Patent application title: PROCESS FOR THE ENZYMATIC REDUCTION OF ENOATES
Inventors:
Steffen Maurer (Dirmstein, DE)
Bemhard Hauer (Fussgonheim, DE)
Melanie Bonnekesse (Mannheim, DE)
Kurt Faber (Graz, AT)
Kurt Faber (Graz, AT)
Clemens Stückler (Graz, AT)
Assignees:
BASF SE
IPC8 Class: AC12P1710FI
USPC Class:
435121
Class name: Micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition preparing heterocyclic carbon compound having only o, n, s, se, or te as ring hetero atoms nitrogen as only ring hetero atom
Publication date: 2012-03-22
Patent application number: 20120070867
Abstract:
A process for the enzymatic reduction of an enoate (1) wherein the
C═C bond of the enoate (1) is stereoselectively hydrogenated in the
presence of an enoate-reductase and an oxidizable co-substrate (2) in a
system which is free of NAD(P)H,
##STR00001##
a.
b. in which
c. A is a ketone radical (--CRO), an aldehyde radical (--CHO), a carboxyl
radical (--COOR), with R═H or optionally substituted
C1-C6-alkyl radical,
d. R1, R2 and R3 are independently of one another H,
--O-C1-C6-alkyl , --O--W with W=a hydroxyl protecting group,
C1-C6-alkyl, which can be substituted, C2-C6-alkenyl,
carboxyl, or an optionally substituted carbo- or heterocyclic, aromatic
or nonaromatic radical, or one of R1, R2 and R3 is a --OH
radical, or R1 is linked to R3 so as to become part of a
4-8-membered cycle, or R1 is linked to R so as to become part of a
4-8-membered cycle, with the proviso that R1, R2 and R3
may not be identical.Claims:
1. A process for the enzymatic reduction of an enoate (1), wherein the
C═C bond of the enoate (1) is stereoselectively hydrogenated in the
presence of an enoate-reductase and an oxidizable co-substrate (2) in a
system which is free of NAD(P)H, ##STR00015## in which A is a ketone
radical (--CRO), an aldehyde radical (--CHO), a carboxyl radical
(--COOR), with R═H or optionally substituted C1-C6-alkyl
radical, R1, R2 and R3 are independently of one another H,
--O-C1-C6-alkyl, --O--W with W=a hydroxyl protecting group,
C1-C6-alkyl, which can be substituted, C2-C6-alkenyl,
carboxyl, or an optionally substituted carbo- or heterocyclic, aromatic
or nonaromatic radical, or one of R1, R2 and R3 is a --OH
radical, or R1 is linked to R3 so as to become part of a
4-8-membered cycle, or R1 is linked to R so as to become part of a
4-8-membered cycle, with the proviso that R1, R2 and R3
may not be identical.
2. The process according to claim 1, wherein the enoate reductase is selected from a reductase (i) comprising at least one of the polypeptide sequences SEQ ID NO:1, 2, 3, 4 or (ii) with a functionally equivalent polypeptide sequence which has at least 80% sequence identity with SEQ ID NO:1, 2, 3 or 4.
3. The process according to claim 1, wherein the enoate (1) has the general formula SEQ ID NO: 1, 2, 3 or 4.
4. The process according to claim 1, wherein the co-substrate (2) is identical with the enoate (1).
5. The process according to claim 1, wherein a molar ratio of enoate (1) to co-substrate (2) is from 1:1 to 1:3.
6. The process according to claim 1, wherein the C═C bond of the enoate (1) is enantioselectively or diastereoselectively hydrogenated.
7. The process according to claim 2, wherein the C═C bond of the enoate (1) is enantioselectively or diastereoselectively hydrogenated.
8. The process according to claim 3, wherein the C═C bond of the enoate (1) is enantioselectively or diastereoselectively hydrogenated.
9. The process according to claim 4, wherein the C═C bond of the enoate (1) is enantioselectively or diastereoselectively hydrogenated.
10. The process according to claim 5, wherein the C═C bond of the enoate (1) is enantioselectively or diastereoselectively hydrogenated.
Description:
[0001] The present invention relates to a novel process for the enzymatic
reduction of enoates.
[0002] The disproportionation of conjugated enones, such as cyclohex-2-enone, has been described as minor catalytic activity for several flavoproteins exhibiting enoate reductase-activity. In the context of these studies, this phenomenon has been generally considered as a side reaction, rather than as a useful transformation. Overall, this reaction constitutes a flavin-dependent hydrogen-transfer, during which an equivalent of [2H] is formally transferred from one enone molecule (being oxidised) onto another one (being reduced). In case of cyclohex-2-enone, this leads to the formation of an equimolar amount of cyclohexanone and cyclohex-2,5-dien-one. The latter spontaneously tautomerises to form phenol, going in hand with the generation of an aromatic system, which provides a large driving force (within a range of 30 kcal/M) for the reaction.
DETAILED DESCRIPTION OF THE INVENTION
[0003] The invention relates to a process for the enzymatic reduction of an enoate (1) wherein the C═C bond of the enoate (1) is stereoselectively hydrogenated in the presence of an enoat-reductase and an oxidizable co-substrate (2) in a system which is free of NAD(P)H,
##STR00002##
in which
[0004] A is a ketone radical (--CRO), an aldehyde radical (--CHO), a carboxyl radical (--COOR), with
[0005] R═H or optionally substituted C1-C6-alkyl radical,
[0006] R1, R2 and R3 are independently of one another H, --O-C1-C6-alkyl , --O--W, with W=a hydroxyl protecting group, C1-C6-alkyl which can be substituted, C2-C6-alkenyl, carboxyl, or an optionally substituted carbo- or heterocyclic, aromatic or nonaromatic radical, or R1 is linked to R3 so as to become part of a 4-8-membered cycle, or R1 is linked to R so as to become part of a 4-8-membered cycle, with the proviso that R1, R2 and R3 may not be identical. Preferably, the C═C bond of the enoate (1) is enantioselectively or diastereoselectively hydrogenated.
