFC-Modul; Column: Reprosil Chiral NR 5 µ 100×4.6 mm; eluent A: CO2; eluent B: 2-propanol; isocratic: 25% B; flow: 4 mL/min; temperature: 37.5° C.; BPR: 100 bar; UV: 254 nm.
Analytical HPLC: Rt = 3.41 min.
1H-NMR (600 MHz, CDCl3) δ [ppm]: 0.000 (2.08), 1.032 (13.48), 1.117 (0.25), 1.124 (0.30), 1.129 (0.47), 1.137 (0.99), 1.147 (0.90), 1.150 (0.65), 1.154 (0.70), 1.157 (0.93), 1.168 (0.97), 1.175 (0.53), 1.180 (0.33), 1.188 (0.30), 1.222 (0.50), 1.230 (0.53), 1.237 (0.38), 1.239 (0.59), 1.244 (0.71), 1.248 (0.70), 1.252 (0.83), 1.256 (0.66), 1.262 (0.78), 1.272 (16.00), 1.322 (0.38), 1.330 (0.43), 1.337 (0.26), 1.344 (0.81), 1.351 (0.86), 1.366 (0.46), 1.373 (0.45), 1.558 (0.71), 1.573 (0.73), 1.578 (0.78), 1.580 (0.67), 1.593 (0.82), 1.596 (0.70), 1.600 (0.71), 1.616 (0.71), 1.723 (0.35), 1.730 (0.38), 1.745 (0.66), 1.752 (0.68), 1.767 (0.71), 1.774 (0.75), 1.788 (0.35), 1.796 (0.36), 1.850 (0.36), 1.854 (0.71), 1.857 (0.73), 1.861 (0.91), 1.869 (0.48), 1.872 (0.82), 1.879 (0.99), 1.884 (0.55), 1.892 (0.77), 1.897 (0.59), 1.909 (0.25), 1.916 (0.27), 1.928 (0.59), 1.933 (0.66), 1.935 (0.65), 1.940 (0.51), 1.950 (0.57), 1.954 (0.62), 1.957 (0.60), 1.961 (0.46), 2.073 (0.30), 2.077 (0.40), 2.081 (0.47), 2.083 (0.44), 2.085 (0.44), 2.092 (0.39), 2.099 (0.40), 2.102 (0.44), 2.104 (0.43), 2.113 (0.27), 2.380 (0.61), 2.388 (0.62), 2.393 (0.63), 2.400 (0.80), 2.408 (0.60), 2.413 (0.59), 2.422 (0.84), 2.426 (0.51), 2.430 (0.43), 2.441 (0.67), 2.446 (0.73), 2.448 (0.72), 2.453 (0.59), 2.504 (0.41), 2.507 (0.45), 2.512 (0.45), 2.515 (0.44), 2.526 (0.65), 2.529 (0.66), 2.535 (0.65), 2.538 (0.61), 2.549 (0.29), 2.552 (0.31), 2.557 (0.27), 2.560 (0.25), 2.623 (5.61), 3.948 (1.17), 3.962 (1.59), 3.977 (1.10), 6.101 (1.45), 6.103 (2.45), 6.107 (1.46), 6.241 (1.54), 6.244 (1.48), 6.258 (1.70), 6.262 (1.62), 7.031 (2.46), 7.048 (2.39), 7.266 (8.11).
1H-NMR (600 MHz, CDCl3) δ [ppm]: 1.03 (s, 3H), 1.11 - 1.20 (m, 2H), 1.21 - 1.28 (m, 1H), 1.27 (s, 3H), 1.31 - 1.38 (m, 1H), 1.59 (ddd, 1H), 1.76 (qd, 1H), 1.84 - 1.97 (m, 3H), 2.06 - 2.12 (m, 1H), 2.37 - 2.46 (m, 2H), 2.50 - 2.57 (m, 1H), 2.78 (s, 1H), 3.96 (t, 1H), 6.10 (t, 1H), 6.25 (dd, 1H), 7.04 (d, 1H).
