HK1213882B

HK1213882B - Process and intermediates for the preparation of pregabalin

Info

Publication number: HK1213882B
Application number: HK16101742.1A
Authority: HK
Inventors: S．德巴尔热; D．T．厄德曼; P．M．奥尼尔; R.库玛; M．J．卡尔米洛维兹
Original assignee: 辉瑞爱尔兰制药公司
Priority date: 2013-03-27
Filing date: 2014-03-25
Publication date: 2018-06-15

Description

Process and intermediates for the preparation of pregabalin

Technical Field

The present invention relates to the preparation of 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone and derivatives thereof for the preparation of (S) - (+) -3-aminomethyl-5-methylhexanoic acid (pregabalin). The present invention also relates to an improved process for the conversion of 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone to pregabalin₂Gamma-amino acids of the binding affinity of the calcium channel subunits.

Background

Pregabalin, or (S) - (+) -3-aminomethyl-5-methyl-hexanoic acid ((S) -II),

is thatIt exhibits anti-seizure activity (as discussed in U.S. Pat. No. 5,563,175) and anti-nociceptive activity (as discussed in U.S. Pat. No.6,001,876). it is hypothesized that the pharmacological activity of pregabalin (II) consists of α -2- (α) binding to calcium channels₂) Caused by a subunit. Pregabalin (II) is also described as having utility under other conditions, for example, physiological conditions associated with psychomotor stimulants, inflammation, gastrointestinal damage, alcoholism, insomnia, and various psychiatric disorders including anxiety, depression, mania, and bipolar disorder.

Many processes for preparing pregabalin have been disclosed. 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone (I)^A) Have been identified as a useful precursor.

It will be appreciated that Compound (I)^A) Like many of the other compounds discussed herein, can be considered cyclic isomers of 4-oxobutanoic acid derivatives. The derivative of 4-oxobutanoic acid can exist in an open chain form or a cyclic form.

These two isomeric forms may coexist in equilibrium and the relative contribution of these two forms will depend on the exact chemistry of the species.

International patent application PCT/US2008/004699 (published as WO2008/127646A2) proposes the use of chemical or enzyme mediated reductive amination to convert 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone (I)^A) Or a ring-opening isomeric form of an ester (XIII, wherein R^AAlkyl) to (II). Suggesting that the use of a transaminase will selectively provide the preferred ((S) -II) enantiomer.

The ester (XIII) is prepared from 4-methylpentanal (III) by alkylation with the appropriate haloacetate in the presence of diisobutylamine. Precursor (I)^A) By ester hydrolysis of the ester (XIII) or by condensation of 4-methylpentanal (III) with glyoxylic acid (IV) and subsequent reduction of the double bond. The use of enzyme-mediated reduction has also been suggested as a means of introducing the desired stereochemistry.

International patent application PCT/IN2010/000140 (published as WO2011/086565) discloses a related method. The condensation product of 4-methylpentanal (III) and glyoxylic acid (IV) is reacted with a chiral amine, for example α -methylbenzylamine, to give the pyrrolone (V). Hydrogenation gives a degree of stereoselectivity and deprotection gives the chiral form of pregabalin (II).

Still further improved syntheses of pregabalin (II) are sought. It is particularly desirable to provide a cost effective and safe method. In particular, it is important to provide a pregabalin (II) synthesis: it can be carried out on a commercial scale, it uses readily available, cheap and safe starting materials and reagents, and it avoids the need for difficult separations.

Summary of The Invention

The invention provides a method for preparing 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone (I)^A) And intermediates useful in these improved processes.

In a first aspect a1, the invention provides a compound according to formula (VI)

In a first embodiment A1E1, the invention provides a compound according to formula (VI), wherein R is selected from:

the presence of hydrogen in the presence of hydrogen,

(C₁-C₆) An alkyl group, a carboxyl group,

(C₁-C₆) A halogenated alkyl group,

(C₁-C₃) Alkoxy (C)₂-C₆) An alkyl group, a carboxyl group,

(C₂-C₆) Alkenyl radical，

(C₃-C₁₀) Cycloalkyl, which may optionally be independently selected from halogen, (C)₁-C₃) Alkyl and (C)₁-C₃)1, 2 or 3 groups of alkoxy,

(C₃-C₁₀) Cycloalkyl (C)₁-C₆) Alkyl, wherein cycloalkyl may optionally be independently selected from halogen, (C)₁-C₃) Alkyl and (C)₁-C₃)1, 2 or 3 groups of alkoxy,

aryl, which may optionally be independently selected from halogen, (C)₁-C₃) Alkyl and (C)₁-C₃)1, 2 or 3 groups of alkoxy,

aryl radical (C)₁-C₆) Alkyl, wherein aryl may optionally be independently selected from halogen, (C)₁-C₃) Alkyl and (C)₁-C₃)1, 2 or 3 groups of alkoxy,

R¹-C (O) -, and

R²-SO₂-；

R¹selected from:

the presence of hydrogen in the presence of hydrogen,

(C₁-C₆) An alkyl group, a carboxyl group,

(C₁-C₆) A halogenated alkyl group,

(C₁-C₃) Alkoxy (C)₂-C₆) An alkyl group, a carboxyl group,

(C₂-C₆) An alkenyl group, which is a radical of an alkenyl group,

(C₃-C₁₀) Cycloalkyl (C)₁-C₆) Alkyl, wherein said cycloalkyl may optionally be independently selected from halogen, (C)₁-C₃) Alkyl and (C)₁-C₃)1, 2 or 3 groups of alkoxy,

aryl, which may optionally be independently selected from halogen, (C)₁-C₃) Alkyl and (C)₁-C₃)1, 2 or 3 radical substitutions of alkoxy, and

and R is²Selected from:

(C₁-C₆) An alkyl group, a carboxyl group,

(C₁-C₆) A halogenated alkyl group,

(C₁-C₃) Alkoxy (C)₂-C₆) An alkyl group, a carboxyl group,

(C₂-C₆) An alkenyl group, which is a radical of an alkenyl group,

aryl radical (C)₁-C₆) Alkyl, wherein aryl may optionally be independently selected from halogen, (C)₁-C₃) Alkyl and (C)₁-C₃)1, 2 or 3 groups of alkoxy.

In a further embodiment A1E2, the invention provides a compound according to embodiment A1E1, wherein R is hydrogen, such that the compound of formula (VI) is according to formula (VI)^A) 5-hydroxy-4- (2-methyl-1-propenyl) -5H-2-furanone.

In another embodiment A1E3, the invention provides a compound of formula (VI)^B) According to embodiment A1E1

Wherein R is chiral (C)₅-C₁₅) A hydrocarbyl group.

In a further embodiment A1E4, the invention provides a compound according to embodiment A1E3, wherein R is selected from (R) -or (S) - α -methylbenzyl, (R) -or (S) -1- (1-naphthyl) ethyl, (R) -or (S) -1- (2-naphthyl) ethyl, menthyl and bornyl, such that the compound of formula (VI) is of formula (VI)^C)-(VI^K) The compound of (1).

In a further embodiment A1E5, the invention provides a compound according to embodiment A1E1, wherein R is R¹-C (O) -or R²-SO₂-andR¹and R²Is chiral (C)₅-C₁₅) A hydrocarbyl group.

In another aspect a2, the invention provides a compound of formula (IX)

The compounds of the formula (IX) may be present as (E) -or (Z) -geometric isomers or as a mixture of both geometric isomers.

In a first embodiment A2E1, the invention provides a compound of formula (IX), wherein:

n is 1 and M⁺Selected from Li⁺、Na⁺、K⁺、Rb⁺、NH₄ ⁺、((C₁-C₃) Alkyl) NH₃ ⁺、((C₁-C₃) Alkyl radical)₂NH₂ ⁺、((C₁-C₃) Alkyl radical)₃NH⁺And ((C)₁-C₃) Alkyl radical)₄N⁺(ii) a Or

n is 2 and M²⁺Selected from Mg²⁺、Ca²⁺And Zn²⁺。

In a further embodiment A2E2, the invention provides a compound according to embodiment A2E1, wherein n is 1 and M⁺Is selected from NH₄ ⁺And ((C)₁-C₃) Alkyl) NH₃ ⁺。

In a further embodiment A2E3, the invention provides a compound according to embodiment A2E1, wherein n is 1 and M⁺Selected from Li⁺、Na⁺And K⁺。

In another aspect a3, the invention provides a compound of formula (VII).

In a first embodiment A3E1, the invention provides a compound of formula (VII) wherein-X-represents a single bond, -CH₂-、-O-、-NH-、-N((C₁-C₃) Alkyl) -, -N (benzyl) -, or

In a further embodiment A3E2, the invention provides a compound according to embodiment A3E1 selected from:

4- (2-methylpropenyl) -5-pyrrolidin-1-yl-5H-furan-2-one;

4- (2-methylpropenyl) -5-piperidin-1-yl-5H-furan-2-one;

4- (2-methylpropenyl) -5-morpholin-4-yl-5H-furan-2-one; and

1, 4-bis- (4- (2-methylpropenyl) -5H-furan-2-one-5-yl) piperazine.

In another aspect a4, the invention provides a compound of formula (VIII).

In a first embodiment A4E1, the invention provides a compound of formula (VIII) wherein-Y-represents a single bond, -CH₂-、-O-、-NH-、-N((C₁-C₃) Alkyl) -, -N (benzyl) -, or

In a further embodiment A4E2, the invention provides a compound according to embodiment A4E1 selected from:

4- (4-methyl-1, 3-pentadien-1-yl) morpholine;

1- (4-methyl-1, 3-pentadien-1-yl) -piperazine,

1- (4-methyl-1, 3-pentadien-1-yl) -4-methylpiperazine,

4-ethyl-1- (4-methyl-1, 3-pentadien-1-yl) -piperazine,

4-benzyl-1- (4-methyl-1, 3-pentadien-1-yl) -piperazine, and

1, 4-bis- (4-methyl-1, 3-pentadien-1-yl) piperazine.

In another aspect A5, the invention provides a compound of formula (VI)^A) A method for producing the compound of (1), which comprises the step of treating the compound of the formula (VII) with water in the presence of an acid catalyst.

In a first embodiment A5E1, the invention provides a compound of formula (VI)^A) A process for the preparation of a compound of (a), comprising the steps of:

(a) preparation of the Compound of formula (VII)

wherein-X-represents a single bond, -CH₂-、-O-、-NH-、-N((C₁-C₃) Alkyl) -, -N (benzyl) -, or

And

(b) treating the compound of formula (VII) with water in the presence of an acid catalyst.

In a further embodiment A5E2, the invention provides a process according to embodiment A5E1, wherein the compound of formula (VII) from step (a) is isolated prior to the hydrolysis step (b).

In a further embodiment A5E3, the instant invention provides a process according to embodiment A5E1, wherein the hydrolysis step (b) is performed directly after step (a) such that formula (VII) or (VII) is not isolated prior to the hydrolysis step (b)^B) The compound of (1).

In a further embodiment A5E4, the invention provides a process according to embodiment A5E1, A5E2 or A5E3 wherein the compound of formula (VII) is prepared by treating the compound of formula (VIII) with glyoxylic acid or a hydrate thereof

wherein-Y-represents a single bond, -CH₂-、-O-、-NH-、-N((C₁-C₃) Alkyl) -, -N (benzyl) -, or

In another aspect A6, the invention provides a process for the preparation of a compound of formula (VI) wherein R is other than hydrogen, R¹-C (O) -and R²-SO₂-, which process comprises treating the compound of formula (VI) with an alcohol R-OH in the presence of an acid catalyst^A) The step of (1).

In an embodiment A6E1, the invention provides a process for the preparation of a compound of formula (VI) wherein R is other than hydrogen, R¹-C (O) -and R²-SO₂-, said method comprising the steps of:

(a) preparation of formula (VI) using a method according to any one of embodiments A5E1, A5E2, A5E3 and A5E4 as defined above^A) A compound of (1); and

(b) treatment of formula (VI) with an alcohol R-OH in the presence of an acid catalyst^A) The compound of (1).

In another aspect a7, in which,the present invention provides a process for the preparation of a compound of formula (VI) wherein R is other than hydrogen, R¹-C (O) -and R²-SO₂The process comprises the step of treating the compound of formula (VII) with an alcohol R-OH in the presence of a stoichiometric amount of acid.

In an embodiment A7E1, the invention provides a process for the preparation of a compound of formula (VI) wherein R is other than hydrogen, R¹-C (O) -and R²-SO₂-, said method comprising the steps of:

(a) preparing a compound of formula (VII); and

(b) treating a compound of formula (VII) with an alcohol R-OH in the presence of a stoichiometric amount of an acid.

In another aspect A8, the invention provides a process for the preparation of a compound of formula (VI) wherein R is R¹-C (O) -, said process comprising the use of an acid chloride R¹-C (O) -Cl or anhydride (R)¹-C(O))₂O treatment formula (VI)^A) The step of (1).

In an embodiment A8E1, the invention provides a process for the preparation of a compound of formula (VI) wherein R is R¹-c (o) -, said method comprising the steps of:

(b) with acid chlorides R¹-C (O) -Cl or anhydride (R)¹-C(O))₂O treatment formula (VI)^A) The compound of (1).

In another aspect A9, the invention provides a process for the preparation of a compound of formula (VI) wherein R is R²-SO₂-, said process comprising the use of a sulfonyl chloride R²-SO₂-Cl-treated formula (VI)^A) The step of (1).

In an embodiment A9E1, the invention provides a process for preparing a compound of formula (VI) wherein R is R²-SO₂-, the processThe method comprises the following steps:

(b) with sulphonyl chlorides R²-SO₂-Cl-treated formula (VI)^A) The compound of (1).

In another aspect a10, the invention provides a process for the preparation of an enamine derivative of 4-methyl-2-pentenal.

In a first embodiment a10E1, the invention provides a process for the preparation of an enamine derivative of 4-methyl-2-pentenal which comprises reacting acetaldehyde with isobutyraldehyde in the presence of a suitable amine.

In another embodiment a10E2, the invention provides a method according to embodiment a10E1, wherein the suitable amine is a secondary amine.

In other embodiments a10E3, the invention provides a method according to embodiment a10E2, wherein the secondary amine is selected from: ((C)₁-C₄) Alkyl radical)₂NH, pyrrolidine, piperidine, morpholine, piperazine, N-methylpiperazine, N-ethylpiperazine and N-benzylpiperazine.

In a further embodiment a10E4, the present invention provides a process according to embodiment a10E3, wherein the secondary amine is selected from pyrrolidine, piperidine, morpholine, and piperazine.

In a further embodiment a10E5, the invention provides a process according to any one of embodiments a10E1, a10E2, a10E3 and a10E4, wherein the reaction is carried out in the presence of an acid catalyst.

In a further embodiment a10E6, the instant invention provides a process according to any one of embodiments a10E1, a10E2, a10E3, a10E4, and a10E5, wherein isobutyraldehyde is combined with the appropriate amine prior to addition of acetaldehyde.

In a further embodiment a10E6, the instant invention provides a process according to any one of embodiments a10E1, a10E2, a10E3, a10E4, and a10E5, wherein isobutyraldehyde and acetaldehyde are added simultaneously to the reaction vessel.

