PROCESS AND INTERMEDIATES FOR THE PREPARATION OF PREGABALIN

Field of the Invention The present invention relates to the manufacture of 5-hydroxy-4-(2-methylpropyl)-3,4- dihydro-5/-/-2-furanone, and derivatives thereof, for use in the manufacture of (S)-(+)-3- aminomethyl-5-methylhexanoic acid (Pregabahn). The invention further relates to an improved process for the conversion of 5-hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2- furanone into Pregabahn. Pregabahn is a γ-amino acid that exhibits binding affinity to the human 00 ₂ 6 calcium channel subunit.

Background to the Invention

Pregabahn, or (S)-(+)-3-aminomethyl-5-methyl-hexanoic acid ((S)-ll),

Pregabahn ((S)-ll)

is the active agent in Lyrica®, which is approved for the treatment of epilepsy, neuropathic pain, fibromyalgia and generalized anxiety disorder. It exhibits anti-seizure activity, as discussed in U.S. Patent US 5,563, 175, and anti-nociceptive activity, as discussed in U.S. Patent US 6,001 ,876. It is hypothesised that the pharmacological activity of Pregabahn (II) is the result of binding to the alpha-2-delta (α ₂ δ) subunit of a calcium channel. Pregabahn (II) is also described as having utility in other conditions, such as physiological conditions associated with psychomotor stimulants, inflammation, gastrointestinal damage, alcoholism, insomnia, and various psychiatric disorders, including anxiety, depression, mania, and bipolar disorder.

A number of methods for manufacturing Pregabahn have been disclosed. 5-Hydroxy-4- (2-methylpropyl)-3,4-dihydro-5/-/-2-furanone (l ^A ) has been identified as a useful precursor.

(|A)

It will be appreciated that compound (l ^A ), in common with a number of the other compounds discussed herein, can be considered to be the cyclized isomer of a 4- oxobutanoic acid derivative. Derivatives of 4-oxobutanoic acid can exist as either the open-chain form, or as the cyclic form.

"Open chain" "Cyclic"

isomer isomer

These two isomeric forms may co-exist in equilibrium, and the relative contributions of the two forms will depend on the precise chemical nature of the species. International Patent Application PCT/US2008/004699 (published as WO2008/127646A2) proposes the conversion of 5-hydroxy-4-(2-methylpropyl)-3,4- dihydro-5/-/-2-furanone (l ^A ) or an ester of the ring-opened isomeric form (XIII, wherein R ^A is alkyl) to (II) using a chemical or an enzyme-mediated reductive amination. It is suggested that the use of a transaminase enzyme will provide selectively the preferred ((S)-ll) enantiomer.

(XIII)

The ester (XIII) is prepared from 4-methylpentanal (III) by alkylation with an appropriate haloacetate in the presence of diisobutylamine. The precursor (l ^A ) is made either by ester hydrolysis of ester (XIII) or by condensation of 4-methylpentanal (III) with glyoxylic acid (IV) and subsequent reduction of the double bond. The use of an enzyme- mediated reduction is also suggested as a way of introducing the desired stereochemistry.

(|A)

International Patent Application PCT/IN2010/000140 (published as WO201 1/086565) discloses a related process. The condensation product of 4-methylpentanal (III) and glyoxylic acid (IV) is reacted with a chiral amine such as a-methylbenzylamine to give a pyrrolone (V). Hydrogenation gives some degree of stereoselectivity, and deprotection gives Pregabalin (II) in chiral form.

((S)-N)

Still further improved syntheses of Pregabalin (II) are sought. It is especially desirable to provide a process which is cost effective and safe. In particular, it is important to provide a synthesis of Pregabalin (II) which can be carried out on a commercial scale, which uses readily available, inexpensive and safe starting materials and reagents, and which avoids the need for difficult separations. Summary of the Invention

The present invention provides improved methods for the manufacture of 5-hydroxy-4- (2-methylpropyl)-3,4-dihydro-5/-/-2-furanone (l ^A ), and intermediates that are useful in these improved methods. In a first aspect A1 , the invention provides a compound according to formula (VI)

(VI)