[0007] One of the rests R1, R2 and R3 may also be a --OH group; however in this case the formula (1) depicts the enol form which is in equilibrium with its keto form (formula 1a), i.e. R1=formyl (see above):
##STR00003##
[0008] A system which is free of NAD(P)H means that no external NAD.sup.+ and/or NADH and/or NADP.sup.- and/or NADPH is added to the system.
[0009] Preferred co-substrates (2) are enoates having a chemical structure which has been described for the enoates (1) above. In a much preferred embodiment the cosubstrate (2) has the identical chemical structure as the enoate (1) used for the specific reaction. In another preferred embodiment the cosubstrate (2) has not the identical chemical structure as the enoate (1) used for the specific reaction.
[0010] Another embodiment of the invention uses cosubstrates (2) which after having been oxidized during the reaction possess a conjugated, preferably an aromatic, electronic system.
[0011] Unless stated otherwise,
[0012] --O-C1-C6-alkyl means in particular --O-methyl, --O-ethyl, --O-propyl, --O-butyl, --O-pentyl or --O-hexyl and the corresponding singly or multiply branched analogs such as --O-isopropyl, --O-isobutyl, --O-sec-butyl, --O-tert-butyl, --O-isopentyl or --O-neopentyl; with preference being given in particular to the --O-C1-C4-alkyl radicals;
[0013] --O--W means a hydroxyl protecting group W which is bound to oxygen in particular such as --O-allyl, --O-benzyl, O-tetrahydropyranyl, --O-tert. Butyldimethylsilyl (TBDMS), --O-tert. Butyldiphenyl-silyl (TBDPS) [0014] C1-C6-alkyl means in particular methyl, ethyl, propyl, butyl, pentyl or hexyl and the corresponding singly or multiply branched analogs such as isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl or neopentyl; with preference being given in particular to the C1-C4-alkyl radicals; [0015] C1-C6-alkyl which can be substituted means in particular methyl, ethyl, propyl, butyl, pentyl or hexyl and the corresponding singly or multiply branched analogs such as isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl or neopentyl; where 1, 2 or 3 hydrogen atoms can be substituted by a group selected from F, Cl, Br, J, OH, O--W, SH,NH2. Preferred are single-substituted C1-C6-alkyls with preference being given in particular to CH2OH and to CH2O--W. [0016] C2-C6-alkenyl means in particular the monounsaturated analogs of the abovementioned alkyl radicals having from 2 to 6 carbon atoms, with preference being given in particular to the corresponding C2-C4-alkenyl radicals, [0017] carboxyl means in particular the group COOH, [0018] carbo- and heterocyclic aromatic or nonaromatic rings mean in particular optionally fused rings having from 3 to 12 carbon atoms and if appropriate from 1 to 4 heteroatoms such as N, S and O, in particular N or O. Examples which may be mentioned are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, the mono- or polyunsaturated analogs thereof such as cyclobutenyl, cyclopentenyl, cyclohexenlyl, cycloheptenyl, cyclohexadienyl, cycloheptadienyl; phenyl and naphthyl; and 5- to 7-membered saturated or unsaturated heterocyclic radicals having from 1 to 4 heteroatoms which are selected from O, N and S, where the heterocycle may optionally be fused to a further heterocycle or carbocycle. Mention should be made in particular of heterocyclic radicals derived from pyrrblidine, tetrahydrofuran, piperidine, morpholine, pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, thiazole, pyridine, pyran, pyrimidine, pyridazine, pyrazine, coumarone, indole and quinoline. The cyclic radicals, but also the abovementioned O-alkyl, alkyl and alkenyl radicals, may optionally be substituted one or more times, such as, for example, 1, 2 or 3 times. Mention should be made as examples of suitable substituents of: halogen, in particular F, Cl, Br; --OH, --SH, --NO2, --NH3, --SO3H, C1-C4-alkyl and C2-C4-alkenyl, C1-C4-alkoxy; and hydroxy-C1-C4-alkyl; where the alkyl and alkenyl radicals are as defined above, and the alkoxy radicals are derived from the above-defined corresponding alkyl radicals.
[0019] The radicals R1 and R3 may also be linked directly to one another so as to form together with the double bond to be reduced a 4-8-, preferably a 5- or 6-membered cycle, for example a cyclopentene or cyclohexene structure which may also be optionally substituted, for example by alkyl, preferably methyl radicals.
[0020] The radicals R1 and R may also be linked directly to one another so as to form together with the double bond to be reduced a 4-8-, preferably a 5- or 6-membered cycle, for example a cyclopentene or cyclohexene structure which may also be optionally substituted, for example by --O-alkyl or alkyl, preferably methoxy or methyl radicals.
[0021] The abovementioned 4-8-membered cycles may be both carbocycles, i.e. only carbon atoms form the cycle, and heterocycles, i.e. heteroatoms such as O; S; N, are present in the cycle. If desired, these carbo- or heterocycles may also still be substituted, i.e. hydrogen atoms are replaced with heteroatoms. For example, N-phenylsuccinimides (see substrate 3 below) are to be considered such substituted heterocycles which are the result of R1 and R forming a cycle.