In another aspect A11, the invention provides a process for the preparation of 3-aminomethyl-5-methylhexanoic acid (II).

In a first embodiment a11E1, the present invention provides a process for preparing 3-aminomethyl-5-methylhexanoic acid (II), or a pharmaceutically acceptable salt thereof, comprising the steps of:

(a) preparation of 5-hydroxy-4- (2-methyl-1-propenyl) -5H-2-furanone (VI)^A)

(b) Reacting said 5-hydroxy-4- (2-methyl-1-propenyl) -5H-2-furanone (VI)^A) Conversion to 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone (I)^A)

And

(c) reacting the 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone (I)^A) Converted into 3-aminomethyl-5-methylhexanoic acid (II).

In a further embodiment a11E2, the invention provides a method according to embodiment a11E1, wherein the 3-aminomethyl-5-methylhexanoic acid (II) is (S) -3-aminomethyl-5-methylhexanoic acid ((S) -II)

Wherein the (S) -3-aminomethyl-5-methylhexanoic acid has an enantiomeric excess of at least 80%.

In a further embodiment a11E3, the invention provides a method according to embodiment a11E1 or a11E2, wherein step (a) comprises a method according to embodiment A5E1, A5E2, A5E3 or A5E4 as described above.

In a further embodiment a11E4, the invention provides a method according to embodiment a11E1, a11E2 or a11E3, wherein step (b) comprises the steps of:

(b1) with metal oxides, hydroxides, carbonates or bicarbonates, ammonia, mono-, di-or tri- (C)₁-C₃) Alkylamine, or tetra- (C)₁-C₃) Treatment of 5-hydroxy-4- (2-methyl-1-propenyl) -5H-2-furanone (VI) with alkylammonium hydroxide^A) To form a salt of formula (IX)

Wherein:

n is 2 and M²⁺Selected from Mg²⁺、Ca²⁺And Zn²⁺；

(b2) Hydrogenating the salt of formula (IX) to obtain a salt of formula (X)

(b3) Treating the salt of formula (X) with an acid.

In a further embodiment a11E5, the invention provides a method according to embodiment a11E1, a11E2 or a11E3, wherein step (b) comprises the steps of:

(b1) mixing 5-hydroxy-4- (2-methyl-1-propenyl) -5H-2-furanone (VI)^A) Into a compound of formula (VI) as defined in embodiment A1E3, wherein R is chiral (C)₅-C₁₅) A hydrocarbyl group;

(b2) hydrogenating the compound of formula (VI) to obtain a compound of formula (XI)

Wherein R is chiral (C)₅-C₁₅) A hydrocarbyl group; and

(b3) treating a compound of formula (XI) with an acid to produce ((S) -I)^A)。

In another aspect A12, the invention provides other methods for the preparation of 3-aminomethyl-5-methylhexanoic acid (II).

In a first embodiment a12E1, the present invention provides a process for preparing 3-aminomethyl-5-methylhexanoic acid (II), or a pharmaceutically acceptable salt thereof, comprising the steps of:

(a) preparation of 5-hydroxy-4- (2-methyl-1-propenyl) -5H-2-furanone (VI)^A)

(b) With ammonia or mono- (C)₁-C₃) Alkylamine treatment of 5-hydroxy-4- (2-methyl-1-propenyl) -5H-2-furanone (VI)^A) To form formula (IX)^A) Salt of (meth) acrylic acid

Wherein n is 1 and M⁺Is selected from NH₄ ⁺And ((C)₁-C₃) Alkyl) NH₃ ⁺

(c) Hydrogenated of formula (IX)^A) Salt to obtain formula (X)^A) Salt of (meth) acrylic acid

(d) Treatment of formula (X) with transaminase or amine oxidase/imine reductase^A) Salt to provide 3-aminomethyl-5-methylhexanoic acid (II).

In a further embodiment a12E2, the invention provides a method according to embodiment a12E1, wherein the 3-aminomethyl-5-methylhexanoic acid (II) is (S) -3-aminomethyl-5-methylhexanoic acid ((S) -II)

In a further embodiment a12E3, the invention provides a method according to embodiment a12E1 or a12E2, wherein step (a) comprises a method according to embodiment A5E1, A5E2, A5E3 or A5E4 as described above.

In another aspect, A13, the invention provides 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone (I)^A) Other methods of preparation of (1).

In a first embodiment A13E1, the invention provides 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone (I)^A) The method of (3), comprising the steps of:

(a) obtaining 3-isobutylidene-2-oxoglutaric acid (XII)^A) Or a cyclized isomer thereof (XII)^B)

(b) Reducing the carbon-carbon double bond and decarboxylating the alpha-keto acid functional group, either sequentially or simultaneously.

In a further embodiment a13E2, the invention provides a method according to embodiment a13E1, wherein the carbon-carbon double bond is reduced prior to decarboxylation of the α -keto acid functional group to provide 3-isobutylidene-2-oxoglutaric acid (XV) or its cyclized isomer (XV)^A)

In a further embodiment a13E3, the invention provides a process according to embodiment a13E1, wherein the α -keto acid functional group is decarboxylated prior to the reduction of the carbon-carbon double bond to provide 3-formyl-5-methyl-3-pentenoic acid (XVI) or its cyclized isomer (XVI)^A)

In a further embodiment a13E4, the invention provides a method according to embodiment a13E1, wherein the α -keto acid functional group is decarboxylated and the carbon-carbon double bond is reduced simultaneously.

In a further embodiment a13E5, the invention provides a method according to embodiment a13E1, a13E2, a13E3 or a13E4, wherein the decarboxylation reaction is carried out in the presence of a decarboxylase.

In a further embodiment a13E6, the invention provides a process according to embodiment a13E1, a13E2, a13E3, a13E4 or a13E5, wherein the reduction of the carbon-carbon double bond is carried out in the presence of an enone reductase (enoate reductase).

In another aspect a14, the invention provides a compound selected from:

3-isobutylidene-2-oxoglutaric acid;

3-isobutyl-2-oxoglutaric acid; and

3-formyl-5-methyl-3-pentenoic acid,

or a salt thereof, (C)₁-C₆) Alkyl esters or cyclic isomers.

In another aspect A15, the present invention provides a process for preparing (S) -3-aminomethyl-5-methylhexanoic acid ((S) -II) or a pharmaceutically acceptable salt thereof

The method comprises the following steps:

(a) preparation of 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone (I) using a method according to any one of embodiments a13E1, a13E2, a13E3, a13E4, a13E5 or a13E6 as defined above^A) And are and

(b) converting the 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone to (S) -3-aminomethyl-5-methylhexanoic acid.

In another aspect a16, the invention provides a method of converting (R) -3-aminomethyl-5-methylhexanoic acid to (S) -3-aminomethyl-5-methylhexanoic acid comprising treating (R) -3-aminomethyl-5-methylhexanoic acid with a transaminase or an amine oxidase/imine reductase.

In another aspect a17, the invention provides a method for increasing the proportion of (S) -3-aminomethyl-5-methylhexanoic acid in a mixture of (R) -and (S) -3-aminomethyl-5-methylhexanoic acid comprising treating the mixture with a transaminase or an amine oxidase/imine reductase.

In another aspect A18, the present invention provides a process useful for the preparation of 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone (I)^A) A transaminase converted to pregabalin.

In a first embodiment a18E1, the invention provides a transaminase having an amino acid sequence which is at least 95% homologous to the following amino acid sequence

MNKPQSWEARAETYSLYGFTDMPSLHX²⁷RGTVVVTHGEGPYX⁴¹VDVX⁴⁵GRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRX¹⁴⁷NAYHGVTAVSASMTGX¹⁶³PX¹⁶⁵NSVFGLPLPGFVHLX¹⁸⁰CPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELX³⁰⁴KRLETAIEAIEEFPHGFTAX³²⁴GHPVGCAIALKAIDWMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSX⁴⁰¹RIANTCX⁴⁰⁸DLGLICX⁴¹⁵X⁴¹⁶X⁴¹⁷GQSVILX⁴²⁴PPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ ID NO.1)

Wherein

x²⁷Selected from glutamine (Q) and glutamic acid (E);

x⁴¹selected from isoleucine (I) and valine (V);

x⁴⁵selected from asparagine (N) and histidine (H);

x¹⁴⁷selected from asparagine (N) and glutamine (Q);

x¹⁶³selected from leucine (L) and methionine (M);

x¹⁶⁵selected from tyrosine (Y) and histidine (H);

x¹⁸⁰selected from threonine (T); glycine (G) and serine (S);

x³⁰⁴selected from alanine (A) and serine (S);

x³²⁴selected from glycine (G) and serine (S);

x⁴⁰¹selected from lysine (K) and glutamic acid (E);

x⁴⁰⁸selected from threonine (T) and glutamine (Q);

x⁴¹⁵selected from serine (S) and alanine (a);

x⁴¹⁶selected from proline (P) and alanine (A);

x⁴¹⁷selected from leucine (L) and methionine (M); and

x⁴²⁴selected from cysteine (C) and serine (S).

In a further embodiment a18E2, the invention provides a transaminase having the amino acid sequence of SEQ id No.1 according to embodiment a18E 1.

In a further embodiment a18E3, the invention provides a transaminase according to embodiment a18E1 or a18E2, wherein:

X²⁷is glutamic acid (E);

X¹⁴⁷is glutamine (Q);

X¹⁶⁵is histidine (H);

X³⁰⁴is serine (S);

X³²⁴is glycine (G);

X⁴⁰¹is lysine (K);

X⁴⁰⁸is glutamine (Q);

X⁴¹⁶is alanine (a);

X⁴¹⁷is methionine (M); and

X⁴²⁴is serine (S).

In a further embodiment a18E4, the invention provides a transaminase according to embodiment a18E2 with an amino acid sequence selected from the group consisting of:

MNKPQSWEARAETYSLYGFTDMPSLHQRGTVVVTHGEGPYIVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAVSASMTGLPYNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELAKRLETAIEAIEEFPHGFTASGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSERIANTCTDLGLICSPMGQSVILCPPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ IDNO.2)；

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYIVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAVSASMTGLPYNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSALGQSVILCPPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ IDNO.3)；

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYVVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSALGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ IDNO.4)；

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYIVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGMPHNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPALSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ IDNO.5)；

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYVVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGLPLPGFVHLGCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICAAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 6); and

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYIVDVHGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGLPLPGFVHLSCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ IDNO.7)。

in another aspect A19, the present invention provides a process for preparing (S) -3-aminomethyl-5-methylhexanoic acid ((S) -II) or a pharmaceutically acceptable salt thereof

Said method comprising treating 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone (I) with a transaminase according to any one of embodiments A18E1, A18E2, A18E3 and A18E4^A) And an amine.

Detailed description of the preferred embodiments

The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group (radial) containing the indicated number of carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, neopentyl and n-hexyl.

The term "alkoxy" refers to a group consisting of an alkyl group as defined above attached to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy and isopropoxy.

The term "alkoxy-alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group in which an alkoxy group replaces an alkyl hydrogen atom. An example of alkoxy-alkyl is 2-methoxyethyl.

The term "haloalkyl" refers to an alkyl group as defined above wherein one or more hydrogen atoms are replaced by fluorine, chlorine, bromine or iodine. When more than one hydrogen atom is replaced, the halogen atoms replaced may be the same or different. Examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chlorodifluoromethyl, 2,2, 2-trifluoroethyl and 3-bromopropyl.

The term "aryl" refers to phenyl or naphthyl.

The term "aryl-alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon radical in which an aryl group replaces an alkyl hydrogen atom. An example of aryl-alkyl is benzyl.

The term "cycloalkyl" refers to a saturated monocyclic or polycyclic carbocyclic ring containing the indicated number of carbon atoms. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Examples of polycyclic cycloalkyl groups include bicyclo [2,2,1] heptyl and bicyclo [3,2,1] octyl.

The term "optionally substituted" with respect to alkyl or aryl means that a hydrogen atom of an alkyl or aryl group may be replaced by one of the listed groups. The substitution may occur at any position in the alkyl or aryl group. When optional substitution is with "one or more groups," then any number of hydrogen atoms of the alkyl or aryl group, up to a maximum number equal to the number of hydrogens present in the alkyl or aryl group, may be replaced, and each replacement is independent of the other.

The term "enantiomeric excess", sometimes abbreviated as "e.e." is a measure of the excess of one enantiomer over its enantiomer for a given sample and is expressed as a percentage. Enantiomeric excess is defined as:

100x(er-1)/(er+1)

where "er" is the ratio of the more enantiomeric to the less enantiomeric.

5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone (I)^A) Is a suitable intermediate in the preparation of 3-aminomethyl-5-methylhexanoic acid (II). Racemization of (I) with ammonia in the presence of a chemical reducing agent^A) The treatment of (a) provides racemic 3-aminomethyl-5-methylhexanoic acid ((R/S) -II) (see WO2008/127646a 2). The reaction is presumed to involve the ring-opened isomer (I)^B)。

Reductive amination using an amine donor in the presence of a transaminase can provide enantiomerically enriched 3-aminomethyl-5-methylhexanoic acid. Suitable amine donors are primary amines such as mono-alkylamines, especially isopropylamine, and alpha-amino acids.

(I^A) Reaction with a suitable amine dehydrogenase/imine reductase in the presence of ammonia may also be a suitable route to pregabalin. A stoichiometric amount of a cofactor, such as NADH or NADPH, may be required, or a second oxidoreductase, such as formate dehydrogenase, may be included to recycle the cofactor.

Wherein dihydrofuranone (I)^A) Having a defined stereochemistry at the C-4 position, which stereochemistry may be retained during the reductive amination reaction. Since pregabalin has an (S) -stereochemistry, it may be preferable to have this stereochemistry already present in dihydrofuranones.

Alternatively, dihydrofuranones (I)^A) Can be used in racemic form. The desired stereoisomer of the product may then be obtained by performing a reductive amination reaction under conditions allowing the stereoselective formation of a single enantiomer, for example by conversion in the presence of a transaminase, or by subjecting the product to a separate resolution step, for example by crystallization with a chiral acid or base.

Dihydrofuranones (I) can be conveniently prepared in racemic or enantiomerically enriched form by the methods shown below^A)。

In the first process, dihydrofuranone (I)^A) By reducing 5-hydroxy-4- (2-methyl-1-propenyl) -5H-2-furanone (VI)^A) And (4) preparation.

This reduction is conveniently accomplished by hydrogenation in the presence of a suitable catalyst. Suitable catalysts include homogeneous and heterogeneous catalysts. The catalyst typically comprises a transition metal such as palladium, platinum, rhodium, ruthenium or nickel, or a salt or oxide thereof. The heterogeneous catalyst comprises finely divided metal and substrate-supported metal and metal oxide, wherein the substrate may be carbon, carbon dioxideSilicon oxide, aluminum oxide or any other suitable inert material. Homogeneous catalysts include phosphine ligand complexes of transition metals. When the phosphine ligand is chiral, then the catalyst is chiral. When an achiral catalyst is used, the product is racemic dihydrofuranone (I)^A). The use of chiral catalysts makes it possible to provide dihydrofuranones (I) in an enantioselective manner^A)。

The selectivity and overall yield of the hydrogenation reaction can be improved when the reaction is carried out in a basic medium. Without being bound by theory, it is believed that furanones in the presence of a base exist predominantly as ring-opened salt (IX).