3,16beta-dihydroxyestra-1 (10),2,4-trien-17-one
Media of the preculture: tryptone (16 g/L), sodium chloride (10 g/L) and yeast extract (10 g/L) in demineralized water. The pH value was maintained at pH 7.3. The medium was sterilized at 121° C. for 20 minutes. Afterwards kanamycin (50 mg/L) was added.
Cell cultivation 1: One preculture (100 mL) was inoculated with the strain E. coli BL21 Star™ (DE3) pETM11-BM3-268; M177Y, A184Y, S72G, V178P, L181Y, L188F (50 µL) containing the desired plasmids and was shaken at 37° C. and 165 rpm for 17 hours. This preculture (100 mL) was used to inoculate one 10 L steel fermenter. The cultivation media was prepared in the fermenter. Tryptone (12 g/L), yeast extract (24 g/L), predigested beef extract (2 g/L), KH2PO4 (2.2 g/L), K2HPO4 (9.4 g/L) and glycerol (87%, 4.6 g/L) were dissolved in demineralized water (9.2 L) and sterilized for 20 minutes at 121° C. in the fermenter. Afterwards kanamycin (0.5 g) in water (20 mL), riboflavin (10 mg) in water (20 mL), thiamine hydrochloride (3.37 g) in water (10 mL) and Oxford trace metal solution (2.5 mL) were added. After the inoculation of the fermenter, the culture was stirred at 315 rpm at 37° C. with an aeration rate of 3.3 L/min at pH 6.6 which was regulated by addition of aqueous sodium hydroxide solution (16%) or aqueous phosphoric acid solution (16%). After 4 hours, an OD550 of 0.7 was reached, the temperature was decreased to 27° C. within 10 minutes and IPTG (1.43 g) in water (40 mL) were added to start the protein expression. After 9.50 hours the aqueous phosphoric acid solution (16%) was substituted for an aqueous glucose solution (50%) for pH regulation. After additional 24 hours an OD550 of 19.6 was reached, the cells were harvested by centrifugation (194.4 g), suspended in buffer (194.4 mL; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 1 × 150 mL, 1 × 100 mL, 2 × 40 mL und 5 × 1 mL.
Cell cultivation 2: One preculture (100 mL) was inoculated with the strain E. coli BL21 Star™ (DE3) pETM11-BM3-268; M177Y, A184Y, S72G, V178P, L181Y, L188F (50 µL) containing the desired plasmids and was shaken at 37° C. and 165 rpm for 17 hours. This preculture (100 mL) was used to inoculate one 10 L steel fermenter. The cultivation media was prepared in the fermenter. Tryptone (12 g/L), yeast extract (24 g/L), predigested beef extract (2 g/L), KH2PO4 (2.2 g/L), K2HPO4 (9.4 g/L) and glycerol (87%, 4.6 g/L) were dissolved in demineralized water (9.2 L) and sterilized for 20 minutes at 121° C. in the fermenter. Afterwards kanamycin (0.5 g) in water (20 mL), riboflavin (10 mg) in water (20 mL), thiamine hydrochloride (3.37 g) in water (10 mL) and Oxford trace metal solution (2.5 mL) were added. After the inoculation of the fermenter, the culture was stirred at 315 rpm at 37° C. with an aeration rate of 3.3 L/min at pH 6.6 which was regulated by addition of aqueous sodium hydroxide solution (16%) or aqueous phosphoric acid solution (16%). After 3.55 hours, an OD550 of 0.69 was reached, the temperature was decreased to 27° C. within 10 minutes and IPTG (1.43 g) in water (40 mL) were added to start the protein expression. After 9 hours the aqueous phosphoric acid solution (16%) was substituted for an aqueous glucose solution (50%) for pH regulation. After additional 24 hours an OD550 of 27.75 was reached, the cells were harvested by centrifugation (231 g), suspended in buffer (231 mL; KH2PO4 (4 g/L), K2HPO4 (12.3 g/L), glycerol (4% (v/v)), glucose (5% (w/v)), EDTA (0.5 mM)), frozen in liquid nitrogen and stored at -80° C. until further use in aliquots of 2 × 150 mL, 1 × 50 mL, 2 × 40 mL und 5 × 1 mL.