[0022] Particularly advantageous embodiments of the invention comprise the enzymatic conversion of the following enoates (1) (substrates) to the corresponding hydrogenated compounds:
##STR00004##
[0023] Preferred enoate-reductases (1):
[0024] In addition, the reductases suitable for the method of the invention (which are occasionally also referred to as enoate reductases) have a polypeptide sequence as shown in SEQ ID NO:1, 2, 3, 4 or a polypeptide sequence which has at least 80% such as, for example, at least 90%, or at least 95% and in particular at least 97%, 98% or 99% sequence identity with SEQ ID NO: 1, 2, 3, 4.
[0025] A polypeptide having SEQ ID NO:1 is known under the name OYE1 from Saccharomyces carlsbergensis (Genbank Q02899).
[0026] A polypeptide having SEQ ID. NO:2 is encoded by the OYE2 gene from baker's yeast (Saccharomyces cerevisiae gene locus YHR179VV) (Genbank Q03558).
[0027] A polypeptide having SEQ ID NO:3 is encoded by the YqjM gene from Bacillus subtilis.
[0028] A polypeptide having SEQ ID NO:4 is encoded by the FCC248 gene from estrogen binding protein.
[0029] The sequence identity is to be ascertained for the purposes described herein by the "GAP" computer program of the Genetics Computer Group (GCG) of the University of Wisconsin, and the version 10.3 using the standard parameters recommended by GCG is to be employed.
[0030] Such reductases can be obtained starting from SEQ ID NO: 1, 2, 3, 4 by targeted or randomized mutagenesis methods known to the skilled worker. An alternative possibility is, however, also to search in microorganisms, preferably in those of the genera Alishewanella, Alterococcus, Aquamonas, Aranicola, Arsenophonus, Azotivirga, Brenneria, Buchnera (aphid Pendosymbionts), Budvicia, Buttiauxella, Candidatus Phlomobacter, Cedecea, Citrobacter, Dickeya, Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella, Gninontella, Hafnia, Klebsiella, Kluyvera, Leclercia, Lemihorella, Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium, Photorhabdus, Plesiomonas, Pragia, Proteus, Providencia, Rahnella, Raoultella, Salmonella, Samsonia, Serratia, Shigella, Sodalis, Tatumella, Trabulsiella, Wigglesworthia, Xenorhabdus, Yersinia or Yokenella, for reductases which catalyze the abovementioned model reaction and whose amino acid sequence already has the required sequence identity to SEQ ID NO: 1, 2, 3, 4 is obtained by mutagenesis methods.
[0031] The reductase can be used in purified or partly purified form or else in the form of the microorganism itself. Methods for obtaining and purifying dehydrogenases from microorganisms are well known to the skilled worker.
[0032] The reaction can be carried out in aqueous or nonaqueous reaction media or in 2-phase systems or (micro)emulsions. The aqueous reaction media are preferably buffered solutions which ordinarily have a pH of from 4 to 8, prefetably from 5 to 8. The aqueous solvent may, besides water, additionally comprise at least one alcohol, e.g. ethanol or isopropanol, or dimethyl sulfoxide.
[0033] Nonaqueous reaction media mean reaction media which comprise less than 1% by weight, preferably less than 0.5% by weight, of water based on the total weight of the liquid reaction medium. The reaction can in particular be carried out in an organic solvent.
[0034] Suitable organic solvents are for example aliphatic hydrocarbons, preferably having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane, halogenated aliphatic hydrocarbons, preferably having one or two carbon atoms, such as dichloromethane, chloroform, tetrachloromethane, dichloroethane or tetrachloroethane, aromatic hydrocarbons such as benzene, toluene, the xylenes, chlorobenzene or dichlorobenzene, aliphatic acyclic and cyclic ethers or alcohols, preferably having 4 to 8 carbon atoms, such as ethanol, isopropanol, diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran or esters such as ethyl acetate or n-butyl acetate or ketones such as methyl isobutyl ketone or dioxane or mixtures thereof. The aforementioned ethers, especially tetrahydrofuran, are particularly preferably used.
[0035] The reduction with reductase can for example be carried out in an aqueous organic reaction medium such as, for example, water/isopropanol in any mixing ratio such as, for example, 1:99 to 99:1 or 10: 90 to 90:10, or an aqueous reaction medium.
[0036] The substrate (1) is preferably employed in the enzymatic reduction in a concentration from 0.1 g/l to 500 g/l, particularly preferably from 1 g/l to 50 g/l, and can be fed in continuously or discontinuously.
[0037] The enzymatic reduction ordinarily takes place at a reaction temperature below the deactivation temperature of the reductase employed and above -10° C. It is particularly preferably in the range from 0 to 100° C., in particular from 15 to 60° C. and specifically from 20 to 40° C., e.g. at about 30° C.
[0038] A possible procedure for example is to mix the substrate (1) with the reductase and if appropriate the solvent thoroughly, e.g. by stirring or shaking: However, it is also possible to immobilize the reductase in a reactor, for example in a column, and to pass a mixture comprising the substrate through the reactor. For this purpose it is possible to circulate the mixture through the reactor until the desired conversion is reached.