Any suitable base may be used as long as it does not interfere with the hydrogenation process, for example by poisoning the catalyst. Examples of suitable bases include bases (e.g., Li, Na, K, and Rb) and alkaline earth metal (e.g., Ca and Mg) oxides, hydroxides, carbonates, and bicarbonates. Other metal salts, such as zinc salts, may also be used. Alkali metal salts may be preferred due to their good solubility and/or low toxicity. Amine bases such as ammonia and primary, secondary and tertiary amines can be used to prepare the ammonium salts. Tetraalkylammonium hydroxides can also be used, resulting in the formation of tetraalkylammonium salts.

Hydrogenation of the salt of formula (IX) provides a salt of formula (X).

After the hydrogenation reaction, the dihydrofuranone (I) is recovered by treating the salt with a suitable acid^A). Alternatively, the salt can be converted directly to 3-aminomethyl-5-methylhexanoic acid (II) by treatment with a transaminase or an amine oxidase/imine reductase. In this case, it may be preferable to use ammonium salt (M)⁺＝NH₄ ⁺) Or primary alkyl ammonium salts (M)⁺alkyl-NH₃ ⁺) Since the ammonium or alkylammonium ions provide the cosubstrate for the enzyme. The use of isopropyl ammonium salts in combination with transaminases is a preferred example.

The furanones of formula (VI) wherein R is not hydrogen can also be reduced by hydrogenation. When R is alkyl, haloalkyl, alkoxyalkyl alkenyl, cycloalkyl-alkyl, aryl or aryl-alkyl, these furanones may be derived from formula (VI)^A) The compound of (1) is prepared by reaction with an alcohol R-OH in the presence of an acid catalyst. When R is R¹when-C (O) -is present, the furanone may be derived from formula (VI)^A) By reaction with an acid anhydride (R)¹-C(O))₂O or acid chlorides R¹-c (o) -Cl, optionally in the presence of a base such as a tertiary amine. When R is R²-SO₂When the furanone is of formula (VI)^A) By reaction with sulfonyl chlorides R²-SO₂-Cl is optionally reacted in the presence of a base such as a tertiary amine.

After the hydrogenation step, the compound of formula (I)^A) The dihydrofuranones of (a) can be obtained from the reduction product by treatment with an acid (when R is alkyl, haloalkyl, alkoxyalkyl alkenyl, cycloalkyl-alkyl, aryl or aryl-alkyl) or a base (when R is R¹-C (O) -or R²-SO₂-time) processing.

The use of a chiral R group can provide a chiral hydrogenation product without the need for a chiral catalyst.

For example, furanones (VI)^A) Reacting with chiral alcohol R-OH to provide chiral ether derivative (VI)^B)。

Suitable chiral alcohols may include α -aryl alcohols such as 1-phenyl ethanol and 1-naphthyl ethanol, toAnd terpene alcohols such as menthol and borneol. Derivative (VI)^B) Can be carried out in an enantioselective manner, followed by treatment of the product obtained with a suitable acid and in the presence of water to give the dihydrofuranone (I) in chiral form^A)。

Furanone (VI)^A) Can be prepared from compounds of formula (VII) by treating compounds of formula (VII) with water in the presence of an acid catalyst

. Suitable acids include mineral acids such as sulfuric acid. Alternatively, the compound of formula (VII) may be treated with an alcohol R-OH to provide directly the compound of formula (VI) wherein R is alkyl, haloalkyl, alkoxyalkyl alkenyl, cycloalkyl-alkyl, aryl or aryl-alkyl.

The compound (VII) of formula (VIII) can be prepared by reacting a dienamine of formula (VIII) with glyoxylic acid or a hydrate thereof.

It will be understood that-X-in formula (VII) corresponds to-Y-in the starting material of formula (VIII), except when-Y-is

When then-X-is

wherein-Y-represents a single bond or-CH₂The compounds of the formula (VIII) of (VIII) have been prepared by reaction of 4-methyl-2-pentenal with pyrrolidine or piperidine (Kienzle, F. et al, Helv. Chim. acta1985,68(5), 1133-39). Other compounds of formula (VIII) may be prepared similarly.

Alternatively, condensation of isobutyraldehyde and acetaldehyde with a catalytic acid in a solvent such as acetonitrile in the presence of a suitable amine such as pyrrolidine, piperidine or morpholine provides the dienamine derivative (VIII).

Y is a single bond, CH₂O, N (alkyl), N (benzyl)

If piperazine is used as amine, a bis-dienamine is obtained.

Compounds of formula (VIII) wherein Y is NH can be obtained by using a mono-protected piperazine as amine followed by a deprotection step.

Acyclic secondary amines such as diethylamine and diisopropylamine may also be used, but cyclic secondary amines are preferred.

This mode of reaction of isobutyraldehyde and acetaldehyde is different from a direct base-catalyzed reaction (e.g. using potassium carbonate as the base: british patent GB834100) which produces only the adduct 2, 2-dimethyl-3-hydroxybutyraldehyde where isobutyraldehyde acts as a nucleophile.

Without wishing to be bound by any particular theory, it is hypothesized that both acetaldehyde and isobutyraldehyde are initially converted to their enamine derivatives. In the presence of an acid catalyst, the more basic isobutylene amine is converted to its imine ion. The electrophilic species preferentially reacts with the less sterically hindered nucleophile, which is acetaldehyde enamine-this ensures that the reaction proceeds around the desired manner.

The process of the present invention is more economical and more suitable for scale-up than the literature methods of achieving this "polar inversion" of normal acetaldehyde reactivity, where acetaldehyde is converted to an O-silylized enol derivative and coupled with isobutyraldehyde under Mukiyama aldol conditions. In the present invention, both coupling partners are activated simultaneously-electronic and steric effects, directing the observed reactivity pattern. Another advantage is that the product dienamine is the desired "activated" form of 4-methyl-2-pentenal for reaction with glyoxylic acid to form the desired 5-aminofuranone (VII).

These dienamine derivatives of formula (VIII) can be isolated and purified, or alternatively they can be treated directly with glyoxylic acid (or a hydrate thereof), which directly provides the furanone derivative (VII).

The furanone derivative (VII) can be isolated and purified. Subsequent treatment with aqueous acid provides furanone (VI)^A)。

In general, the transformations shown above provide short paths for the preparation of pregabalin using inexpensive and safe starting materials.

In an alternative embodiment, dihydrofuranone (I) is prepared from 3-isobutylidene-2-oxoglutarate (XII), which is readily obtained from the condensation of isobutyraldehyde with 2-oxoglutarate (. alpha. -ketoglutarate) (XIV).

The conversion of the diacid (XII) to the dihydrofuranone (I) requires a decarboxylation step and a reduction step. The two treatment steps may be carried out separately, in which case the decarboxylation step or the reduction step may be the first step, or the two processes may be carried out simultaneously. When the reduction step is first carried out, intermediate (XV) will be produced. When the decarboxylation step is first carried out, intermediate (XVI) will be produced.

The reduction step may be performed chemically, for example by hydrogenation, but is preferably achieved using enzyme mediated reduction, for example by treatment with enone reductase. The decarboxylation step is preferably carried out by treating the compound with a decarboxylase.

When performing enzyme-mediated transformation, the enzyme may be an isolated enzyme, including an enzyme immobilized on a carrier, which may be a partially isolated enzyme preparation such as a cell homogenate, or it may be a non-isolated enzyme, in which case a whole cell preparation is used. The cells may include cells that naturally express the desired enzyme and cells that have been manipulated to express the desired enzyme.

Formula (I)^A) The enzyme-mediated reductive amination of the compounds of (a) is reversible, so that treatment of 3-aminomethyl-5-methylhexanoic acid (II) with a transaminase or an amine oxidase/imine reductase can lead toTo form dihydrofuranone (I)^A). The ring-opened isomer of the compound is compound (I)^B) It is epimerizable. In this regard, it is possible to convert ((R) -II) to ((S) -II) or to increase the optical purity of a mixture of ((R) -II) and ((S) -II) using such an enzyme.

Examples

The invention is illustrated by the following non-limiting examples in which the following abbreviations and definitions are used:

bp boiling point

CPME cyclopentyl methyl ether

d double peak

DIW deionized water

dd doublet of doublets

eq or eq

ee enantiomeric excess of e.e.or

ES⁺Positive mode electrospray ionization

EtOAc ethyl acetate

EtOH ethanol

GC gas chromatography

GC/MS gas chromatography/Mass Spectrometry

HOAc acetic acid

HPLC high performance liquid chromatography

Hr or h hours

¹H NMR proton nuclear magnetismResonance spectrum

L liter

LCMS liquid chromatography/Mass Spectrometry

M multiplet

mbar

MeOH methanol

min for

mL of

mmol millimole

mol mole of

Mp melting Point

MTBE methyl tert-butyl ether

NAD⁺Nicotinamide adenine dinucleotide (oxidized)

NADP⁺Nicotinamide adenine dinucleotide phosphate (oxidized)

NADH nicotinamide adenine dinucleotide (reduced)

NADPH Nicotinamide adenine dinucleotide phosphate (reduced)

PLP pyridoxal phosphate

parts per million ppm

pTsOH p-toluenesulfonic acid

q quartet peak

qNMR quantitative nuclear magnetic resonance spectrum

R_fRetention factor

RT Room temperature

s single peak

t triplet peak

Thiamine diphosphate ThDP

TLC thin layer chromatography

TsOH toluene sulfonic acid (═ pTsOH)

UPLC ultra-high performance liquid chromatography

XRD X-ray diffraction crystallography

Chemical shift

Commercial chemicals were used as received unless otherwise stated. Thin layer chromatography was performed on pre-coated plastic plates (Merck silica 60F254) and using UV light and KMnO₄And (5) dipping and developing. Proton (C)¹H) And carbon (C)¹³C) NMR spectra were recorded on a Varian INOVA300MHz spectrometer. Chemical shifts are quoted relative to tetramethylsilane and are referenced to residual solvent peaks as appropriate. Unless otherwise indicated, chiral HPLC analysis was performed using an Agilent 1200HPLC system and data was processed using Chemstation software, or using galixie software with Varian semi-preparative/analytical HPLC.

Example 1

Preparation of 4- (2-methyl-1-propenyl) -5-morpholino-5H-2-furanone from 4-methyl-2-pentenal

A50% solution of glyoxylic acid (29.6g, 0.2mol) in water was added to a two-phase stirred mixture of morpholine (17.8g, 0.2mol) and heptane (75mL) which had been pre-cooled to 0-10 ℃. The temperature was maintained below 10 ℃. The mixture was warmed to 20 ℃ and 4-methyl-2-pentenal (19.6g, 0.2mol) was added. The mixture was stirred at 45 ℃ for 20 h. A large amount of solid was formed. Water (100mL) was added at ambient temperature and the mixture was stirred for 2 h. The mixture was extracted with cyclopentyl methyl ether (100mL), and the organic solution was washed twice with water (100mL) and concentrated to leave 30.4g of crude product. The solid was purified by recrystallization from methanol (100mL) to afford 17.7g (40%) of pure 4- (2-methyl-1-propenyl) -5-morpholino-5H-2-furanone.

GC/MS:m/z＝223

¹H NMR:6.0(s,1H)；5.9(s,1H)；5.5(s,1H),3.7(d,4H),2.7(d,4H),2.00(s,3H),1.95(s,3H)。

Example 2

Preparation of 4- (4-methyl-1, 3-pentadien-1-yl) morpholine

Isobutyraldehyde (46.90g, 0.65mol, 1.43eq.) was stirred in acetonitrile (300 mL). Morpholine (56.63g, 0.65mol, 1.43eq.) followed by pTsOH (8.63g, 0.1 eq.) was slowly added to the isobutyraldehyde solution at room temperature. A solution of acetaldehyde (20g, 0.454mol) in acetonitrile (100mL) was added dropwise over 1h, monitored at an internal temperature of 50 ℃. After the addition was complete, the mixture was stirred at 50 ℃ for 30min, then cooled to room temperature, followed by evaporation of the solvent in vacuo to yield an orange oil (123.3 g). The content of crude product was determined to be 40% by quantitative NMR using benzyl benzoate as internal standard, which gave a 65% yield of the desired dienamine.

GC/MS:m/z＝167

¹H NMR:6.01(d,1H)；5.69(d,1H)；5.31(dd,1H),3.70(m,4H),2.89(m,4H),1.71(s,3H),1.63(s,3H)。

Example 3

Preparation of 4- (2-methyl-1-propenyl) -5-morpholino-substituted starting from crude 4- (4-methyl-1, 3-pentadien-1-yl) morpholine 5H-2-furanones

The crude 4- (4-methyl-1, 3-pentadien-1-yl) morpholine dienamine from example 2 (123.3g, 40% assay) was dissolved in methanol (300ml) at room temperature. After complete dissolution, glyoxylic acid (50% by weight, 60g, 1.1eq) was added and the resulting biphasic mixture was stirred at 50 ℃ for 18 h. The reaction mixture was cooled to room temperature and the solvent was removed by rotary evaporation. The residue was partitioned between ethyl acetate (200mL) and saturated sodium carbonate solution (200 mL). The aqueous phase was extracted with ethyl acetate (100ml) and the combined organics were washed with brine and evaporated to a thick oil which solidified on standing (89.0g, 67% by qNMR content, 60% yield).

GC/MS：m/z＝223

NMR: as in example 1.

Example 4

4- (2-methyl-1-propenyl) -5-morpholino-5H-2-furanone is prepared in one pot from isobutyraldehyde and acetaldehyde.

Isobutyraldehyde (102.90g, 1.43mol) was stirred in acetonitrile (600 mL). Morpholine (124.3g, 1.43mol) followed by pTsOH (19.0g, 0.1 eq) was slowly added to the isobutyraldehyde solution at room temperature. A solution of acetaldehyde (44.05g, 1.0mol) in acetonitrile (150mL) was added dropwise over 1hr, monitoring the internal temperature at 50 ℃. After the addition was complete, the mixture was stirred at 50 ℃ for 30min and then cooled to <10 ℃. Glyoxylic acid (50% by weight, 211.3g, 1.43mol) was added and the resulting biphasic mixture was stirred at 50 ℃ for 18 h. The reaction mixture was cooled to room temperature and the solvent was removed by rotary evaporation. The residue was partitioned between ethyl acetate (1L) and saturated sodium carbonate solution (1L). The aqueous phase was extracted with ethyl acetate and the combined organics were washed with brine and evaporated to a thick oil which solidified on standing (166 g). The crude product was triturated with MTBE at room temperature. The product was collected by filtration and washed with MTBE to give pure 4- (2-methyl-1-propenyl) -5-morpholino-5H-2-furanone (84g, 37% yield) which was the same as the material prepared in example 1. Analysis of the crude solid (166g) indicated about 50% yield.