To a 10 L steel fermenter which contained KH2PO4 (40 g), K2HPO4 (123 g), Pluronic® PE 8100 (1 mL), demineralized water (9.0 L), aqueous glucose solution (50 %, 200 mL) and aqueous EDTA solution (0.5 M, 10 mL) androsta-1,4-diene-3,17-dione (500 mg, 1.75 mmol) dissolved in DMF (10 mL) was added and the mixture was stirred at 315 rpm at 27° C. with an aeration rate of 3.3 L/min. Cells in buffer (330 mL of cell cultivation 1 and 150 ml of cell cultivation 2) were added, and the biotransformation was maintained at an oxygen partial pressure of 50% by varying the stirring rate to up to 1200 rpm. The pH value was maintained at pH 7.4 by addition of an aqueous solution of sodium hydroxide (16%). After 4 hours the biotransformation was extracted with 4-methyl-2-pentanone. The organic layer was concentrated to give an oil (5.65 g) which was dissolved in methanol, and was concentrated to give an oil (3.73 g).
The crude product was further purified by flash chromatography using silica gel (dichloromethane/ethanol gradient) and by preparative HPLC to give the title compound (54 mg).
Instrument: Waters Autopurificationsystem; Column: XBrigde C18 5 µ, 100×30 mm; eluent A: water + 0.1 vol % formic acid; eluent B: acetonitrile; gradient: 0.0-0.5 min 23 % B (35-70 mL/min), 0.5-5.5 min 23-58% B; flow: 70 mL/min; temperature: 25° C.; DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µ, 50×2.1 mm; eluent A: water + 0.1 vol % formic acid; eluent B: acetonitrile; gradient: 0-2.6 min 1-99% B, 2.6-3.0 min 99% B; flow: 0.8 mL/min; temperature: 60° C.; DAD scan: 210-400 nm.
Analytical HPLC: Rt = 0.84 min.
1H-NMR (600 MHz, Pyridin-d5) δ [ppm]: 1.04 (s, 3H), 1.28 - 1.41 (m, 2H), 1.45 - 1.62 (m, 3H), 1.75 (ddd, 1H), 1.86 - 1.93 (m, 1H), 1.98 - 2.08 (m, 1H), 2.21 - 2.27 (m, 1H), 2.28 - 2.36 (m, 1H), 2.45 - 2.53 (m, 1H), 2.80 - 2.92 (m, 2H), 4.30 (t, 1H), 7.03 (d, 1H), 7.11 (dd, 1H), 7.28 (d, 1H), 7.91 (s, 1H), 11.24 (s, 1H).