[0039] During this reaction, the flavin-cofactor is recycled internally and no external cofactor, such as NADH or NADPH, which are commonly used to recycle reduced flavoproteins are required. In these classic nicotinamide-dependent systems, C═C-bonds are reduced at the expense of an external hydride donor, such as formate, glucose, glucose-6-phosphate or phosphite, which requires a second (dehydrogenase) enzyme, such as FDH, GDH, G-6-PDH [i] or phosphite-DH [ii], respectively. This technology is generally denoted as `coupled-enzyme-approach` and depends on the concurrent operation of two independent redox enzymes for substrate-reduction and co-substrate-oxidation, resp.
[0040] In order to avoid the use of a second nicotinamide-dependent redox enzyme, the disproportionation of enones can be envisaged to function via a more simple system, denoted as `coupled-substrate-approach`, which solely depends on a single flavoprotein. Thereby, the use of (i) an additional redox-enzyme and (ii) an additional redox-cofactor, such as NAD(P)H, can be omitted.
EXPERIMENTAL SECTION
[0041] During an initial screening, a set of cloned and overexpressed enoate reductases was tested for their catalytic activity in the disproportionation of cyclohex-2-enone. To our delight, the desired disproportionation activity was observed in a variety of OYE homologs, most prominent in YqjM, OYE1, OYE2 and estrogen-binding protein.
Example 1
[0042] General procedure for the screening for enzymatic disproportionation of cyclohex-2-enone An aliquot of the isolated enzyme OPR1, OPR3, YqjM, OYE1, OYE2, OYE3, Zymonas mobilis ER, NEM-Red, MOR-Red and PETN-Red (protein purity >90%, protein content 90-110 μg/mL) was added to a Tris-HCl buffer solution (0.8 mL, 50 mM, pH 7.5) containing cyclohex-2-enone (10 mM). The mixture was shaken at 30° C. and 120 rpm for 24 h and the products were extracted with EtOAc (2×0.5 mL). The combined organic phases were dried (Na2SO4) and the resulting samples were analyzed on achiral GC. Products were identified by comparison with authentic reference materials via co-injection on GC-MS and achiral GC. Column: 6% Cyanopropyl-phenyl phase capillary column (Varian CP-1301, 30 m, 0.25 mm, 0.25 μm), detector temperature 250° C., split ratio 30:1; temperature program: 80° C.: hold 2 min.; rise to 120° C. with 5° C./min. Retention times: cyclohex-2-enone 2.97 min, cyclohexanone 2.43 min, phenol 4.98 min.
TABLE-US-00001 ##STR00005## ##STR00006## ##STR00007## Enzyme a Conv. [%] OPR1 <1 OPR3 <1 YqjM 85 OYE1 92 OYE2 75 OYE3 7 Zym-mob ER 7 NEM-Red <1 MOR-Red <1 PETN-Red 0 FCC248b 45 FCC249c 13 a OPR1, OPR3 = oxophytodienoate reductase isoenzymes 1 and 3, resp., from tomato [iii]; YqjM = OYE-homolog from Bacillus subtilis [iv]; OYE1-3 = OYEs from yeasts [v]; Zym-mob ER = Zy-momonas mobilis enoate reductase [vi]; MOR-Red = morphinone reductase [vii]; NEM-Red = N-ethylmaleimide reductase; PETN-Red = pentaerythritol tetranitrate reductase [viii]; bFCC249 = E. coli expressing native estrogen-binding protein [ix]; cFCC248 = E. coli expressing synthetic estrogen-binding protein, both preparations were employed as crude cell-free extract [x].
[0043] Taking these relative activities as a lead, further experiments were performed using the three `champions`, YqjM, OYE1 and OYE2.
[0044] In order to turn the scrambling-like non-directed hydrogen-transfer reaction occurring between two identical cyclohexenone molecules into a useful directed redox process, where one substrate is dehydrogenated/oxidised, while another is hydrogenated/reduced, two suitable enone substrates--one only being oxidised, the other only being reduced--have to be coupled. During our previous studies on NAD(P)H-coupled enone reduction, we observed that alpha-substituted cyclic enones were quickly reduced, whereas alkyl-substituents in the beta-position severely impeded the reaction rate. Hence, we envisaged to couple an alpha- and a beta-substituted enone as substrates, being reduced and oxidised, resp.
[0045] In order to check the feasibility of this protocol, we investigated the disproportionation of 2-(1) and 3-methylcyclohex-2-enone (2); under identical conditions as for cyclohex-2-enone. With all
TABLE-US-00002 1a [%] 1b [%] 2a [%] 2b [%] Enzyme 24 h 72 h 24 h 72 h 24 h 72 h 24 h 72 h OYE1 10 12 6 9 4 9 6 14 OYE2 7 9 3 7 2 4 3 8 YqjM 7 7 3 5 0 0 18 30
of the enzymes tested, the relative rate of disproportionation for 1 was higher than those for 2, meaning that the C═C-bond of alpha-methylcyclohex-2-enone was faster reduced than its beta-substituted analog 2. This difference was most pronounced for YqjM.
##STR00008##
[0046] Encouraged by these results, we next attempted the coupled-substrate hydrogen-transfer between 2-(1) and 3-methylcyclohex-2-enone (2) as substrates to be reduced and oxidised, resp., in a directed fashion.
[0047] The results of these experiments provided a clear proof-of-principle:
[0048] (i) Depending on the enzyme, the desired reduced alpha-methyl derivative 1a was formed in up to 38% conversion, the oxidised beta-methyl analog 2b was detected in roughly equimolar amounts.
