U.S. patent application number 13/375979 was filed with the patent office on 2012-03-22 for process for the enzymatic reduction of enoates.
This patent application is currently assigned to BASF SE. Invention is credited to Melanie Bonnekesse, Kurt Faber, Bemhard Hauer, Steffen Maurer, Clemens Stuckler.
Application Number | 20120070867 13/375979 |
Document ID | / |
Family ID | 43298230 |
Filed Date | 2012-03-22 |
United States Patent
Application |
20120070867 |
Kind Code |
A1 |
Maurer; Steffen ; et
al. |
March 22, 2012 |
PROCESS FOR THE ENZYMATIC REDUCTION OF ENOATES
Abstract
A process for the enzymatic reduction of an enoate (1) wherein
the C.dbd.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.dbd.H
or optionally substituted C.sub.1-C.sub.6-alkyl radical, d.
R.sup.1, R.sup.2 and R.sup.3 are independently of one another H,
--O-C.sub.1-C.sub.6-alkyl , --O--W with W=a hydroxyl protecting
group, C.sub.1-C.sub.6-alkyl, which can be substituted,
C.sub.2-C.sub.6-alkenyl, carboxyl, or an optionally substituted
carbo- or heterocyclic, aromatic or nonaromatic radical, or one of
R.sup.1, R.sup.2 and R.sup.3 is a --OH radical, or R.sup.1 is
linked to R.sup.3 so as to become part of a 4-8-membered cycle, or
R.sup.1 is linked to R so as to become part of a 4-8-membered
cycle, with the proviso that R.sup.1, R.sup.2 and R.sup.3 may not
be identical.
Inventors: |
Maurer; Steffen; (Dirmstein,
DE) ; Hauer; Bemhard; (Fussgonheim, DE) ;
Bonnekesse; Melanie; (Mannheim, DE) ; Faber;
Kurt; (Graz, AT) ; Stuckler; Clemens; (Graz,
AT) |
Assignee: |
BASF SE
Ludwigshafen
DE
|
Family ID: |
43298230 |
Appl. No.: |
13/375979 |
Filed: |
May 31, 2010 |
PCT Filed: |
May 31, 2010 |
PCT NO: |
PCT/EP2010/057511 |
371 Date: |
December 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61227794 |
Jul 23, 2009 |
|
|
|
Current U.S.
Class: |
435/121 ;
435/142; 435/146; 435/147; 435/148; 435/149 |
Current CPC
Class: |
C12N 9/001 20130101;
C12P 7/40 20130101; C12Y 103/01031 20130101; C12P 7/26 20130101;
C12P 7/62 20130101; C12P 7/22 20130101; C12P 7/46 20130101; C12P
7/42 20130101; C12P 7/24 20130101; C12P 17/10 20130101 |
Class at
Publication: |
435/121 ;
435/147; 435/148; 435/149; 435/142; 435/146 |
International
Class: |
C12P 17/10 20060101
C12P017/10; C12P 7/42 20060101 C12P007/42; C12P 7/38 20060101
C12P007/38; C12P 7/44 20060101 C12P007/44; C12P 7/24 20060101
C12P007/24; C12P 7/26 20060101 C12P007/26 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 4, 2009 |
EP |
09161888.4 |
Jul 17, 2009 |
EP |
09165744.5 |
Claims
1. A process for the enzymatic reduction of an enoate (1), wherein
the C.dbd.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.dbd.H or
optionally substituted C.sub.1-C.sub.6-alkyl radical, R.sup.1,
R.sup.2 and R.sup.3 are independently of one another H,
--O-C.sub.1-C.sub.6-alkyl, --O--W with W=a hydroxyl protecting
group, C.sub.1-C.sub.6-alkyl, which can be substituted,
C.sub.2-C.sub.6-alkenyl, carboxyl, or an optionally substituted
carbo- or heterocyclic, aromatic or nonaromatic radical, or one of
R.sup.1, R.sup.2 and R.sup.3 is a --OH radical, or R.sup.1 is
linked to R.sup.3 so as to become part of a 4-8-membered cycle, or
R.sup.1 is linked to R so as to become part of a 4-8-membered
cycle, with the proviso that R.sup.1, R.sup.2 and R.sup.3 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.dbd.C bond of
the enoate (1) is enantioselectively or diastereoselectively
hydrogenated.