Example 5

Preparation of 1, 4-bis- (4-methyl-1, 3-pentadien-1-yl) piperazine

A solution of piperazine (43.0g, 0.5mol) and 4-toluenesulfonic acid monohydrate (4.4g, 0.023mol) in acetonitrile (600mL) was prepared. The solution was heated to 50 ℃ and isobutyraldehyde (100mL, 1.10mol) was added over 10 min. The solution turned orange-red in color and a transient white precipitate appeared. A solution of acetaldehyde (30.8g, 0.70mol) in acetonitrile (30mL) was then added over 3h by syringe pump at 50 ℃. The resulting suspension was stirred at 50 ℃ for 0.5 h. The solvent was removed and the residual solid was separated from methanol (500ml) at-5 ℃. It was filtered and washed with cold methanol and dried to provide 52.9g (60%) of 1, 4-bis- (4-methyl-1, 3-pentadien-1-yl) piperazine.

GC/MS:m/z＝246.

¹H NMR:6.00(d,2H)；5.70(d,2H)；5.28(dd,2H)；2.92(s,8H)；1.73(s,6H)；1.68(s,6H).¹³C NMR(CDCl₃):140.6(C),126.3(CH),123.4(CH),100.2(CH),48.2(CH₂),25.8(CH₃),18.1(CH₃)。

Example 6

Preparation of 5,5' - (piperazine-1, 4-diyl) bis (4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one) from 1, 4-bis- (4-methyl-1, 3-pentadien-1-yl) piperazine.

1, 4-bis- (4-methyl-1, 3-pentadien-1-yl) piperazine (37.3g, 0.151 mol; see example 5) was added to methanol (300 ml). The temperature was adjusted to 34 ℃ and a 50% solution of glyoxylic acid (44.9g, 0.302mol) in water was added rapidly (within 5 min). Stirring the obtained suspension at 45 ℃ for 15 h; cooled to 0-10 ℃ for 2h and filtered. The solid was washed with methanol (100mL) and dried to afford pure 5,5' - (piperazine-1, 4-diyl) bis (4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one) (32.3g, 60%). An additional 5% can be obtained from the mother liquor by concentration.

¹H NMR:5.96(d,2H),5.87(d,2H),5.56(m,2H),2.71(s,8H),2.07-1.90(m,12H).¹³C NMR(CDCl₃):172.3(C),159.2(C),150.9(C),116.6(CH),115.4(CH),99.2(CH),46.7(CH₂),28.2(CH₃),21.4(CH₃)。

The reaction can also be carried out in isopropanol, acetonitrile-water, toluene-water or in heptane-water in similar yields.

Example 7

Preparation of 5,5' - (piperazine-1, 4-diyl) bis (4- (2-methylpropan-1-en-1-yl) -1-one from isobutyraldehyde and acetaldehyde in one pot Yl) furan-2 (5H) -one).

P-toluenesulfonic acid (6.5g, 0.03mol) was added to a solution of piperazine (59g, 0.69mol) in acetonitrile (240 mL). The reaction was heated to 50 ℃ and stirred until solid dissolution was observed, followed by the addition of isobutyraldehyde (138mL, 1.51 mol). The reaction was maintained at 50 ℃ before a solution of acetaldehyde (60mL, 1.07mol) in acetonitrile (30mL) was added to the vessel by syringe pump over 3 h. After the addition was complete, the reaction was stirred for a further 30 minutes before a 50% w/w solution of glyoxylic acid (148g, 1.0mol) in water was added to the reaction mixture over a period of 5min, followed by the addition of water (50 ml). The reaction was then heated to 70 ℃ for 2h and then to 50 ℃ overnight. The reaction was then cooled to 5 ℃ and held for 30 minutes. 5,5' - (piperazine-1, 4-diyl) bis (4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one) was precipitated and isolated by filtration and washed with acetonitrile (2X 100mL) to give the desired product (126g, 93.5% content assay, 66% yield from acetaldehyde).

Example 8

Solvent-free preparation of 5,5' - (piperazine-1, 4-diyl) bis (4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one) Prepare for

Isobutyraldehyde (79g, 100mL, 1.1mol) was added to a 3-neck round-bottom flask and argon was flushed through the system. Piperazine (43g, 0.5mol) and TsOH-H₂O (4.4 g; 0.023mol) is split into a mixture containing piperazine (10.75g) and TsOH-H₂4 equal parts of O (1.1 g). The addition of the first portion of piperazine (10.75g) resulted in an exotherm from 24 ℃ to 35 ℃. A first portion of TsOH (1.1g) was then added. The reaction was stirred until all the piperazine was dissolved (t ═ 35 ℃), after which a second portion of piperazine (10.75g) was added, followed by TsOH (1.1g) (t ═ 41 ℃). Stirring was continued until all the piperazine was dissolved (t ═ 41 ℃), then a third portion of piperazine (10.75g) was added, followed by TsOH (1.1 g). After the 3 rd addition, a clear solution was produced, followed by the addition of a fourth portion of piperazine (10.75g) and TsOH-H₂O, then TsOH (1.1g) (t 52 ℃). After the addition was complete the reaction was stirred at 50 ℃ for 30 min.

In another flask (50mL), acetaldehyde (30.8g, 39mL, 0.7mol) was added and placed in an ice-water bath (0-2 ℃). An acetaldehyde flask was connected to the 3-neck flask via a septum by a cannula. A stream of argon was then passed through the acetaldehyde flask at a rate to ensure that the addition of acetaldehyde was complete within 3 h. After all the acetaldehyde was added, the reaction was stirred at 50 ℃ for 30min, then cooled to 40 ℃ and 50% glyoxylic acid (104g, 78mL) was added dropwise over 45min at a rate to maintain the temperature below 50 ℃. Water (100mL) was then added and the reaction heated at 70 ℃ for 5 h. The reaction was cooled to 4 ℃ in an ice-water bath. The precipitated product was filtered and the filter cake was washed with cold water (100 mL). Drying at 50 ℃ in vacuo gave 88.9g (71%) of the desired product. The precipitate contained 78.5% of the title compound (HPLC assay).

Example 9

5,5' - (piperazine-1, 4-diyl) bis (4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one) by simultaneous addition Into alternative preparation of two aldehydes

Piperazine (236.6g) was added to a clean 3L dry vessel equipped with a thermometer, addition funnel (100mL) and reflux condenser. pTsOH (25.3g) was added to the vessel followed by acetonitrile (550 mL). The stirrer was turned on and the vessel was inertized with nitrogen. Isobutyraldehyde (150mL, 27% of the total charge) was added to the stirred piperazine/pTsOH slurry and a temperature rise to about 43 ℃ was observed. The white suspension is then heated to 50 ℃ (+/-5 ℃). A pre-mixed ice-cold solution of isobutyraldehyde (400mL, 73% of the total charge) and ice-cold acetaldehyde (260mL) was added to a clean dry 1L vessel, and the mixture was kept in an ice bath. The isobutyraldehyde/acetaldehyde mixture was added as 100mL aliquots to the contents of a 3L vessel at 50 ℃ over a period of 5-6 h. The contents of the reaction flask changed from a white suspension to a wine-red solution during the addition of acetaldehyde, and finally an orange suspension. After the addition was complete, the suspension was stirred at 50 ℃ for about 0.5 to 1.0 h.

An aqueous solution of glyoxylic acid (50% wt/wt) was added to the suspension over a period of 10-15min and the temperature was raised to about 75 ℃. Crystallization of 5,5' - (piperazine-1, 4-diyl) bis (4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one) from solution was observed. The suspension was stirred at 70 ℃ (+/5 ℃) for 6h and then cooled to ambient temperature with stirring. The batch was then cooled to-5 to 0 ℃ and held for 3-4h, after which the suspension was filtered, the filter cake was washed with cold methanol (1X 500mL) and then with 2X 500mL of methanol at ambient temperature. The washed product was dried in a vacuum oven at 40-50 ℃ to constant weight to give 474g of 98% pure 5,5' - (piperazine-1, 4-diyl) bis (4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one) (70%).

Example 10

From 4- (2-methyl-1-propenyl) -5-morpholino-2 (5H) furanone or 5,5' - (piperazine-1, 4-diyl) bis (4- Preparation of 5-hydroxy-4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one (5H) -a ketone.

10% w/w aqueous sulfuric acid (110g) was added to 4- (2-methyl-1-propenyl) -5-morpholino-2- (5H) -furanone (20.0g, 0.896mol) and the mixture was stirred at reflux for 4H or until TLC (100:3 CPME: HOAc) indicated completion of the reaction. The mixture was maintained in suspension at any time. It was then cooled to 5 ℃, held for 2h, and filtered. The white solid was washed with water and dried to afford 5-hydroxy-4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one (12.9g, 93%).

Alternatively, a 10% w/w aqueous solution of sulfuric acid (412g) was added to a vessel containing 5,5' - (piperazine-1, 4-diyl) bis (4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one) (60g, 0.167 mol). The contents were stirred and then heated to reflux. The reaction was kept at reflux until the starting material was consumed, as determined by dissolution. After the reaction was complete, the batch was cooled to 35 ℃ at which time the target compound began to crystallize. The slurry was further cooled to 0-5 ℃ and then transferred to a filter. The product was filtered, washed with water (2 × 100mL), then dried under vacuum at <50 ℃ to give 5-hydroxy-4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one (42.0g, 81%).

GC/MS:m/z＝154

¹H NMR(CDCl₃):6.10(s,1H)；5.92(s,1H)；5.88(s,1H)；5.35(bs,1H,O-H)；1.98(s,3H)；1.93(s,3H).¹³C NMR(CDCl₃):172.8(C),161.4(C),152.3(C),115.3(CH),114.9(CH),99.5(CH),28.2(CH₃),21.4(CH₃)。

Example 11

5-hydroxy-4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one was prepared in one pot from isobutyraldehyde and acetaldehyde.

A mixture of piperazine (43.0g, 0.5mol) and isobutyraldehyde (200mL) was refluxed under Dean-Stark until the theoretical amount of water (18mL) was collected. Excess isobutyraldehyde was allowed to slip off to leave a crystalline mass of 1, 4-bis (2-methylpropen-1-yl) piperazine. It was dissolved in acetonitrile (1.2L) and additional isobutyraldehyde (100mL, 1.0mol) was added. Additional piperazine (4.4g) was added followed by p-toluenesulfonic acid (4.4g, 23.2 mmol). The temperature was adjusted to 40 ℃ and a solution of acetaldehyde (44g, 1.0mol) in acetonitrile (40ml) was added over 4 h. The reaction mixture was stirred at 40 ℃ for 1h and at ambient temperature for 4 h. Acetonitrile was decanted and the residue was suspended in toluene (400 mL). A mixture of 50% glyoxylic acid (148g) and water (150ml) was added over 0.5 h. The mixture was then stirred at 45 ℃ for 15 h. A solid precipitate was produced. Dilute sulfuric acid (10%, 1L) was added and the mixture was refluxed for 3 h. A two-phase mixture results. The toluene phase was separated. It contained 5-hydroxy-4- (2-methylpropen-1-yl) -2-furanone in about 50% yield (based on acetaldehyde) by assay.

Example 12

Preparation of 5-methoxy-4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one

4- (2-methyl-1-propenyl) -5-morpholino-2- (5H) -furanone (7.70g, 34.5mmol) was washed with H₂SO₄(4mL, 2.2eq.) in MeOH (50mL) was stirred at reflux for 3h until the starting material disappeared (by GC). The reaction mixture was cooled to room temperature. The solvent was then evaporated in vacuo and the residue diluted with EtOAc (50 mL). By H₂The organic phase was washed with O (3 × 50mL) then the solvent was evaporated in vacuo to give 5-methoxy-4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one as an orange oil (5.50g, 95%), which was used without purification.

Alternatively, 5' - (piperazine-1, 4-diyl) bis (4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one) (130g, 363mmol) and MeOH (690mL) were added with mechanical stirring to a 2L round bottom flask to form a suspension. Concentrated H is added dropwise under stirring at 20-25 DEG C₂SO₄(43mL, 762mmol), a slight exotherm was observed, and the nature of the solid present changed from dispersed and readily mixed to a thicker slurry. The reaction mixture was refluxed for 5 h. Complete dissolution was observed after 0.5h (orange liquid). LCMS after 5h showed-99% of the desired product. The mixture was cooled to room temperature and left to stand for crystallization of piperazine sulfate. The precipitate was filtered and the filter cake was washed with ice cold MeOH (400 mL). The filtrate was concentrated in vacuo (40 ℃ bath temperature) and the residue partitioned between water (50mL) and MTBE (300 mL). The aqueous layer was separated and extracted further (2x300mL MTBE). The combined organic extracts were extracted with saturated aq₃(200mL) washing. The MTBE layer was added under stirring to Na₂SO₄Dried for 1.5H, filtered and evaporated to give 5-methoxy-4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one as an orange oil (120g, 98%).

Distillation at 105 ℃ in bp 100 under a vacuum of 0.2-0.3 mbar.GC/MS:m/z＝168；¹H NMR(400MHz,CDCl₃)5.59(1H,s)5.88-5.86(1H,m)5.75(1H,d,J＝0.6)3.53(3H,s)2.01-2.00(3H,m)1.97-1.96(3H,m).¹³C NMR(CDCl₃):171.4(C),159.2(C),151.9(C),115.8(CH),115.2(CH),104.4(CH),56.1(CH₃),28.2(CH₃),21.4(CH₃)。

The following compounds were prepared from 4- (2-methyl-1-propenyl) -5-morpholino-2 (5H) -furanone according to the first of the above methods by replacing methanol with 3-methylbutanol (isoamyl alcohol), n-pentanol (amyl alcohol) or n-butanol:

example 12A

5- (3-Methylbutoxy) -4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one

Distilling at 140 ℃ under the vacuum of 0.4mbar and bp 139-; GC/MS, m/z is 224;¹H-NMR(400MHz,CDCl₃)5.94(1H,s)5.87-5.84(1H,m)5.80(1H,s)3.86-3.80(1H,m)3.70-3.64(1H,m)2.00(3H,s)1.96(3H,s)1.77-1.66(1H,m)1.57-1.50(2H,m)0.93-0.91(3H,m)0.91-0.89(3H,m)

example 12B

4- (2-methylpropan-1-en-1-yl) -5-pentoxyfuran-2 (5H) -one

Distillation at 134 ℃ in bp 123-; GC/MS, m/z is 224;¹H-NMR(400MHz,CDCl₃)5.94(s,1H)；5.86(s,1H)；5.80(s,1H)；3.84-3.75(m,1H)；3.68-3.59(m,1H)；2.01(s,3H)；1.95(s,3H)；1.69-1.59(m,2H)；1.38-1.29(m,4H)；0.95-0.85(m,3H)

example 12C

5-butoxy-4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one

At 0.05mbDistillation at bp 136-; GC/MS, m/z is 210;¹H-NMR(400MHz,CDCl₃)5.94(s,1H)；5.86(s,1H)；5.80(s,1H)；3.85-3.76(m,1H)；3.69-3.60(m,1H)；2.00(s,3H)；1.95(s,3H)；1.68-1.58(m,2H)；1.45-1.32(m,2H)；0.93(t,3H,J＝7.4Hz)

example 13

Preparation of 5-methoxy-4- (2-methylpropyl) -dihydrofuran-2 (3H) -one

A solution of 5-methoxy-4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one (100g, see example 12) in MTBE (700mL), 5g (5 wt%) of 10% Pd/C was added to the hydrogenation vessel. 1atm of hydrogen was introduced and the pressure was kept constant during the reaction. After 7h at 20 ℃ the reaction was passed through a pad of celite (H-30 mm) and rinsed with MTBE (3 × 50 mL.) the filtrate was washed with 1M NaHCO₃The solution (200mL), water, brine, washed in Na₂SO₄And drying. After removal of the solvent, 5-methoxy-4- (2-methylpropyl) -dihydrofuran-2 (3H) -one was obtained as a colorless liquid (98g, 96%).