[0049] (ii) In contrast, only trace amounts of the corresponding cross-hydrogen-transfer products, which would be expected from undesired oxidation of 1 and reduction of 2 were found, indicating that the mono-directional hydrogen-transfer indeed worked as envisaged.
[0050] (iii) Investigation of the optical purity and absolute configuration of 1a revealed that the product was formed in the same selective fashion as in the classic reduction-mode using NAD(P)H-recycling, ensuring that the chiral induction process of the enzymes was unchanged [xi].
TABLE-US-00003 ##STR00009## ##STR00010## ##STR00011## Enzyme 1a [%] 2b [%] 1b [%] 2a [%] OYE1 27 [85 (R)]a 18% <1 <1 OYE2 16 [80 (R)]a 9% <1 <1 YqjM 38 [91 (R)]a 39% <1 <1 aEnantiomeric excess [%] and absolute configuration.
[0051] Coupled-substrate C═C-bond reduction of 2-methylcyclohex-2-enone (1) using 3-methylcyclohex-2-enone (2) as hydrogen donor.
[0052] An aliquot of the isolated enzyme YqjM, OYE1, OYE2, (protein purity >90%, protein content 90-110 μg/mL) was added to a Tris-HCl buffer solution (0.8 mL, 50 mM, pH 7.5) containing the substrate 1 (110 mM) and the co-substrate 2 (10 mM). The mixture was shaken at 30 ° C. and 120 rpm for 24 h and products were extracted with EtOAc (2×0.5 mL). The combined organic phases were dried (Na2SO4) and the resulting samples were analyzed on achiral GC. Products were identified by comparison with authentic reference materials via co-injection on GC-MS and achiral GC. Column: 14% cyanopropyl-phenyl phase capillary column (J&W Scientific DB-1701, 30 m, 0.25 mm, 0.25 μm), detector temperature 250° C., split ratio 30:1. Temperature program: 110° C., hold 5 min, rise to 200° C. with 10° C./min, hold 2 min. 2-Methylcyclohexenone (1) 4.38 min; 2-methylcyclohexanone (1a); 3.70 min; 3-methylcyclohexenone (2) 6.27 min; 3-methylphenol (2b) 7.90 min; 2-methylphenol (1b) 7.02 min; 3-methylcyclohexanone (2a) 3.63 min.
[0053] In order to drive the reduction of 1 further towards completion, increasing amounts of co-substrate 2 were employed (cf. scheme above). As can be deduced from the amounts of reduction product 1a formed, elevated co-substrate concentrations had little effect, which is presumably due to enzyme inhibition caused by elevated cosubstrate concentrations. This phenomenon is also common for the asymmetric bioreduction of carbonyl compounds catalysed by alcohol dehydrogenases using the coupled-substrate method.
TABLE-US-00004 Ratio of 1:2 Enzyme 1:1 1:1.5 1:2 OYE1 12 11 10 OYE2 8 7 5 YqjM 26 27 27
[0054] In order to verify this hypothesis, the reaction was performed with a 1:1 ratio of 1 and 2 using increasing amounts of enzyme, added at intervals of 24 h. In this case, the conversion could be significantly improved, which underscores the above mentioned co-substrate inhibition.
TABLE-US-00005 Enzyme portiona Enzyme 1 2 3 OYE1 11% 19% 27% OYE2 6% 13% 19% YqjM 24% 48% 65% aAmounts of reduction product 1a formed by addition of equal amounts of enzyme (100 mL each) at intervals of 24 h.
[0055] Monitoring the reaction over time showed that the process was mainly limited by the catalytic power of the enzyme employed. The conversion steadily increased, indicating that the enzyme remained catalytically active up to 72 h, which proved that the inhibition was largely reversible (FIG. 1).
[0056] FIG. 1 shows the time course of reduction of 1 using 2 as hydrogen-donor (cf. scheme p. 8).
[0057] Aiming to extend the applicability of nicotinamide-free C═C-bond reduction system, we subjected two further substrates (3, 4), which are known to be reduced by enoate reductases in combination with NAD(P)H-recycling, to the hydrogen-transfer protocol in presence of equimolar amounts of beta-methylcyclohex-2-enone (2) as hydrogen donor. In both cases, the reduction proceeded smoothly and furnished the corresponding (R)-configurated products 3a and 4a in the same enantiomeric composition as the nicotinamide-driven process. Among the enzymes tested. YqjM was clearly best.
TABLE-US-00006 ##STR00012## 3a 4a Enzyme [%] e.e. [%] [%] e.e. [%] OYE1 2 n.d. 4 n.d. OYE2 1 n.d. 3 n.d. YqjM 17 >99 (R) 22 76 (R) n.d. = not determined.
[0058] Since the use of equimolar amounts of 3-methylcyclohex-2-enone (2) as co-substrate would be economically disastrous, a cheaper alternative for a hydrogen donor was sought. After attempts using 1-indanone and hydroquinone failed, cyclohexane-1,4-dione (5)--yielding 1,4-dihydroxybenzene (hydroquinone, 5a)--as oxidation product was found to provide a suitable alternative. Substrate 3 showed even enhanced conversion as compared to betamethylcyclohex-2-enone (2) as co-substrate.
TABLE-US-00007 ##STR00013## ##STR00014## 3a Enzyme [%] e.e. [%] OYE1 3 n.d. OYE2 4 n.d. YqjM 20 >99 (R) n.d. = not determined.