7. The process according to claim 2, wherein the C.dbd.C bond of
the enoate (1) is enantioselectively or diastereoselectively
hydrogenated.
8. The process according to claim 3, wherein the C.dbd.C bond of
the enoate (1) is enantioselectively or diastereoselectively
hydrogenated.
9. The process according to claim 4, wherein the C.dbd.C bond of
the enoate (1) is enantioselectively or diastereoselectively
hydrogenated.
10. The process according to claim 5, wherein the C.dbd.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.dbd.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.dbd.H or optionally substituted C.sub.1-C.sub.6-alkyl
radical,
[0006] R.sup.1, R.sup.2 and R.sup.3 are independently of one
another H, --O-C.sub.1-C.sub.6-alkyl , --O--W, with W=a hydroxyl
protecting group, C.sub.1-C.sub.6-alkyl which can be substituted,
C.sub.2-C.sub.6-alkenyl, carboxyl, or an optionally substituted
carbo- or heterocyclic, aromatic or nonaromatic radical, or R.sup.1
is linked to R.sup.3 so as to become part of a 4-8-membered cycle,
or R.sup.1 is linked to R so as to become part of a 4-8-membered
cycle, with the proviso that R.sup.1, R.sup.2 and R.sup.3 may not
be identical. Preferably, the C.dbd.C bond of the enoate (1) is
enantioselectively or diastereoselectively hydrogenated.
[0007] One of the rests R.sup.1, R.sup.2 and R.sup.3 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.
R.sup.1=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-C.sub.1-C.sub.6-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-C.sub.1-C.sub.4-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] C.sub.1-C.sub.6-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
C.sub.1-C.sub.4-alkyl radicals; [0015] C.sub.1-C.sub.6-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 C.sub.1-C.sub.6-alkyls
with preference being given in particular to CH.sub.2OH and to
CH.sub.2O--W. [0016] C.sub.2-C.sub.6-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 C.sub.2-C.sub.4-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,
--NO.sub.2, --NH.sub.3, --SO.sub.3H, C.sub.1-C.sub.4-alkyl and
C.sub.2-C.sub.4-alkenyl, C.sub.1-C.sub.4-alkoxy; and
hydroxy-C.sub.1-C.sub.4-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 R.sup.1 and R.sup.3 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 R.sup.1 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
R.sup.1 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.degree. C. It is particularly preferably in
the range from 0 to 100.degree. C., in particular from 15 to
60.degree. C. and specifically from 20 to 40.degree. C., e.g. at
about 30.degree. 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.dbd.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 .mu.g/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.degree. C. and 120 rpm for 24 h and the
products were extracted with EtOAc (2.times.0.5 mL). The combined
organic phases were dried (Na.sub.2SO.sub.4) 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 .mu.m), detector
temperature 250.degree. C., split ratio 30:1; temperature program:
80.degree. C.: hold 2 min.; rise to 120.degree. C. with 5.degree.
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 .sup.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 FCC248.sup.b 45
FCC249.sup.c 13 .sup.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]; .sup.bFCC249 = E.
coli expressing native estrogen-binding protein [ix]; .sup.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.dbd.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)].sup.a 18% <1 <1 OYE2 16
[80 (R)].sup.a 9% <1 <1 YqjM 38 [91 (R)].sup.a 39% <1
<1 .sup.aEnantiomeric excess [%] and absolute configuration.
[0051] Coupled-substrate C.dbd.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 .mu.g/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 .degree. C. and 120 rpm for 24 h and products were
extracted with EtOAc (2.times.0.5 mL). The combined organic phases
were dried (Na.sub.2SO.sub.4) 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 .mu.m), detector
temperature 250.degree. C., split ratio 30:1. Temperature program:
110.degree. C., hold 5 min, rise to 200.degree. C. with 10.degree.
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 portion.sup.a Enzyme 1 2 3 OYE1 11% 19% 27%
OYE2 6% 13% 19% YqjM 24% 48% 65% .sup.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.dbd.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 amount.sup.a 1:1
1:1.5 1:2 1 x 2 x 3 x 3a [%] 17 20 20 12 20 25 .sup.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.dbd.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
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
* * * * *