GC/MS:m/z＝172

¹H NMR(400MHz,CDCl₃)5.25(d, J ═ 5.0Hz,0.53H, major isomer), 5.04(d, J ═ 2.4Hz,0.26H, minor isomer), 3.48(s,0.92H, minor isomer), 3.46(s,1.66H, major isomer), 2.78(dd, J ═ 17.7,8.6Hz,0.33H), 2.59-2.42 (m,1.27H, major isomer), 2.42-2.34(m,0.33H, minor isomer), 2.29(dd, J ═ 16.7,11.8Hz,0.63H, major isomer), 2.15(dd, J ═ 17.7,4.6Hz,0.33H, minor isomer), 1.67-1.31 (m,2.63H isomers), 1.28-1.19H, 0.86H, minor isomer (m, 0.86H, 0.6H, minor isomer), two isomers (m, 2.33H, 0.33H, 86H, 0.6H, minor isomer), 1.13H, 2.4.33H, or a pharmaceutically acceptable salt thereofBody).¹³C NMR(CDCl₃) 177.7(C, major isomer), 176.04(C, minor isomer), 110.1(CH, minor isomer), 106.1(CH, major isomer), 57.0 (CH)₃Minor isomer), 56.6 (CH)₃Major isomer), 41.2 (CH)₂Minor isomer), 39.1 (CH)₂Minor isomer), 38.3 (CH)₂Major isomer), 37.1 (CH)₂Major isomer), 33.9(CH, minor isomer), 32.8(CH, major isomer), 26.0(CH, major isomer), 25.8(CH, minor isomer), 23.0(CH₃Major isomer), 22.9 (minor isomer), 22.7 (major isomer), 22.6 (minor isomer).

By using the same general procedure and starting from the compounds of examples 12A, 12B and 12C, respectively, the following compounds were also obtained:

example 13A

5- (3-Methylbutoxy) -4- (2-methylpropyl) -dihydrofuran-2 (3H) -one

GC/MS:m/z＝228；¹H-NMR(400MHz,CDCl₃)5.35(0.86H,d,J＝5.0Hz)5.13(0.14H,d,J＝2.7Hz)3.87-3.79(m,1H)3.57-3.45(m,1H)2.58-2.37(2H,m)2.35-2.27(0.79H,m)2.20-2.11(0.21H,m)1.74-1.62(1H,m)1.61-1.44(4H,m)1.42-1.31(1H,m)0.95-0.86(12H,m)

Example 13B

4- (2-methylpropyl) -5-pentoxydihydrofuran-2 (3H) -one

GC/MS:m/z＝228；¹H-NMR(400MHz,CDCl₃)5.35(0.77H,d,J＝5.0Hz)5.13(0.23H,d,J＝2.6Hz,)3.83-3.75(1H,m)3.55-3.40(1H,m)2.59-2.37(2H,m)2.32(0.73H,dd,J＝16.6,11.8Hz)2.15(0.27H,dd,J＝17.7,5.0Hz)1.67-1.45(4H,m)1.41-1.25(5H,m)0.96-0.86(9H,m)

Examples13C

5-butoxy-4- (2-methylpropyl) -dihydrofuran-2 (3H) -one

GC/MS:m/z＝214；¹H-NMR(400MHz,CDCl₃)5.35(0.8H,d,J＝5.0Hz)5.13(0.2H,d,J＝2.6Hz,)3.85-3.75(1H,m)3.56-3.41(1H,m)2.59-2.38(2H,m)2.31(0.76H,dd,J＝16.5,11.7Hz)2.15(0.24H,dd,J＝17.6,5.0Hz)1.66-1.45(4H,m)1.43-1.30(3H,m)0.95-0.87(9H,m)

Example 14

Preparation and hydrogenation of 5- (L-menthoxy) -4- (2-methyl-1-propenyl) -2(5H) -furanone

A mixture of 5-hydroxy-4- (2-methyl-1-propenyl) -2-furanone (59.5g, 0.386mol), L-menthol (89.5g, 1.5eq.) and methanesulfonic acid (1.5g) was stirred at 70-80 deg.C under vacuum (to remove water) for 100 h. The liquid mass was poured (hot) into acetonitrile (450mL) and the title compound was isolated by cooling, filtration and washing. Yield 76.2g (67%). Suitable crystals for XRD grow by slow evaporation of acetone solution. The configuration on the acetal carbon is (R).

Hydrogenation of 5- (L-menthoxy) -4- (2-methyl-1-propenyl) -2-furanone in ethyl acetate using the conditions of example 13 gives quantitative yields of the saturated derivative. By passing¹H NMR, the compound is a 1:1 mixture of diastereomers.

Example 15

Preparation of 5-acetoxy-4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one

A suspension of 5-hydroxy-4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one (19g, 0.123mol) in ethyl acetate (100mL) was treated with solid sodium carbonate (13.1g, 0.123mol) and tetrabutylammonium hydrogen sulfate (0.5 g). Acetic anhydride (18.8g, 1.5eq.) was added in one portion. A mildly exothermic reaction ensued. The mixture was stirred overnight, water (100mL) was added, and the organic phase was separated (pH of aqueous phase 6.0). The ethyl acetate phase was washed with water and concentrated to leave a solid (24.6g, 100%). This was purified by TLC (100:3 CPME: acetic acid). If the reaction is carried out in isopropyl acetate, the pure compound can be isolated in about 70% yield by cooling the isopropyl acetate solution after water washing.

m/z：196

Example 16

Hydrogenation of 5-acetoxy-4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one

The material of example 34 was hydrogenated under the same conditions as outlined in example 13 to yield 5-acetoxy-4- (2-methylpropyl) -3, 4-dihydro-2 (5H) -furanone as the diastereomer in > 90% yield 1:1 of a mixture. The product was accompanied by about 5% 4- (2-methylpropyl) -3, 4-dihydrofuran-2 (5H) -one. This product can be hydrolyzed as in example 30 to give 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone/3-formyl-5-methylhexanoic acid.

Example 17

By 5-hydroxy-4- (2-methylpropan-1-en-1-yl)) Preparation of furan-2 (5H) -one by hydrogenation in alkaline solution ^A5-hydroxy-4- (2-methylpropyl) dihydrofuran-2 (3H) -one (I)

5-hydroxy-4- (2-methylpropen-1-yl) -2-furanone (3.35g, 21.7mmol) and water (20mL) are added to the hydrogenator. Potassium hydroxide (1.21g, 1eq) was then added to the vessel and the contents heated to 40 ℃ until dissolution occurred. A 10% Pd on carbon catalyst (0.67g) was added to the vessel and the reactor contents were subsequently hydrogenated at 40 ℃ and 5barg hydrogen pressure. After completion of the reaction, the reaction was cooled and the catalyst was removed by filtration. The pH was adjusted to pH2 by adding 36% hydrochloric acid and the aqueous layer was washed with toluene to extract the desired product. The combined toluene extracts were concentrated to give the title compound (3.17g, 92%).

Example 18

Hydrogenation of 5-hydroxy-4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one in neutral solution

5-hydroxy-4- (2-methylpropen-1-yl) -2-furanone is hydrogenated with 10% w/w Pd/C catalyst in 6mL of water per gram of starting material. (22h, 40 ℃ C., 10 bar). After filtration, the oil phase separated from the water. This is 5-hydroxy-4- (2-methylpropyl) dihydrofuran-2 (3H) -one (I)^A) And a 1:1 mixture of 4- (2-methylpropyl) -dihydrofuran-2-one. Compound (I)^A) Can be easily separated and purified by acid-base extraction. This reaction was repeated with 2-propanol in an organic solvent to yield relatively pure (I)^A)。

Example 19

From 5,5' - (piperazine-1, 4-diyl) bis (4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one) in one pot ^APreparation of 5-hydroxy-4- (2-methylpropyl) dihydrofuran-2 (3H) -one (I)

5,5' - (piperazine-1, 4-diyl) bis (4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one) (50.0g, 0.14mol) was added to water (300mL) containing sulfuric acid (16.0g, 0.163 mol). Isopropyl acetate (200mL) was added. 50% water-wet 5% palladium on carbon (3.0g) was added and the mixture was hydrogenated at ambient temperature (5bar hydrogen) for 15 h. The reaction mixture was filtered from the catalyst and washed with isopropyl acetate (300mL) -this was also used to wash the vessel. The organic phase was separated, washed with water (100mL) and concentrated on a rotary evaporator to afford 38.8g of a clear oil. This is the desired compound (I)^A) And a mixture of over-reduced lactones (4- (2-methylpropyl) -dihydrofuran-2-one). It was easily purified by extraction with aqueous potassium carbonate and washing with toluene. The potassium carbonate extract is acidified with formic acid to give the desired product (I)^A)(25.6g，58％)。

¹H NMR (addition of K)₂CO₃D of (A)₂O):9.5(1H),2.8(m,2H),2.40(m,2H),1.55(m,2H),1.30(m,2H),0.95(m,6H)。

Example 20

Preparation of 3-isobutylidene-2-oxoglutaric acid monopotassium salt.

To a 2L round bottom bottle was added 300g of alpha-ketoglutaric acid and 450mL of ice water. 314g of potassium hydroxide in 450mL of water was added with stirring and cooling, maintaining the flask temperature below 25 ℃. The reactor was placed under nitrogen and isobutyraldehyde was added at 0.2mL/min over 50 hrs. The resulting two-phase yellow solution was separated and washed with 100mL MTBE. The lower aqueous phase was adjusted to pH 3.2-3.4 and concentrated to 1/3 volumes under vacuum at 65 ℃. After cooling to <5 ℃, the resulting solid was collected and washed with several small volumes of water to give, after drying, 230g of a white solid containing 60% of 3-isobutylidene-2-oxoglutarate monopotassium salt and the remainder KCl.

¹H NMR (D at pH 8-10,400 MHz)₂O)6.4(d,1H),3.3(s,2H),2.7(m,1H),0.96(d,6H)。¹³C NMR (D at pH 8-10,400 MHz)₂O)23(2CH₃),31(CH),35(CH₂),133(C),164(CH),170(C),176(C),182(C)。

Example 21

Preparation of 3- (2-methylpropyl) -2-oxoglutarate monopotassium salt

An aqueous solution of 3-isobutylidene-2-oxoglutaric acid monopotassium salt, produced as in example 20, was adjusted to pH 6-8, 5% Pd/C was added and the solution was brought to 10bar H₂Hydrogenation was carried out at 25 ℃ for 10 hours. The catalyst was removed by filtration and the pH of the solution was adjusted to pH 3.8. Cooling the mixture to<Filtered and washed with a small amount of cold water at 5 ℃. The resulting white crystals were dried under vacuum at 40 ℃ to give the title compound, 286g, in 58% yield over 2 steps.

¹H NMR (D at pH 8-10,400 MHz)₂O)3.5(m,1H),2.5(dd,1H),2.3(dd,1H),1.6-1.5(m,2H)1.3-1.2(m,1H)0.9(d,6H)。¹³C NMR (D at pH 8-10,400 MHz)₂O)21.7(CH₃),22.2(CH₃),25.5(CH),38.4(CH₂),39.3(CH₂),43.4(CH),167.8(C),170.6(C),180.7(C)。

Example 22

Expression of decarboxylase in E.coli

PubMed was used to search for literature on decarboxylases with broad spectrum activity. The program KEGG (kyoto gene and genome encyclopedia) was used to search for microbial decarboxylases with activity on compounds that have some similarity in structure to 3- (2-methylpropyl) -2-oxoglutarate or 3-isobutylidene-2-oxoglutarate. Seven classes of decarboxylases were selected for the study based on reports of activity for compounds with some structural similarities. The decarboxylase is glutamate decarboxylase, diaminopimelate decarboxylase, indolylacetate decarboxylase, branched-chain alpha-keto acid decarboxylase, aromatic-L-amino acid decarboxylase, lysine decarboxylase, and benzoylformate decarboxylase. 41 sequences of the confirmed or putative decarboxylase genes were selected. The genes were codon optimized for expression in e.coli, synthesized by geneart (germany), DNA2.0(Menlo Park, CAUSA) or Blue Heron Biotechnology (Bothell, WA USA), cloned into the expression vector pSTRC52(Pfizer inc., USA) and placed into the expression strain e.coli BDG62(Pfizer inc., USA) (table 1 below). Both genes were amplified by PCR from genomic DNA from the appropriate organism and cloned into the same expression vector and E.coli strain. Four decarboxylases were purchased from Sigma and tested for activity on 3- (2-methylpropyl) -2-oxoglutarate.

Each E.coli strain containing the cloned decarboxylase gene was grown overnight in LB broth with the appropriate antibiotic. A small amount (50. mu.L-100. mu.L) of overnight seed culture was used to inoculate 4.0mL of Terrific Broth medium with the appropriate antibiotic in20 mm diameter culture tubes. Cultures were grown in a shaking incubator at 32 ℃ and 300 rpm. After 5h growth IPTG was added to a final concentration of 0.4mM to induce expression of the enzyme. The culture was returned to the incubator and grown for another 19 h.

Example 23

Decarboxylation of potassium 3- (2-methylpropyl) -2-oxoglutarate and 3-isobutylidene with a decarboxylase-2-oxoglutaric acid Potassium salt

The decarboxylation of 3- (2-methylpropyl) -2-oxoglutarate (XV) and 3-isobutylidene-2-oxoglutarate (XII) was tested for the recombinant decarboxylase using E.coli cells as prepared in example 22. Reaction (1mL) at 37 ℃ using decarboxylase (60mg wet cells), 50mM 3- (2-methylpropyl) -2-oxoglutarate or 3-isobutylidene-2-oxoglutarate, ThDP (0.1mM) and MgSO₄(2.5mM) was carried out in potassium phosphate buffer (100mM, pH 6.4). Decarboxylation of 3- (2-methylpropyl) -2-oxoglutarate and 3-isobutylidene-2-oxoglutarate was determined by UPLC assay. An aliquot of the reaction (0.1mL) was treated with 0.5mL potassium phosphate buffer (100mM, adjusted to pH2.2 with phosphoric acid) and 0.3mL of 2, 4-dinitrophenylhydrazine solution (20mM in 1M HCl: acetonitrile, 3:1, v/v) at 50 ℃ for 30 min. The derivatized sample was diluted with 0.5mL acetonitrile, filtered and analyzed by UPLC on an Agilent Eclipse Plus C18(100mm x3.0mm,1.8 μm) column, eluting with 0.1% trifluoroacetic acid in water acetonitrile (55:45, v/v) at 1.1 mL/min. The column was maintained at 40 ℃ and the effluent at 360nm and ES⁺And (4) monitoring on a mass spectrum. A positive result is indicated by the presence of a peak of 3-formyl-5-methylhexanoic acid from 3- (2-methylpropyl) -2-oxoglutarate or 3-formyl-5-methylhexane (methylhex) -3-enoic acid from 3-isobutylidene-2-oxoglutarate. The results of the analysis are shown in table 1.