[0059] Upon closer examination using YqjM, this reaction showed similar effects of reversible co-substrate inhibition, as indicated by the data below. In line with previous observations using 2 as hydrogen donor, the conversion gradually increased from 0 to 25% over a period of 7 days.
TABLE-US-00008 Conditions Ratio of 3:5 Enzyme amounta 1:1 1:1.5 1:2 1 x 2 x 3 x 3a [%] 17 20 20 12 20 25 aEqual amounts of enzyme (100 mL each) were added at intervals of 24 h.
[0060] Although the overall performance of this novel substrate-coupled C═C-bond reduction system has not yet reached the standard of nicotinamide-driven reactions, it has the following advantages compared to the following existing technologies:
[0061] (i) it depends only on a single flavoprotein and neither requires a second (dehydrogenase) recycling enzyme, nor a nicotinamide cofactor, and
[0062] (ii) it has clear advantages to competitive alternative systems, such as the light-driven FAD-recycling [xii] and the electrochemical reduction via a (transition)metal-dependent mediator [0063] i. H. Yamamoto, A. Matsuyama, in: Biocatalysis in the pharmaceutical and biotechnology industry: R. N. Patel, ed., CRC Press, Boca Raton, 2007, pp. 623-44; C. Wandrey, Chem. Rec. 2004, 4, 254-65: U. Kragl, D. Vasic-Racki, C. Wandrey, Indian J. Chem., Sect. B 1993, 32B, 103-117. [0064] ii. J. M. Vrtis, A. K. White, W. W. Metcalf, W. A. van der Donk, Angew. Chem. Int. Ed. 2002, 41, 3391-3; T. W. Johannes, R. D. Woodyer, H. Zhao, Biotechnol. Bioeng. 2006, 96, 18-26. [0065] iii. C. Breithaupt, J. Strassner, U. Breitinger, R. Huber, P. Macheroux, A. Schaller, T. Clausen, Structure 2001, 9, 419-29. [0066] iv. K. Kitzing, T. B. Fitzpatrick, C. Wilken, J. Sawa, G. P. Bourenkov, P. Macheroux, T. Clausen, J. Biol. Chem. 2005, 280, 27904-13. [0067] v. M. Hall. C. Stueckler, B. hauer, R. Stuermer, T. Friedrich, M. Breuer, W. Kroutil, K. Faber, Eur. J. Org. Chem. 2008, 1511-6. [0068] vi. A. Muller, B. Hauer, B. Rosche. Biotechnol. Bioeng. 2007, 98, 22-9. [0069] vii. F. Barna, H. L. Messiha, C. Petosa, N. C. Bruce, N. S. Scrutton, P. C. E. Moody, J. Biol. Chem. 2002, 277, 30976-83; H. L., Messiha, A. W. Munroe, N. C. Bruce, I. Barsukov, N. S. Scrutton. J. Biol. Chem. 2005, 280, 10695-709. [0070] viii. R. E. Williams, D. A. Rathbone, N. S. Scrutton, N. C. Bruce, Appl. Environ. Microbiol. 2004, 70, 3566-74. [0071] ix. J. Buckman, S. M. Miller, Biochemistry 1998, 37, 14326-36. [0072] x. Estrogen binding protein was cloned into E. coli by Nina Baudendistel at BASF AG. [0073] xi. M. Hall, C. Stueckler, H. Ehammer, E. Pointner, G. Oberdorfer, K. Gruber, B. Hauer, R. Stuermer, P. Macheroux, W. Kroutil, K. Faber, Adv. Synth. Catal. 2008, 350, 411-8; M. Hall, C. Stueckler, B. Hauer, R. Stuermer, T. Friedrich, M. Breuer, W. Kroutil, K. Faber, Eur. J. Org. Chem. 2008, 1511-6. [0074] xii. A. Taulieber, F. Schulz, F. Hollmann, M. Rusek. M. T. Reetz. ChemBioChem 2008, 9, 565-72; F. Hollmann, A. Taglieber, F. Schulz, M. T. Reetz, Angew. Chem. Int. Ed. 2007, 46, 2903-2906.