1H-NMR (600 MHz, Pyridin-d5) δ [ppm]: -0.002 (1.01), 0.000 (2.61), 0.965 (0.35), 1.038 (16.00), 1.292 (0.33), 1.303 (0.42), 1.313 (0.94), 1.323 (1.00), 1.333 (1.12), 1.344 (1.18), 1.353 (0.90), 1.355 (0.90), 1.365 (1.11), 1.370 (1.07), 1.374 (1.06), 1.378 (0.87), 1.388 (0.84), 1.396 (0.76), 1.461 (0.25), 1.467 (0.28), 1.483 (0.83), 1.489 (0.69), 1.504 (1.07), 1.508 (1.19), 1.524 (2.72), 1.539 (1.21), 1.545 (1.23), 1.553 (0.73), 1.557 (0.76), 1.561 (0.70), 1.571 (1.25), 1.574 (1.22), 1.590 (1.08), 1.593 (1.04), 1.608 (0.40), 1.612 (0.36), 1.722 (0.73), 1.737 (0.96), 1.741 (1.08), 1.744 (0.91), 1.757 (1.08), 1.759 (0.97), 1.764 (0.84), 1.779 (0.67), 1.875 (0.85), 1.881 (0.87), 1.886 (0.98), 1.890 (0.83), 1.896 (0.93), 1.901 (0.84), 1.907 (0.81), 2.007 (0.29), 2.026 (1.65), 2.031 (1.03), 2.042 (1.15), 2.046 (1.18), 2.051 (0.70), 2.224 (0.72), 2.241 (1.12), 2.253 (0.50), 2.260 (0.54), 2.302 (1.04), 2.313 (0.84), 2.317 (0.78), 2.324 (1.11), 2.329 (1.01), 2.474 (0.74), 2.482 (0.89), 2.487 (0.99), 2.494 (1.19), 2.502 (0.94), 2.506 (0.82), 2.515 (0.68), 2.813 (0.44), 2.823 (0.52), 2.841 (1.32), 2.849 (1.37), 2.858 (0.95), 2.869 (0.89), 2.879 (0.83), 2.888 (0.90), 2.907 (0.31), 2.917 (0.27), 3.615 (0.54), 4.287 (0.83), 4.301 (1.48), 4.314 (0.78), 5.038 (1.80), 7.025 (2.83), 7.029 (2.71), 7.099 (1.47), 7.103 (1.36), 7.113 (1.74), 7.117 (1.46), 7.212 (11.96), 7.270 (2.43), 7.284 (2.07), 7.580 (5.57), 7.908 (0.88), 8.729 (10.46), 11.240 (0.77).
1 alpha,2alpha-1,2-epoxyandrost-4-ene-3,17-dione
For the preparation of the title compound see Example 25. The crude product was further purified by flash chromatography using silica gel (dichloromethane/ethanol gradient) and by preparative HPLC to give the title compound (5.8 mg).
Instrument: Waters Autopurificationsystem; Column: XBrigde C18 5 µ, 100×30 mm; eluent A: water + 0.1 vol % formic acid; eluent B: acetonitrile; gradient: 0.0-0.5 min 23% B (35-70 mL/min), 0.5-5.5 min 23-58% B; flow: 70 mL/min; temperature: 25° C.; DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µ, 50×2.1 mm; eluent A: water + 0.1 vol % formic acid; eluent B: acetonitrile; gradient: 0-2.6 min 1-99% B, 2.6-3.0 min 99 % B; flow: 0.8 mL/min; temperature: 60° C.; DAD scan: 210-400 nm.
Analytical HPLC: Rt = 1.06 min.
1H-NMR (600 MHz, CHLOROFORM-d) δ [ppm]: 0.95 (s, 3H), 1.13 (qd, 1H), 1.23 - 1.36 (m, 4H), 1.42 (s, 3H), 1.54 - 1.68 (m, 2H), 1.84 - 1.94 (m, 2H), 1.88 - 1.98 (m, 1H), 2.05 - 2.15 (m, 2H), 2.22 - 2.31 (m, 1H), 2.36 - 2.43 (m, 1H), 2.48 (dd, 1H), 3.40 (dd, 1H), 3.67 (d, 1H), 5.76 (s, 1H).