TABLE 1

^a-: no suitable products were detected

^bNT: not tested

^c+: the corresponding product was detected

Example 24

Preparation of 3-formyl-5-methylhexa-3-enoic acid

Into a 250mL round bottom flask was added 80g of a cell concentrate comprising Escherichia coli expressing Pseudomonas putida (Pseudomonas putida) benzoyl formate decarboxylase (see example 23). To this solution was added a pH 6.2 adjusted solution of 3-isobutylidene-2-oxoglutarate monopotassium salt (10g, see example 20) in 50mL of water, as well as 0.5g of magnesium sulfate and 0.5g of thiamine pyrophosphate. The resulting pH 6.2 slurry was heated to 50 ℃ and stirred for 145hrs, maintaining the pH adjusted between 6.2 and 7.2 with concentrated HCl. The reaction mixture was cooled to room temperature and centrifuged. The decanted aqueous solution was washed with 50mL of MTBE with minimal agitation. The pH of the aqueous phase was adjusted to 4 and filtered through celite. The filtrate was extracted three times with 50mL MTBE with minimal stirring. MTBE was dried over anhydrous sodium sulfate and concentrated to a viscous red oil. The oil was extracted with several portions of hot hexane; the combined hexanes were cooled to <0 deg.C, the resulting crystals were isolated and dried in air to give 3-formyl-5-methylhexa-3-enoic acid as a white solid (0.6 g).

¹H NMR (D at pH 8-10,400 MHz)₂O)9.2(s,1H),6.6(d,1H),3.1(s,2H),2.7(m,1H),1(d,6H)

Example 25

Expression of enone reductase homologs in E.coli

The DNA sequence corresponding to the gene of tomato (Lycopersicon esculentum) (tomato) 12-oxodienoic acid reductase 1 (12-oxodienoic acid reductase 1) (OPR1) was retrieved from the Genbank database (accession number ACQ9XG54) and synthesized by GeneArt (Germany). The sequences were codon optimized for expression in e.coli and subcloned into e.coli expression plasmid pSTRC18(Pfizer inc., USA). The protein sequence is shown below. The OPR1 expression construct was transformed into BL21(DE3) escherichia coli (Stratagene, Agilent Technologies, Santa Clara, CA, USA) as directed and the overnight culture was incubated in LB + streptomycin medium. The LB culture was used to inoculate an expression culture (LB, M9Y or TB), which was incubated at 37 deg.C (210 rpm). After the culture reached the appropriate biomass concentration (OD 1at A600), IPTG (1mM) was added and the culture was incubated for a further 20h (30 ℃, 210 rpm). Cells were harvested by centrifugation (4000 Xg, 30min, 4 ℃) and stored at-20 ℃.

The BLASTP program was used to search the NCBI non-redundant protein sequence database for gene sequences related to 12-oxophytadienic acid reductase 1(OPR1) from tomato. Thirty-eight sequences of the relevant genes were selected, codon optimized for expression in e.coli, and subcloned into the pET28b (+) e.coli expression plasmid (Novagen, emdcchemicals, Gibbstown, NJ, USA). The OPR1 related expression construct was transformed into BL21(DE3) escherichia coli (Stratagene, Agilent Technologies, Santa Clara, CA, USA) as directed and the overnight culture was grown in LB + kanamycin medium. The LB cultures were used to inoculate expression cultures grown in overnight expression ready TB medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). The culture was incubated at 30 ℃ for 20 h. Cells were harvested by centrifugation (4000 Xg, 30min, 4 ℃) and stored at-20 ℃.

Tomato (tomato) 12-oxophytadienoate reductase 1 protein sequence:

tomato (tomato) 12-oxophytodienoic acid reductase 1 codon optimized sequence:

ATGGAAAACAAAGTTGTGGAAGAAAAACAGGTTGATAAAATCCCGCTGATGAGCCCGTGTAAAATGGGTAAATTCGAGCTGTGTCATCGCGTTGTACTGGCACCGCTGACTCGTCAGCGTTCTTATGGTTACATTCCGCAGCCGCACGCAATCCTGCATTACTCTCAGCGCAGCACCAACGGTGGCCTGCTGATCGGTGAAGCAACCGTGATCAGCGAAACTGGCATCGGTTACAAAGATGTGCCGGGTATCTGGACGAAAGAGCAGGTTGAGGCCTGGAAACCGATCGTCGACGCGGTGCATGCCAAAGGTGGTATTTTCTTTTGTCAGATCTGGCACGTTGGTCGTGTATCCAACAAAGATTTTCAGCCGAACGGCGAAGATCCGATTTCCTGTACTGACCGCGGCCTGACCCCGCAGATCCGTTCCAACGGCATTGACATTGCCCACTTCACCCGTCCACGTCGCCTGACTACTGACGAGATTCCGCAGATCGTGAACGAGTTCCGCGTTGCAGCGCGTAATGCTATTGAAGCGGGTTTCGATGGCGTCGAGATTCATGGTGCCCACGGTTACCTGATCGACCAATTCATGAAAGACCAAGTTAACGACCGCAGCGATAAGTATGGCGGTTCTCTGGAGAACCGTTGTCGCTTCGCGCTGGAAATCGTTGAAGCAGTAGCCAACGAGATTGGCTCCGACCGTGTTGGTATCCGTATCTCTCCATTCGCACACTACAACGAAGCGGGCGACACTAACCCGACCGCACTGGGCCTGTATATGGTGGAGAGCCTGAATAAATACGACCTGGCGTATTGTCACGTGGTCGAGCCGCGCATGAAAACCGCCTGGGAAAAGATTGAGTGCACCGAAAGCCTGGTGCCGATGCGTAAAGCCTACAAAGGCACCTTCATCGTAGCTGGTGGCTACGACCGTGAAGACGGTAACCGCGCTCTGATCGAAGACCGTGCCGACCTGGTTGCGTACGGTCGTCTGTTCATCAGCAACCCAGACCTGCCGAAGCGTTTTGAACTGAACGCTCCGCTGAACAAATACAACCGTGACACTTTCTACACTTCCGACCCGATCGTTGGTTACACCGATTACCCGTTTCTGGAAACTATGACTTAATAA(SEQ ID NO.9)

example 26

Reduction of (E) -3-Formyl-5-methylhexa-3-enoic acid

The recombinant enone reductase was tested for the reduction of (E) -3-formyl-5-methylhexa-3-enoic acid using e.coli cells as prepared in example 25. The reaction (0.5mL) was carried out at 30 ℃ using E.coli cells (100mg wet cells/mL), NADPH (10mM), NADH (10mM) and (E) -3-formyl-5-methylhexa-2-enoic acid (10mM) in potassium phosphate buffer (100mM, pH 7.0). After 16h, acetonitrile (0.5ml) was added to each reaction and the resulting mixture was centrifuged (2000rpm x5 min). An aliquot (0.1mL) of the resulting supernatant was treated with 0.1mL potassium phosphate buffer (100mM, adjusted to pH2.2 with phosphoric acid) and 0.225mL 2, 4-dinitrophenylhydrazine solution (20mM in 1M HCl: acetonitrile, 3:1, v/v) at 50 ℃ for 30 min. The derivatized sample was diluted with 0.225mL acetonitrile and analyzed by HPLC on a Phenomenex Lux 5. mu. amylose-2 column (250mm x 4.6mm id), eluting with 0.1% trifluoroacetic acid in water acetonitrile (65:35, v/v) at 2 mL/min. The column was maintained at 50 ℃ and the effluent was monitored at 360 nm. The results of the HPLC analysis are shown in Table 2.

TABLE 2

Example 27

Reduction of (E) -3-formyl-5-methylhexa-3-enoic acid with recombinant reductase and formate dehydrogenase

With NAD⁺Evaluation of recombinant enone reductase by Formate dehydrogenase (E) -3-formyl-5-methylhexa-3-enoic acid reduction of enone reductase in E.coli cells as described in example 25 Formate dehydrogenase is expressed in E.coli cells by transformation of pET26b formate dehydrogenase expression construct into BL21(DE3) E.coli (Stratagene, Agilent Technologies, Santa Clara, Calif., USA) as directed and growth of overnight culture in LB + kanamycin medium LB culture is used to inoculate expression culture grown in overnight expression immediate TB medium + kanamycin (Novagen, EMDChemics, Gibbstown, NJ, USA.) the culture is incubated at 30 ℃ for 20h, cells are harvested by centrifugation (4000 × g, 30min, 4 ℃) and stored in-20 ℃ oxophytic acid reductase 3 (FEQ 9W 9) by centrifugation (D M) and growth of DNA from Escherichia coli cells expressed in Strain reductase gene expression medium, strain DNA growth medium, strain growth medium is used to inoculate DNA growth in strain culture grown in LB + kanamycin (ATCC) as directed by growth of Escherichia coli strain, strain growth of Escherichia coli cells (ATCC, strain growth medium, strain growth in strain, strain

Reaction (0.5mL) at 30 ℃ using enone reductase (40mg wet cells/m)L), formate dehydrogenase (80mg wet cells/mL), NAD⁺(0.02mM), ammonium formate (30mM) and (E) -3-formyl-5-methylhexa-3-enoic acid (20mM) were carried out in potassium phosphate buffer (100mM, pH 7.0). After 24h, the reaction mixture was acidified with 0.025mL of 4N hydrochloric acid and extracted with 1mL of ethyl acetate. An aliquot (0.5mL) of the ethyl acetate extract (0.5mL) was dried over anhydrous sodium sulfate and treated with methanol (0.02mL) and (trimethylsilyl) diazomethane (0.01mL of a 2M solution in ether) to derivatize the carboxylic acid moiety to its corresponding methyl ester. The derivatized sample was passed through a Chiraldex^TMGC analysis on a G-TA column (30M x 0.25.25 mm column, column temperature: 135 ℃ isothermal, injector temperature: 200 ℃, carrier gas: helium, flow rate about 1mL/min) gave the results shown in Table 3.

TABLE 3

Example 28

Reduction of (E) -3-formyl-5-methylhexa-3-enoic acid with recombinant reductase

NADP⁺And Lactobacillus brevis (X-zyme) alcohol dehydrogenase (X-zyme) to evaluate the reduction of (E) -3-formyl-5-methylhexa-3-enoic acid to recombinant enone reductase. Enone reductase was expressed in E.coli cells as described in example 25. A variant of oxophytadienoate reductase 3(OPR3) (D74M) was prepared as described in example 27. Reaction (0.5mL) Using enone reductase (40mg wet cells/mL), Lactobacillus brevis alcohol at 30 deg.CCatalase (32U/mL) NADP⁺(0.02mM), 2-propanol (3 vol%) and (E) -3-formyl-5-methylhexa-2-enoic acid (20mM) were carried out in potassium phosphate buffer (100mM, pH 7.0). After 24h, the reaction mixture was acidified with 0.025mL of 4N HCl and extracted with 1mL of ethyl acetate. An aliquot (0.5mL) of the ethyl acetate extract (0.5mL) was dried over anhydrous sodium sulfate and treated with methanol (0.02mL) and (trimethylsilyl) diazomethane (0.01mL of a 2M solution in ether) to derivatize the carboxylic acid moiety to its corresponding methyl ester. The derivatized sample was passed through a Chiraldex^TMGC analysis on a G-TA column (30M x 0.25.25 mm column, column temperature: 135 ℃ isothermal, injector temperature: 200 ℃, carrier gas: helium, flow rate about 1mL/min) gave the results shown in Table 4.

TABLE 4

Example 29

Preparation of 3-formyl-5-methylhexanoic acid

7.5mL of potassium phosphate buffer (0.1M, pH7.0) and 8.4mg of NADP were added to the reaction vessel⁺0.2mL of Lactobacillus brevis alcohol dehydrogenase (32U/mL, X-zyme), 0.3mL of 2-propanol, pentaerythritol tetranitrate reductase (2 mL of a 200mg/mL suspension of E.coli cells in potassium phosphate buffer) and 156mg of (E) -3-formyl-5-methylhexa-3-enoic acid, and stirring at 40 ℃ after 6.75h, the reaction mixture was centrifuged and the supernatant was adjusted to pH2 with 4N HCl and extracted with ethyl acetate (2 × 10 mL). the ethyl acetate extract was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to give 141mg of a colorless oil (89% yield).

¹H NMR (D at pH 8-10)₂O)2.53–2.43(m,2H),2.34–2.28(m,1H),1.53–1.42(m,1H),1.41–1.34(m,1H),1.20–1.12(m,1H),0.76(d,3H),0.74(d,3H)。

Example 30

Bioconversion of 3-formyl-5-methylhexanoic acid to pregabalin using recombinant ω -transaminase.

Transamination of 3-formyl-5-methylhexanoic acid to form pregabalin was evaluated with various recombinant transaminases. Recombinant ω -transaminase from Vibrio fluvialis (Vibrio fluvialis), Rhodobacter sphaeroides (Rhodobacter sphaeroides) and Paracoccus denitrificans (Paracoccus denitificans) was expressed in E.coli as follows: the pET28b ω -transaminase expression construct was transformed into BL21(DE3) E.coli (Stratagene, Agilent technologies, Santa Clara, Calif., USA) as directed and the overnight culture was grown in LB + kanamycin medium. LB cultures were used to inoculate expression cultures grown in overnight expression ready TB + kanamycin medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). The cultures were incubated at 30 ℃ for 20h, and the cells were harvested by centrifugation (4000 Xg, 30min, 4 ℃) and stored at-20 ℃.

The reaction (0.5mL) was carried out at 30 ℃ in potassium phosphate buffer (100mM, pH7.0), pyridoxal phosphate (2mM), isopropylamine (150mM), 3-formyl-5-methylhexanoic acid (50mM), and omega-transaminase (40mg wet cells/mL) from Vibrio fluvialis, Rhodobacter sphaeroides, or Paracoccus denitrificans (Paracoccus densilicans). After 24h, the mixture was washed with 0.4mL of acetonitrile: the reaction sample (0.1mL) was diluted with water (1: 1, v/v). An aliquot (0.05mL) of the diluted reaction sample was treated with saturated aqueous sodium bicarbonate (0.01mL) and Marfey's reagent (N- α - (2, 4-dinitro-5-fluorophenyl) alaninamide, 0.2mL of a 5g/L solution in acetonitrile) at 40 ℃. After 1h, the derivatization reaction was quenched with 0.01mL of 1N aqueous hydrochloric acid and diluted with 0.23mL of acetonitrile. The derivatized reaction samples were analyzed by UPLC (column: BEHC18, 50mm x2.1mm id, gradient elution: 70% A: 30% B to 55% A: 45% B (A ═ 1% triethylamine (pH 3 using phosphoric acid); B ═ acetonitrile) over 5min, flow rate: 0.8mL/min, column temperature: 30 ℃, detection: 210-.