Sequence CWU
1
41399PRTsaccharomyces carlsbergensis 1Ser Phe Val Lys Asp Phe Lys Pro Gln
Ala Leu Gly Asp Thr Asn Leu1 5 10
15Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg Ala
Val 20 25 30Ile Pro Pro Leu
Thr Arg Met Arg Ala Leu His Pro Gly Asn Ile Pro 35
40 45Asn Arg Asp Trp Ala Val Glu Tyr Tyr Thr Gln Arg
Ala Gln Arg Pro 50 55 60Gly Thr Met
Ile Ile Thr Glu Gly Ala Phe Ile Ser Pro Gln Ala Gly65 70
75 80Gly Tyr Asp Asn Ala Pro Gly Val
Trp Ser Glu Glu Gln Met Val Glu 85 90
95Trp Thr Lys Ile Phe Asn Ala Ile His Glu Lys Lys Ser Phe
Val Trp 100 105 110Val Gln Leu
Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Asn Leu Ala 115
120 125Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp
Asn Val Phe Met Asp 130 135 140Ala Glu
Gln Glu Ala Lys Ala Lys Lys Ala Asn Asn Pro Gln His Ser145
150 155 160Leu Thr Lys Asp Glu Ile Lys
Gln Tyr Ile Lys Glu Tyr Val Gln Ala 165
170 175Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val
Glu Ile His Ser 180 185 190Ala
Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser Asn Thr 195
200 205Arg Thr Asp Glu Tyr Gly Gly Ser Ile
Glu Asn Arg Ala Arg Phe Thr 210 215
220Leu Glu Val Val Asp Ala Leu Val Glu Ala Ile Gly His Glu Lys Val225
230 235 240Gly Leu Arg Leu
Ser Pro Tyr Gly Val Phe Asn Ser Met Ser Gly Gly 245
250 255Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala
Tyr Val Ala Gly Glu Leu 260 265
270Glu Lys Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His Leu Val
275 280 285Glu Pro Arg Val Thr Asn Pro
Phe Leu Thr Glu Gly Glu Gly Glu Tyr 290 295
300Glu Gly Gly Ser Asn Asp Phe Val Tyr Ser Ile Trp Lys Gly Pro
Val305 310 315 320Ile Arg
Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg Glu Glu
325 330 335Val Lys Asp Lys Arg Thr Leu
Ile Gly Tyr Gly Arg Phe Phe Ile Ser 340 345
350Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu
Asn Lys 355 360 365Tyr Asp Arg Asp
Thr Phe Tyr Gln Met Ser Ala His Gly Tyr Ile Asp 370
375 380Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp
Asp Lys Lys385 390
3952399PRTSaccharomyces cerevisiae 2Pro Phe Val Lys Asp Phe Lys Pro Gln
Ala Leu Gly Asp Thr Asn Leu1 5 10
15Phe Lys Pro Ile Lys Ile Gly Asn Asn Glu Leu Leu His Arg Ala
Val 20 25 30Ile Pro Pro Leu
Thr Arg Met Arg Ala Gln His Pro Gly Asn Ile Pro 35
40 45Asn Arg Asp Trp Ala Val Glu Tyr Tyr Ala Gln Arg
Ala Gln Arg Pro 50 55 60Gly Thr Leu
Ile Ile Thr Glu Gly Thr Phe Pro Ser Pro Gln Ser Gly65 70
75 80Gly Tyr Asp Asn Ala Pro Gly Ile
Trp Ser Glu Glu Gln Ile Lys Glu 85 90
95Trp Thr Lys Ile Phe Lys Ala Ile His Glu Asn Lys Ser Phe
Ala Trp 100 105 110Val Gln Leu
Trp Val Leu Gly Trp Ala Ala Phe Pro Asp Thr Leu Ala 115
120 125Arg Asp Gly Leu Arg Tyr Asp Ser Ala Ser Asp
Asn Val Tyr Met Asn 130 135 140Ala Glu
Gln Glu Glu Lys Ala Lys Lys Ala Asn Asn Pro Gln His Ser145
150 155 160Ile Thr Lys Asp Glu Ile Lys
Gln Tyr Val Lys Glu Tyr Val Gln Ala 165
170 175Ala Lys Asn Ser Ile Ala Ala Gly Ala Asp Gly Val
Glu Ile His Ser 180 185 190Ala
Asn Gly Tyr Leu Leu Asn Gln Phe Leu Asp Pro His Ser Asn Asn 195
200 205Arg Thr Asp Glu Tyr Gly Gly Ser Ile
Glu Asn Arg Ala Arg Phe Thr 210 215
220Leu Glu Val Val Asp Ala Val Val Asp Ala Ile Gly Pro Glu Lys Val225
230 235 240Gly Leu Arg Leu
Ser Pro Tyr Gly Val Phe Asn Ser Met Ser Gly Gly 245
250 255Ala Glu Thr Gly Ile Val Ala Gln Tyr Ala
Tyr Val Leu Gly Glu Leu 260 265
270Glu Arg Arg Ala Lys Ala Gly Lys Arg Leu Ala Phe Val His Leu Val
275 280 285Glu Pro Arg Val Thr Asn Pro
Phe Leu Thr Glu Gly Glu Gly Glu Tyr 290 295
300Asn Gly Gly Ser Asn Lys Phe Ala Tyr Ser Ile Trp Lys Gly Pro
Ile305 310 315 320Ile Arg
Ala Gly Asn Phe Ala Leu His Pro Glu Val Val Arg Glu Glu
325 330 335Val Lys Asp Pro Arg Thr Leu
Ile Gly Tyr Gly Arg Phe Phe Ile Ser 340 345
350Asn Pro Asp Leu Val Asp Arg Leu Glu Lys Gly Leu Pro Leu
Asn Lys 355 360 365Tyr Asp Arg Asp