1H-NMR (600 MHz, CHLOROFORM-d) δ [ppm]: 0.000 (1.78), 0.880 (0.44), 0.935 (0.39), 0.949 (14.78), 0.988 (1.17), 1.093 (0.36), 1.100 (0.42), 1.114 (0.85), 1.121 (0.85), 1.136 (0.89), 1.143 (0.90), 1.157 (0.45), 1.164 (0.41), 1.238 (0.73), 1.244 (0.81), 1.255 (2.21), 1.279 (1.39), 1.288 (1.42), 1.303 (1.60), 1.309 (1.87), 1.318 (0.94), 1.327 (1.16), 1.419 (16.00), 1.545 (0.66), 1.560 (1.16), 1.565 (1.06), 1.576 (0.92), 1.581 (1.72), 1.587 (1.04), 1.596 (1.54), 1.602 (1.28), 1.610 (1.26), 1.617 (1.71), 1.633 (1.26), 1.638 (1.20), 1.654 (0.67), 1.662 (0.58), 1.849 (0.39), 1.856 (0.43), 1.868 (1.11), 1.875 (1.61), 1.880 (1.14), 1.887 (1.23), 1.895 (1.53), 1.900 (2.16), 1.918 (0.88), 1.925 (1.35), 1.936 (0.62), 1.940 (0.70), 1.950 (0.73), 1.956 (0.56), 1.961 (0.61), 1.971 (0.54), 2.013 (0.39), 2.072 (0.90), 2.086 (1.84), 2.093 (0.67), 2.103 (1.67), 2.119 (1.48), 2.134 (0.65), 2.245 (0.61), 2.252 (0.74), 2.257 (0.60), 2.267 (0.85), 2.271 (0.99), 2.278 (0.76), 2.291 (0.41), 2.371 (0.67), 2.380 (0.75), 2.393 (1.15), 2.402 (1.07), 2.414 (0.57), 2.423 (0.47), 2.454 (1.10), 2.468 (1.06), 2.486 (0.96), 2.501 (0.92), 2.625 (0.45), 3.393 (1.58), 3.396 (1.81), 3.398 (1.89), 3.402 (1.65), 3.662 (2.62), 3.668 (2.55), 5.756 (2.73), 7.264 (14.93).
19-hydroxy-1 alpha,2alpha-1 ,2-epoxyandrost-4-ene-3,17-dione
For the preparation of the title compound see Example 25. The crude product was further purified by flash chromatography using silica gel (dichloromethane/ethanol gradient) and twice by preparative HPLC to give the title compound (3.7 mg).
Instrument: Waters Autopurificationsystem; Column: XBrigde C18 5 µ, 100×30 mm; eluent A: water + 0.1 vol % formic acid; eluent B: acetonitrile; gradient: 0.0-0.5 min 23% B (35-70 mL/min), 0.5-5.5 min 23-58% B; flow: 70 mL/min; temperature: 25° C.; DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µ, 50×2.1 mm; eluent A: water + 0.1 vol % formic acid; eluent B: acetonitrile; gradient: 0-2.6 min 1-99% B, 2.6-3.0 min 99% B; flow: 0.8 mL/min; temperature: 60° C.; DAD scan: 210-400 nm.
Instrument: Waters Autopurificationsystem; Column: XBrigde C18 5 µ, 100×30 mm; eluent A: water + 0.1 vol % formic acid; eluent B: acetonitrile; gradient: 0.0-0.5 min 20 % B (35-70 mL/min), 0.5-5.5 min 20-30 % B; flow: 70 mL/min; temperature: 25° C.; DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µ, 50×2.1 mm; eluent A: water + 0.1 vol % formic acid; eluent B: acetonitrile; gradient: 0-2.6 min 1-99% B, 2.6-3.0 min 99% B; flow: 0.8 mL/min; temperature: 60° C.; DAD scan: 210-400 nm.
Analytical HPLC: Rt = 0.82 min.
1H-NMR (600 MHz, CHLOROFORM-d) δ [ppm]: 0.95 (s, 3H), 1.12 - 1.21 (m, 1H), 1.23 - 1.37 (m, 3H), 1.54 - 1.65 (m, 2H), 1.84 - 2.01 (m, 4H), 2.06 - 2.18 (m, 2H), 2.33 - 2.41 (m, 2H), 2.43 - 2.52 (m, 2H), 3.38 (dd, 1H), 3.95 (d, 1H), 4.16 (t, 1H), 4.29 (d, 1H), 5.92 (s, 1H).