TABLE 5

Example 31

The recombinant variant of Vibrio fluvialis was tested for reductive amination of 3-formyl-5-methylhexanoic acid to form pregabalin. The variant of vibrio fluvialis ω -transaminase (accession number AEA39183) is expressed in e.coli as follows: QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA, USA) was used as directed to generate Vibrio fluvialis omega-transaminase variants. Primers were ordered from Integrated DNA Technologies (Coralville, IA, USA). The pET28b ω -transaminase expression construct was transformed into BL21(DE3) E.coli (Stratagene, Agilent technologies, Santa Clara, Calif., USA) as directed and the overnight culture was grown in LB + kanamycin medium. LB cultures were used to inoculate expression cultures grown in overnight expression ready TB + kanamycin medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). The cultures were incubated at 30 ℃ for 20h, and the cells were harvested by centrifugation (4000 Xg, 30min, 4 ℃) and stored at-20 ℃.

The reaction (0.5mL) was carried out at 30 ℃ using pyridoxal phosphate (2mM), isopropylamine (300mM), 3-formyl-5-methylhexanoic acid (100mM), and Vibrio fluvialis ω -transaminase wild-type or variant (40mg wet cells/mL) in potassium phosphate buffer (100mM, pH 7.0). After 28h, the mixture was washed with 0.4mL of acetonitrile: the reaction sample (0.1mL) was diluted with water (1: 1, v/v). An aliquot (0.05mL) of the diluted reaction sample was treated with saturated aqueous sodium bicarbonate (0.01mL) and Marfey's reagent (N- α - (2, 4-dinitro-5-fluorophenyl) alaninamide, 0.2mL of a 5g/L solution in acetonitrile) at 40 ℃. After 1h, the derivatization reaction was quenched with 0.01mL of 1N aqueous hydrochloric acid and diluted with 0.23mL of acetonitrile. The derivatized reaction samples were analyzed by UPLC (column: BEHC18, 50mm x2.1mm id, gradient elution: 70% A: 30% B to 55% A: 45% B (A ═ 1% triethylamine (pH 3 using phosphoric acid); B ═ acetonitrile) over 5min, flow rate: 0.8mL/min, column temperature: 30 ℃, detection: 210-.

TABLE 6

Note-1: entries 25 to 33-reactions were performed using 400mM of 3-formyl-5-methylhexanoic acid, 3mM PLP, 800mM IPM at 45 ℃.

2：Vfat 888：

DNA sequence

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACCAGCGTGGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTAACAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGTACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGAACTGGCGAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCAGCGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCGAGCGTATCGCTAACACCTGTACCGACCTGGGCCTGATCTGTAGCCCGATGGGTCAGTCCGTTATCCTGTGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA(SEQ ID NO.10)

Amino acid sequence

MNKPQSWEARAETYSLYGFTDMPSLHQRGTVVVTHGEGPYIVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAVSASMTGLPYNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELAKRLETAIEAIEEFPHGFTASGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSERIANTCTDLGLICSPMGQSVILCPPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ IDNO.2)

Example 32

(E) Enzymatic reduction of-3-formyl-5-methylhexa-2-enoic acid to 3-formyl-5-methylhexanoic acid and conversion with omega Promozyme in-situ conversion to pregabalin

(E) Reduction of-3-formyl-5-methylhexa-3-enoic acid to 3-formyl-5-methylhexanoic acid using pentaerythritol tetranitrate reductase and in situ conversion to pregabalin was evaluated with various ω -transaminases. Recombinant ω -transaminase from vibrio fluvialis, rhodobacter sphaeroides and paracoccus denitrificans is expressed in e.coli as follows: the pET28b ω -transaminase expression construct was transformed into BL21(DE3) E.coli (Stratagene, Agilent Technologies, Santa Clara, Calif., USA) as directed and the overnight culture was grown in LB + kanamycin medium. LB cultures were used to inoculate expression cultures grown in overnight expression ready TB + kanamycin medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). The cultures were incubated at 30 ℃ for 20h, and the cells were harvested by centrifugation (4000 Xg, 30min, 4 ℃) and stored at-20 ℃.

Reaction (0.5mL) at 30 ℃ using pentaerythritol tetranitrate reductase (40mg wet cells/mL), Lactobacillus brevis alcohol dehydrogenase (32U/mL), NADP⁺(0.02mM), 2-propanol (3 vol%), pyridoxal phosphate (2mM), isopropylamine (100mM), (E) -3-formyl-5-methylhexa-2-enoic acid (20mM) and a ω -transaminase from Vibrio fluvialis, Pseudomonas rhodochronas or Paracoccus denitrificans (40mg wet cells/mL) were carried out in potassium phosphate buffer (100mM, pH 7.0). after 43h, the reaction sample (0.1mL) was diluted with 0.1mL of acetonitrile: water (1: 1, v/v). after treatment of an aliquot of the diluted reaction sample (0.1mL) with saturated aqueous sodium bicarbonate solution (0.01mL) and Marfey reagent (N- α - (2, 4-dinitro-5-fluorophenyl) alaninamide, 0.4mL of 5g/L solution in acetonitrile), after 1h derivatization was terminated with 0.1mL of 1N-HCl aqueous solution.the derivatized reaction sample was passed through a UPC (BEC: 32 min, the pH of the derivatized reaction sample was eluted through a gradient column (35% A: 35 mM, 30% triethylamine (30% A: 60: 32 mM), and the flow rate was measured using a gradient of acetonitrile (30% A: 35% to 400mM, 30% triethylamine (30% A: 60.5% B) as indicated in 1 nm).

TABLE 7

Example 33

(E) Enzymatic reduction of-3-formyl-5-methylhexa-2-enoic acid to 3-formyl-5-methylhexanoic acid and use of river arcs In-situ conversion of bacteria omega-transaminase into pregabalin

(E) The reduction of-3-formyl-5-methylhexan-3-enoic acid to 3-formyl-5-methylhexanoic acid using pentaerythritol tetranitrate reductase and in situ conversion to pregabalin was evaluated with a variant of vibrio fluvialis transaminase. The variant of vibrio fluvialis ω -transaminase (accession number AEA39183) is expressed in e.coli as follows: QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA, USA) was used as directed to generate Vibrio fluvialis omega-transaminase variants. Primers were ordered from Integrated DNA Technologies (Coralville, IA, USA). The pET28b ω -transaminase expression construct was transformed into BL21(DE3) E.coli (Stratagene, Agilent technologies, Santa Clara, Calif., USA) as directed and the overnight culture was grown in LB + kanamycin medium. LB cultures were used to inoculate expression cultures grown in overnight expression ready TB + kanamycin medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). The cultures were incubated at 30 ℃ for 20h, and the cells were harvested by centrifugation (4000 Xg, 30min, 4 ℃) and stored at-20 ℃.

Reaction (0.5mL) at 30 ℃ using pentaerythritol tetranitrate reductase (40mg wet cells/mL), Lactobacillus brevis alcohol dehydrogenase (32U/mL), NADP⁺(0.1mM), 2-propanol (3 vol%), pyridoxal phosphate (2mM), isopropylamine (300mM), (E) -3-formyl-5-methylhexa-2-enoic acid (100mM), and Vibrio fluvialis ω -transaminase wild-type or variant (40mg wet cells/mL) were performed in potassium phosphate buffer (100mM, pH 7.0). After 48h, the mixture was quenched with 0.18mL of acetonitrile: reaction sample (0) was diluted with water (1: 1, v/v)02 mL.) an aliquot of the diluted reaction sample (0.1mL) was treated with saturated aqueous sodium bicarbonate (0.01mL) and Marfey reagent (N- α - (2, 4-dinitro-5-fluorophenyl) alaninamide, 0.4mL of a 5g/L solution in acetonitrile) at 40 deg.C after 1h the derivatization reaction was stopped with 0.1mL of 1N aqueous hydrochloric acid solution the derivatized reaction sample was analyzed by UPLC (column: BEHC18, 50mm x2.1mm id, gradient elution, 70% A: 30% B to 55% A: 45% B (A ═ 1% triethylamine (pH 3 using phosphoric acid); B ═ acetonitrile) over 5min at a flow rate of 0.8mL/min, column temperature: 30 deg.C, detection: 210-.

TABLE 8

Example 34

Substitutional variants of vibrio fluvialis omega-transaminase

The following other recombinant variants of Vibrio fluvialis ω -transaminase were expressed in E.coli as follows: the pET28b ω -transaminase expression construct was transformed into BL21(DE3) E.coli (Stratagene, Agilent technologies, Santa Clara, Calif., USA) as directed and the overnight culture was grown in LB + kanamycin medium. LB cultures were used to inoculate expression cultures grown in overnight expression ready TB + kanamycin medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). The cultures were incubated at 30 ℃ for 20h, and the cells were harvested by centrifugation (4000 Xg, 30min, 4 ℃) and stored at-20 ℃.

Example 34 a: vcat 906

DNA sequence:

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACgAGCGT GGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTaacAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGTACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCggcGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCaaaCGTATCGCTAACACCTGTcagGACCTGGGCCTGATCTGTAGCgCGCTGGGTCAGTCCGTTATCCTGTGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA(SEQ IDNO.11)

amino acid sequence:

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYIVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAVSASMTGLPYNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSALGQSVILCPPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ IDNO.3)

example 34 b: vflat 999

DNA sequence:

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACGTGGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAACACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGCTGGGTCAGTCCGTTATCCTGAGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA(SEQ ID NO.12)

amino acid sequence:

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYVVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSALGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ IDNO.4)

example 34 c: vcat 1010

DNA sequence:

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTATGCCGCACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGCACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAACACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGAGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA(SEQ ID NO.13)

amino acid sequence:

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYIVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGMPHNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPALSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ IDNO.5)

example 34 d: vcat 1020

DNA sequence:

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACGTGGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGGGTTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAACACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGAGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA(SEQ ID NO.14)

amino acid sequence:

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYVVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGLPLPGFVHLGCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICAAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ IDNO.6)

example 34 e: vcat 1030

DNA sequence:

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACATCGTGGACGTCCACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGAGCTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAACACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGAGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA(SEQ ID NO.15)

amino acid sequence:

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYIVDVHGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGLPLPGFVHLSCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA(SEQ IDNO.7)

example 35

Bioconversion of 3-isobutylidene-2-oxoglutarate to pregabalin

Pseudomonas putida benzoyl decarboxylase, Enterobacter cloacae pentaerythritol tetranitrate reductase, Lactobacillus brevis alcohol dehydrogenase and Vibrio fluvialis omega-transaminase were cloned into an expression vector pDSTRC52(Pfizer Inc., USA), and put into an expression strain Escherichia coli BDG62(Pfizer Inc., USA). The cultures were grown and enzyme production was induced as described in example 22. Enzyme expression was determined by polyacrylamide gel electrophoresis using Novex gel and stain (Invitrogen Corporation Carlsbad, California).

The reaction (1.0mL) was carried out at 40 ℃ in one chamber using Pseudomonas putida decarboxylase, Vibrio fluvialis transaminase, Lactobacillus brevis alcohol dehydrogenase, Enterobacter cloacae pentaerythritol tetranitrate reductase and NADP (0.1mM) in potassium phosphate buffer (100mM, pH 6.4). 200 μ L of 1M 3-isobutylidene-2-oxoglutarate, 100 μ L of 1mM ThDp +25mM MgSO4, 40 μ L of 50mM PLP, 30 μ L isopropanol, 250 μ L of 2M isopropylamine. Adjusting the pH to 6.4 for 24h, and then mixingpH adjusted to 6.8 for an additional 24h an aliquot of the reaction (0.5mL) was treated with 1M aqueous sodium bicarbonate (0.05mL) and Marfey's reagent (N- α - (2, 4-dinitro-5-fluorophenyl) alaninamide, 0.5mL of a 5g/L solution in acetonitrile) at 40 deg.C after 1h the derivatization reaction was stopped with 0.05mL of 1N aqueous hydrochloric acid solution the derivatized reaction sample was filtered and analyzed by UPLC (column: Agilent Eclipse Plus C18 column (100 mm. times.3.0 mm,1.8 μ M), eluted with 0.1% trifluoroacetic acid in water: acetonitrile (60:40, v/v) at 1.3mL/min, the column was maintained at 30 deg.C and the effluent at 340nm and ES was maintained⁺And (4) monitoring on a mass spectrum. This reaction produced a small amount of pregabalin, 82% of which was the desired S-isomer.

Example 36

Bioconversion of 3- (2-methylpropyl) -2-oxoglutarate to pregabalin

Pseudomonas putida benzoyl decarboxylase and Vibrio fluvialis omega-transaminase were cloned into the expression vector pDSTRC52(Pfizer Inc., USA) and placed into the expression strain Escherichia coli BDG62(Pfizer Inc., USA). The cultures were grown and enzyme production was induced as described in example 22. Enzyme expression was determined by polyacrylamide gel electrophoresis using Novex gel and stain (Invitrogen Corporation Carlsbad, California).

Reaction (2.0mL) Using 500. mu.L of culture (42.5mg dry cell weight) (Pseudomonas putida decarboxylase and Vibrio fluvialis omega-transaminase cloned into one plasmid), 400. mu.L of 0.5M 3- (2-methylpropyl) -2-oxoglutarate, 200. mu.L 10 × ThDP (final 0.1mM) and MgSO 40 ℃ from a 24-hour fermentor₄(final 2.5mM), 80. mu.L of 50mM PP (final 2mM), 300. mu.L of 2M isopropylamine (final 0.3M) were performed in potassium phosphate buffer (100mM, pH 6.4). The pH was adjusted to 6.4 and incubated at 45 ℃ for 24 hours, then the pH was adjusted to 6.8 for additional24 h. an aliquot of the reaction (0.5mL) was treated with 1M aqueous sodium bicarbonate (0.05mL) and Marfey's reagent (N- α - (2, 4-dinitro-5-fluorophenyl) alaninamide, 0.5mL of a 5g/L solution in acetonitrile) at 40 deg.C.after 1h, the derivatization reaction was stopped with 0.05mL of 1N aqueous hydrochloric acid solution, the derivatized reaction sample was filtered and analyzed by UPLC (column: Agilent Eclipse U.S. C18 column (100 mm. times.3.0 mm,1.8 μ M), eluted with 0.1% trifluoroacetic acid in water: acetonitrile (60:40, v/v) at 1.3mL/min, the column was maintained at 30 deg.C and the effluent at 340nm and ES⁺And (4) monitoring on a mass spectrum. This reaction produced 100. mu.g/mL of pregabalin, 65% of which was the desired S-isomer.