Thr Phe Tyr Lys Met Ser Ala Glu Gly Tyr Ile Asp 370
375 380Tyr Pro Thr Tyr Glu Glu Ala Leu Lys Leu Gly Trp
Asp Lys Asn385 390 3953338PRTBacillus
subtilis 3Met Ala Arg Lys Leu Phe Thr Pro Ile Thr Ile Lys Asp Met Thr
Leu1 5 10 15Lys Asn Arg
Ile Val Met Ser Pro Met Cys Met Tyr Ser Ser His Glu 20
25 30Lys Asp Gly Lys Leu Thr Pro Phe His Met
Ala His Tyr Ile Ser Arg 35 40
45Ala Ile Gly Gln Val Gly Leu Ile Ile Val Glu Ala Ser Ala Val Asn 50
55 60Pro Gln Gly Arg Ile Thr Asp Gln Asp
Leu Gly Ile Trp Ser Asp Glu65 70 75
80His Ile Glu Gly Phe Ala Lys Leu Thr Glu Gln Val Lys Glu
Gln Gly 85 90 95Ser Lys
Ile Gly Ile Gln Leu Ala His Ala Gly Arg Lys Ala Glu Leu 100
105 110Glu Gly Asp Ile Phe Ala Pro Ser Ala
Ile Ala Phe Asp Glu Gln Ser 115 120
125Ala Thr Pro Val Glu Met Ser Ala Glu Lys Val Lys Glu Thr Val Gln
130 135 140Glu Phe Lys Gln Ala Ala Ala
Arg Ala Lys Glu Ala Gly Phe Asp Val145 150
155 160Ile Glu Ile His Ala Ala His Gly Tyr Leu Ile His
Glu Phe Leu Ser 165 170
175Pro Leu Ser Asn His Arg Thr Asp Glu Tyr Gly Gly Ser Pro Glu Asn
180 185 190Arg Tyr Arg Phe Leu Arg
Glu Ile Ile Asp Glu Val Lys Gln Val Trp 195 200
205Asp Gly Pro Leu Phe Val Arg Val Ser Ala Ser Asp Tyr Thr
Asp Lys 210 215 220Gly Leu Asp Ile Ala
Asp His Ile Gly Phe Ala Lys Trp Met Lys Glu225 230
235 240Gln Gly Val Asp Leu Ile Asp Cys Ser Ser
Gly Ala Leu Val His Ala 245 250
255Asp Ile Asn Val Phe Pro Gly Tyr Gln Val Ser Phe Ala Glu Lys Ile
260 265 270Arg Glu Gln Ala Asp
Met Ala Thr Gly Ala Val Gly Met Ile Thr Asp 275
280 285Gly Ser Met Ala Glu Glu Ile Leu Gln Asn Gly Arg
Ala Asp Leu Ile 290 295 300Phe Ile Gly
Arg Glu Leu Leu Arg Asp Pro Phe Phe Ala Arg Thr Ala305
310 315 320Ala Lys Gln Leu Asn Thr Glu
Ile Pro Ala Pro Val Gln Tyr Glu Arg 325
330 335Gly Trp4408PRTArtificial SequenceEstrogen binding
protein 4Met Thr Ile Glu Ser Thr Asn Ser Phe Val Val Pro Ser Asp Thr Lys1
5 10 15Leu Ile Asp Val
Thr Pro Leu Gly Ser Thr Lys Leu Phe Gln Pro Ile 20
25 30Lys Val Gly Asn Asn Val Leu Pro Gln Arg Ile
Ala Tyr Val Pro Thr 35 40 45Thr
Arg Phe Arg Ala Ser Lys Asp His Ile Pro Ser Asp Leu Gln Leu 50
55 60Asn Tyr Tyr Asn Ala Arg Ser Gln Tyr Pro
Gly Thr Leu Ile Ile Thr65 70 75
80Glu Ala Thr Phe Ala Ser Glu Arg Gly Gly Ile Asp Leu His Val
Pro 85 90 95Gly Ile Tyr
Asn Asp Ala Gln Ala Lys Ser Trp Lys Lys Ile Asn Glu 100
105 110Ala Ile His Gly Asn Gly Ser Phe Ser Ser
Val Gln Leu Trp Tyr Leu 115 120
125Gly Arg Val Ala Asn Ala Lys Asp Leu Lys Asp Ser Gly Leu Pro Leu 130
135 140Ile Ala Pro Ser Ala Val Tyr Trp
Asp Glu Asn Ser Glu Lys Leu Ala145 150
155 160Lys Glu Ala Gly Asn Glu Leu Arg Ala Leu Thr Glu
Glu Glu Ile Asp 165 170
175His Ile Val Glu Val Glu Tyr Pro Asn Ala Ala Lys His Ala Leu Glu
180 185 190Ala Gly Phe Asp Tyr Val
Glu Ile His Gly Ala His Gly Tyr Leu Leu 195 200
205Asp Gln Phe Leu Asn Leu Ala Ser Asn Lys Arg Thr Asp Lys
Tyr Gly 210 215 220Cys Gly Ser Ile Glu
Asn Arg Ala Arg Leu Leu Leu Arg Val Val Asp225 230
235 240Lys Leu Ile Glu Val Val Gly Ala Asn Arg
Leu Ala Leu Arg Leu Ser 245 250
255Pro Trp Ala Ser Phe Gln Gly Met Glu Ile Glu Gly Glu Glu Ile His
260 265 270Ser Tyr Ile Leu Gln
Gln Leu Gln Gln Arg Ala Asp Asn Gly Gln Gln 275
280 285Leu Ala Tyr Ile Ser Leu Val Glu Pro Arg Val Thr
Gly Ile Tyr Asp 290 295 300Val Ser Leu
Lys Asp Gln Gln Gly Arg Ser Asn Glu Phe Ala Tyr Lys305
310 315 320Ile Trp Lys Gly Asn Phe Ile
Arg Ala Gly Asn Tyr Thr Tyr Asp Ala 325
330 335Trp Pro Glu Phe Lys Thr Leu Ile Asn Asp Leu Lys
Asn Asp Arg Ser 340 345 350Ile
Ile Gly Phe Ser Arg Phe Phe Thr Ser Asn Pro Asp Leu Val Glu 355
360 365Lys Leu Lys Leu Gly Lys Pro Leu Asn
Tyr Tyr Asn Arg Glu Glu Phe 370 375
380Tyr Lys Tyr Tyr Asn Tyr Gly Tyr Asn Ser Tyr Asp Glu Ser Glu Lys385
390 395 400Gln Val Ile Gly
Lys Pro Leu Ala 405
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