1H-NMR (600 MHz, CDCl3) δ [ppm]: 0.881 (0.18), 0.904 (0.57), 0.927 (0.28), 0.948 (16.00), 1.136 (0.33), 1.143 (0.36), 1.157 (0.87), 1.164 (0.89), 1.179 (0.96), 1.186 (0.96), 1.200 (0.48), 1.208 (0.57), 1.240 (0.68), 1.247 (0.73), 1.263 (1.38), 1.269 (1.30), 1.287 (1.12), 1.291 (0.93), 1.297 (0.82), 1.308 (0.99), 1.315 (0.97), 1.318 (0.96), 1.326 (1.21), 1.331 (0.88), 1.336 (0.89), 1.347 (1.18), 1.364 (0.77), 1.369 (0.76), 1.414 (0.40), 1.545 (0.55), 1.552 (0.60), 1.568 (1.36), 1.574 (1.53), 1.590 (2.54), 1.597 (2.84), 1.606 (4.35), 1.621 (2.17), 1.627 (1.63), 1.642 (0.73), 1.881 (0.80), 1.887 (1.28), 1.893 (0.88), 1.904 (0.80), 1.909 (1.20), 1.915 (0.87), 1.926 (0.50), 1.937 (1.32), 1.945 (1.51), 1.952 (1.50), 1.958 (2.52), 1.963 (2.30), 1.970 (1.32), 1.981 (1.80), 2.076 (0.82), 2.090 (1.33), 2.108 (1.19), 2.115 (0.59), 2.123 (2.28), 2.138 (1.12), 2.143 (0.92), 2.148 (0.85), 2.367 (1.13), 2.373 (1.49), 2.388 (1.21), 2.394 (1.41), 2.399 (0.97), 2.435 (0.78), 2.443 (0.82), 2.458 (1.29), 2.464 (2.12), 2.478 (1.61), 2.487 (0.59), 2.495 (1.14), 2.510 (1.01), 2.623 (0.54), 3.377 (1.81), 3.380 (2.08), 3.383 (2.14), 3.387 (1.91), 3.942 (2.83), 3.949 (2.78), 4.146 (0.55), 4.163 (1.05), 4.179 (0.71), 4.278 (2.45), 4.297 (1.82), 5.920 (3.16), 7.266 (11.03).
3,15beta-dihydroxyestra-1 (10),2,4-trien-17-one
For the preparation of the title compound see Example 25. The crude product was further purified by flash chromatography using silica gel (dichloromethane/ethanol gradient) and by preparative HPLC to give the title compound (0.8 mg).
Instrument: Waters Autopurificationsystem; Column: XBrigde C18 5 µ, 100x30 mm; eluent A: water + 0.1 vol % formic acid; eluent B: acetonitrile; gradient: 0.0-0.5 min 25% B (35-70 mL/min), 0.5-5.5 min 25-35% B; flow: 70 mL/min; temperature: 25° C.; DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 µ, 50x2.1 mm; eluent A: water + 0.1 vol % formic acid; eluent B: acetonitrile; gradient: 0-2.6 min 1-99% B, 2.6-3.0 min 99% B; flow: 0.8 mL/min; temperature: 60° C.; DAD scan: 210-400 nm.
Analytical HPLC: Rt = 0.85 min.
1H-NMR (600 MHz, Pyr) δ [ppm]: 0.000 (1.30), 1.267 (0.19), 1.452 (0.59), 1.458 (0.76), 1.469 (0.83), 1.477 (0.91), 1.492 (4.48), 1.530 (0.23), 1.551 (0.43), 1.574 (0.61), 1.596 (0.42), 1.618 (0.31), 2.018 (0.47), 2.037 (0.49), 2.137 (0.40), 2.155 (0.41), 2.172 (0.16), 2.301 (0.26), 2.318 (0.42), 2.336 (0.26), 2.352 (0.45), 2.379 (0.51), 2.496 (0.25), 2.666 (0.30), 2.676 (0.32), 2.697 (0.48), 2.707 (0.47), 2.816 (0.89), 2.833 (0.26), 2.848 (0.98), 2.859 (0.55), 2.879 (0.32), 2.887 (0.31), 2.898 (0.31), 4.782 (0.56), 4.989 (1.05), 6.502 (0.65), 7.002 (1.01), 7.106 (0.53), 7.118 (0.60), 7.211 (16.00), 7.313 (0.74), 7.327 (0.66), 7.578 (7.55), 8.731 (12.51), 11.201 (0.27).
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| Number | Date | Country | Kind |
|---|---|---|---|
| 20155122.3 | Feb 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/052295 | 2/1/2021 | WO |