Example 37

Preparation via hydrolysis and enzymatic transamination of 5-methoxy-4- (2-methylpropyl) -dihydrofuran-2 (3H) -one (S) -pregabalin

5-methoxy-4- (2-methylpropyl) -dihydrofuran-2 (3H) -one (2.58g, 15mmol, see example 13) was suspended in DIW (5.2g) and cooled in an ice/water bath. Aq KOH (50% w/w, 1.77g, 1.05eq) was added dropwise over 5min via syringe. The reaction was removed from the ice/water bath and stirred at room temperature for 90 min. The pH was adjusted to 7.0 using formic acid. The reaction mixture is then used as starting material in a subsequent transaminase reaction.

Transaminase solution (12.5g), PLP (35mg), DIW (15mL) and 4.0M isopropylamine HCl aq soln (7.5mL, 30mmol) were added to a 100mL flask and heated to 45 ℃. Hydrolysis reaction was added in one portion followed by DIW (6mL, used as a container rinse). The pH of the reaction was adjusted to 7.25 with pure isopropylamine and the reaction was stirred at 45 ℃ until the reaction was complete. The reaction mixture was then heated to an internal temperature of 55 ℃ and the pH was adjusted to 4.0 with 95% formic acid. Darco carbon (125mg) was added and the mixture was cooled to room temperature followed by cooling on ice/water for 20 minutes. The mixture was then filtered through Whatman paper No. 3. The filtrate was concentrated to one third of its weight and then heated to 55 ℃. The pH of the solution was then adjusted to pH7.5 with 50% KOH, after which the solution was cooled to room temperature and then cooled to 0-5 ℃ in an ice/water bath. Precipitation of the product was observed on cooling. The slurry was filtered and washed with DIW/EtOH (10mL, 1:1, 0 ℃). The white precipitate was dried in a vacuum oven (45 ℃) for 12h to give (S) -pregabalin in 61% yield, 98.6% w/w purity and 99.8% ee (preferred S-isomer).

Example 38

Preparation via hydrolysis and enzymatic transamination of 5-butoxy-4- (2-methylpropyl) -dihydrofuran-2 (3H) -one (S) -pregabalin.

5-butoxy-4- (2-methylpropyl) -dihydrofuran-2 (3H) -one (3.21g, 15mmol, see example 13C) was suspended in DIW (8.5g) and cooled in an ice/water bath. Aq KOH (50% w/w, 2.02g,1.2eq) was added dropwise over 5min via syringe. The reaction was removed from the ice/water bath and stirred at room temperature for 90 min. The pH was then adjusted to 7.0 using formic acid and the reaction mixture was then used as starting material in the subsequent transaminase reaction.

Transaminase solution (12.5g), PLP (35mg), DIW (15mL) and 4.0M isopropylamine HCl solution (7.5mL, 30mmol) were added to a 100mL flask and heated to 45 ℃. The hydrolysis reaction was added in one portion (see above) followed by DIW (6mL, used as a vessel rinse). The pH of the reaction was adjusted to 7.25 with pure isopropylamine and the reaction was stirred at 45 ℃.

The reaction mixture was heated to an internal temperature of 55 ℃ and the pH was adjusted to 4.0 with 95% formic acid. Darco carbon (125mg) was added and the mixture was cooled to room temperature followed by cooling on ice/water for 20 minutes. The mixture was then filtered through Whatman paper No. 3. The filtrate was concentrated to one third of its weight and then heated to 55 ℃. The pH of the solution was then adjusted to pH7.5 with 50% KOH, after which the solution was cooled to room temperature and then cooled to 0-5 ℃ in an ice/water bath. Precipitation of the product was observed on cooling. The slurry was filtered and washed with DIW/EtOH (10mL, 1:1, 0 ℃). The white precipitate was dried in a vacuum oven (45 ℃) for 12 hours to give (S) -pregabalin in 51% yield, 98.4% w/w purity and 99.9% ee of the preferred S-isomer.

Example 39

Preparation of (S) -prorey from 5-hydroxy-4- (2-methylpropyl) -dihydrofuran-2 (3H) -one via enzymatic transamination Barlin

5-hydroxy-4- (2-methylpropyl) -dihydrofuran-2 (3H) -one (2.0g,12.5mmol) was suspended in DIW (8.5g) and cooled in an ice/water bath. Added portionwise over the course of 5min (0.863g,6.3 mmol). The reaction was removed from the ice/water bath and stirred at room temperature for 90 min. The pH was adjusted to 7.0 using formic acid and the reaction mixture was then used as starting material in the subsequent transaminase reaction.

Transaminase solution (10.4g), PLP (30mg), DIW (12.5mL) and aq 4.0M isopropylamine HCl aq soln (6.3mL, 30mmol) were added to a 100mL flask and heated to 45 ℃. Hydrolysis reaction was added in one portion followed by DIW (5mL, used as a container rinse). The pH of the reaction was adjusted to 7.25 with pure isopropylamine and the reaction was stirred at 45 ℃. The reaction mixture was then heated to an internal temperature of 55 ℃ and the pH was adjusted to 4.0 with 95% formic acid. Darco carbon (125mg) was added and the mixture was cooled to room temperature followed by cooling on ice/water for 20 minutes. The mixture was then filtered through Whatman paper No. 3. The filtrate was concentrated to one third of its weight and then heated to 55 ℃. The pH of the solution was then adjusted to pH7.5 with 50% KOH, after which the solution was cooled to room temperature and then cooled to 0-5 ℃ in an ice/water bath. Precipitation of the product was observed on cooling. The slurry was filtered and washed with DIW/EtOH (10mL, 1:1, 0 ℃). The white precipitate was dried in a vacuum oven (45 ℃) for 12 hours to give (S) -pregabalin in 61% yield, 98.3% w/w purity and 99.9% ee of the preferred S-isomer.

Example 40

Preparation of (R/S) -one via reductive amination of 5-hydroxy-4- (2-methylpropan-1-en-1-yl) furan-2 (5H) -one 3-aminomethyl-5-methylhexanoic acid

A0.01M solution of 5-hydroxy-4- (2-methylprop-1-en-1-yl) furan-2 (5H) -one in 30% aqueous ammonia was hydrogenated in the presence of 10 mol% Raney nickel catalyst (10bar, ambient temperature) for 48H. The catalyst was filtered and the solution was concentrated to leave a solid. The product was isolated by adding hydrochloric acid to a methanol suspension and found to be pure 3-aminomethyl-5-methylhexanoic acid hydrochloride.

Using palladium as a catalyst yields a mixture of 3-aminomethyl-5-methylhexanoic acid and the corresponding secondary amine 3- [ (2-carboxymethyl-4-methyl-pentylamino) -methyl ] -5-methyl-hexanoic acid.

EXAMPLE 41

Conversion of R-3-aminomethyl-5-methylhexanoic acid to 5-hydroxy-4- (2-methylpropyl) -3, 4-di-n using transaminase Hydrogen-5H-2-furanones

A solution of R-3-aminomethyl-5-methylhexanoic acid in d.i. water at ph7.5/45 ℃ was stirred with a whole cell preparation or transaminase lysate containing pyridoxal phosphate (PLP) and acetone. The isopropylamine produced was removed via a nitrogen purge. Analysis of the solution after 24H showed the presence of 3-aminomethyl-5-methyl-hexanoic acid with a lower e.e. and the compound 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone. The decrease in chiral purity of 5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone is due to the selective conversion of (4S) -5-hydroxy-4- (2-methylpropyl) -3, 4-dihydro-5H-2-furanone to S-pregabalin.

Example 42

Deracemisation of rac-pregabalin using a transaminase

A solution of racemic pregabalin in DIW, pH7.5, was treated with the appropriate transaminase lysate. PLP was added and the reaction was carried out at 45 ℃ under a nitrogen purge for 12 h. Then isopropylamine was added and the reaction was continued for an additional 12 h. The product was isolated as in example 38 to give enantiomerically enriched pregabalin.

It is also possible to use a suitable amine oxidase/imine reductase system to effect the transformations exemplified in examples 41 and 42 with a cofactor such as NADP.

Sequence listing

SEQ ID NO.1

General Vflat aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHX²⁷RGTVVVTHGEGPYX⁴¹VDVX⁴⁵GRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRX¹⁴⁷NAYHGVTAVSASMTGX¹⁶³PX¹⁶⁵NSVFGLPLPGFVHLX¹⁸⁰CPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELX³⁰⁴KRLETAIEAIEEFPHGFTAX³²⁴GHPVGCAIALKAIDWMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSX⁴⁰¹RIANTCX⁴⁰⁸DLGLICX⁴¹⁵X⁴¹⁶X⁴¹⁷GQSVILX⁴²⁴PPFILTEAQMDEMFDKLEKALDKVFAEVA

X27 is selected from glutamine (Q) and glutamic acid (E); x41 is selected from isoleucine (I) and valine (V); x45 is selected from asparagine (N) and histidine (H); x147 is selected from asparagine (N) and glutamine (Q); x163 is selected from leucine (L) and methionine (M); x165 is selected from tyrosine (Y) and histidine (H); x180 is selected from threonine (T); glycine (G) and serine (S); x304 is selected from alanine (A) and serine (S); x324 is selected from glycine (G) and serine (S); x401 is selected from lysine (K) and glutamic acid (E); x408 is selected from threonine (T) and glutamine (Q); x415 is selected from serine (S) and alanine (a); x416 is selected from proline (P) and alanine (a); x417 is selected from leucine (L) and methionine (M); and X424 is selected from cysteine (C) and serine (S).

SEQ ID NO.2

Vcat 888aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHQRGTVVVTHGEGPYIVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAVSASMTGLPYNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELAKRLETAIEAIEEFPHGFTASGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSERIANTCTDLGLICSPMGQSVILCPPFILTEAQMDEMFDKLEKALDKVFAEVA

SEQ ID NO.3

Vcat 906aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYIVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAVSASMTGLPYNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSALGQSVILCPPFILTEAQMDEMFDKLEKALDKVFAEVA

SEQ ID NO.4

Vcat 999aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYVVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSALGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA

SEQ ID NO.5

Vcat 1010aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYIVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGMPHNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPALSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA

SEQ ID NO.6

Vcat 1020aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYVVDVNGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGLPLPGFVHLGCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICAAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA

SEQ ID NO.7

Vcat 1030aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHERGTVVVTHGEGPYIVDVHGRRYLDANSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGLPLPGFVHLSCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQDLGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA

SEQ ID NO.8

Tomato (tomato) 12-oxophytadienoate reductase 1 protein sequence:

MENKVVEEKQVDKIPLMSPCKMGKFELCHRVVLAPLTRQRSYGYIPQPHAILHYSQRSTNGGLLIGEATVISETGIGYKDVPGIWTKEQVEAWKPIVDAVHAKGGIFFCQIWHVGRVSNKDFQPNGEDPISCTDRGLTPQIRSNGIDIAHFTRPRRLTTDEIPQIVNEFRVAARNAIEAGFDGVEIHGAHGYLIDQFMKDQVNDRSDKYGGSLENRCRFALEIVEAVANEIGSDRVGIRISPFAHYNEAGDTNPTALGLYMVESLNKYDLAYCHVVEPRMKTAWEKIECTESLVPMRKAYKGTFIVAGGYDREDGNRALIEDRADLVAYGRLFISNPDLPKRFELNAPLNKYNRDTFYTSDPIVGYTDYPFLETMT

SEQ ID NO.9

tomato (tomato) 12-oxophytodienoic acid reductase 1 codon optimized sequence:

ATGGAAAACAAAGTTGTGGAAGAAAAACAGGTTGATAAAATCCCGCTGATGAGCCCGTGTAAAATGGGTAAATTCGAGCTGTGTCATCGCGTTGTACTGGCACCGCTGACTCGTCAGCGTTCTTATGGTTACATTCCGCAGCCGCACGCAATCCTGCATTACTCTCAGCGCAGCACCAACGGTGGCCTGCTGATCGGTGAAGCAACCGTGATCAGCGAAACTGGCATCGGTTACAAAGATGTGCCGGGTATCTGGACGAAAGAGCAGGTTGAGGCCTGGAAACCGATCGTCGACGCGGTGCATGCCAAAGGTGGTATTTTCTTTTGTCAGATCTGGCACGTTGGTCGTGTATCCAACAAAGATTTTCAGCCGAACGGCGAAGATCCGATTTCCTGTACTGACCGCGGCCTGACCCCGCAGATCCGTTCCAACGGCATTGACATTGCCCACTTCACCCGTCCACGTCGCCTGACTACTGACGAGATTCCGCAGATCGTGAACGAGTTCCGCGTTGCAGCGCGTAATGCTATTGAAGCGGGTTTCGATGGCGTCGAGATTCATGGTGCCCACGGTTACCTGATCGACCAATTCATGAAAGACCAAGTTAACGACCGCAGCGATAAGTATGGCGGTTCTCTGGAGAACCGTTGTCGCTTCGCGCTGGAAATCGTTGAAGCAGTAGCCAACGAGATTGGCTCCGACCGTGTTGGTATCCGTATCTCTCCATTCGCACACTACAACGAAGCGGGCGACACTAACCCGACCGCACTGGGCCTGTATATGGTGGAGAGCCTGAATAAATACGACCTGGCGTATTGTCACGTGGTCGAGCCGCGCATGAAAACCGCCTGGGAAAAGATTGAGTGCACCGAAAGCCTGGTGCCGATGCGTAAAGCCTACAAAGGCACCTTCATCGTAGCTGGTGGCTACGACCGTGAAGACGGTAACCGCGCTCTGATCGAAGACCGTGCCGACCTGGTTGCGTACGGTCGTCTGTTCATCAGCAACCCAGACCTGCCGAAGCGTTTTGAACTGAACGCTCCGCTGAACAAATACAACCGTGACACTTTCTACACTTCCGACCCGATCGTTGGTTACACCGATTACCCGTTTCTGGAAACTATGACTTAATAA

SEQ ID NO.10

vflat 888DNA sequence

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACCAGCGTGGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTAACAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGTACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGAACTGGCGAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCAGCGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCGAGCGTATCGCTAACACCTGTACCGACCTGGGCCTGATCTGTAGCCCGATGGGTCAGTCCGTTATCCTGTGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA

SEQ ID NO.11

Vcat 906DNA sequence

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACgAGCGTGGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTaacAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGTACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCggcGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCaaaCGTATCGCTAACACCTGTcagGACCTGGGCCTGATCTGTAGCgCGCTGGGTCAGTCCGTTATCCTGTGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA

SEQ ID NO.12

Vbat 999DNA sequence

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACGTGGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAACACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGCTGGGTCAGTCCGTTATCCTGAGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA

SEQ ID NO.13

Vcat 1010DNA sequence

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTATGCCGCACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGCACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAACACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGAGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA

SEQ ID NO.14

Vcat 1020DNA sequence

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACGTGGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGGGTTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAACACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGAGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA

SEQ ID NO.15

Vcat 1030DNA sequence

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTTCACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAGGGCCCATACATCGTGGACGTCCACGGTCGCCGTTACCTGGACGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAAACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAACTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGAGCTGTCCGCACTATTGGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAACACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGAGCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA

Claims

1. According to formula (VI)^A) 5-hydroxy-4- (2-methyl-1-propenyl) -5H-2-furanone of (5-hydroxy-4)