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

hydrogen,

(Ci-C ₆ )alkyl,

(Ci-C ₆ )haloalkyl,

(Ci-C ₃ )alkoxy(C2-C ₆ )alkyl,

(C ₂ -C ₆ )alkenyl,

(C ₃ -Cio)cycloalkyl, which may optionally be substituted with 1, 2 or 3 groups independently selected from halo, (Ci-C3)alkyl, and (Ci-C3)alkyloxy, (C3-Cio)cycloalkyl(Ci-C6)alkyl, wherein the cycloalkyi group may optionally be substituted with 1, 2 or 3 groups independently selected from halo, (d- C ₃ )alkyl, and (Ci-C ₃ )alkyloxy,

aryl, which may optionally be substituted with 1, 2 or 3 groups independently selected from halo, (Ci-C3)alkyl, and (Ci-C3)alkyloxy,

aryl(Ci-C ₆ )alkyl, wherein the aryl group may optionally be substituted with 1, 2 or 3 groups independently selected from halo, (Ci-C ₃ )alkyl, and (d- C ₃ )alkyloxy,

R ¹ -C(O)-, and

R ² -SO ₂ -;

R ¹ is selected from:

hydrogen,

(Ci-C ₆ )alkyl,

(Ci-C ₆ )haloalkyl,

(Ci-C ₃ )alkoxy(C ₂ -C ₆ )alkyl,

(C ₂ -C ₆ )alkenyl,

(C ₃ -Cio)cycloalkyl, which may optionally be substituted with 1, 2 or 3 groups independently selected from halo, (Ci-C ₃ )alkyl, and (Ci-C ₃ )alkyloxy, (C3-Cio)cycloalkyl(Ci-C6)alkyl, wherein the cycloalkyl group may optionally be substituted with 1, 2 or 3 groups independently selected from halo, (d-

C ₃ )alkyl, and (Ci-C ₃ )alkyloxy,

aryl, which may optionally be substituted with 1, 2 or 3 groups independently selected from halo, (Ci-C3)alkyl, and (Ci-C3)alkyloxy, and

aryl(Ci-C ₆ )alkyl, wherein the aryl group may optionally be substituted with 1, 2 or

3 groups independently selected from halo, (Ci-C ₃ )alkyl, and (d-

C ₃ )alkyloxy,

and R ² is selected from:

(Ci-C ₆ )alkyl,

(Ci-C ₆ )haloalkyl,

(C ₁ -C ₃ )alkoxy(C ₂ -C ₆ )alkyl,

(C ₂ -C ₆ )alkenyl,

(C ₃ -Cio)cycloalkyl, which may optionally be substituted with 1, 2 or 3 groups independently selected from halo, (Ci-C ₃ )alkyl, and (Ci-C ₃ )alkyloxy,

(C ₃ -Cio)cycloalkyl(Ci-C6)alkyl, wherein the cycloalkyl group may optionally be substituted with 1, 2 or 3 groups independently selected from halo, (d-

C ₃ )alkyl, and (Ci-C ₃ )alkyloxy,

aryl, which may optionally be substituted with 1, 2 or 3 groups independently selected from halo, (Ci-C ₃ )alkyl, and (Ci-C ₃ )alkyloxy, and

aryl(Ci-C ₆ )alkyl, wherein the aryl group may optionally be substituted with 1, 2 or

3 groups independently selected from halo, (Ci-C ₃ )alkyl, and (d-

C ₃ )alkyloxy.

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 5- hydroxy-4-(2-methyl-1-propenyl)-5/-/-2-furanone according to formula (VI ^A ).

(VIA) In a further embodiment A1 E3, the invention provides a compound according to embodiment A1 E1 of formula (VI ^B

(VIB)

wherein R* is a chiral (C5-C 15) hydrocarbon group.

In a further embodiment A1 E4, the invention provides a compound according to embodiment A1 E3 wherein R* is selected from (R)- or (S)-a-methylbenzyl, (R)- or (S)-1 - (l -naphthyl)ethyl, (R)- or (S)-1 -(2-naphthyl)ethyl, menthyl and bornyl, such that the com

(V|H)

In a further embodiment A1 E5, the invention provides a compound according to embodiment A1 E1 wherein R is R ¹ -C(0)- or R ² -S0 ₂ - and R ¹ and R ² are chiral (C ₅ -C ₁₅ ) hydrocarbon groups.

In another aspect A2, the vention provides a compound of formula (IX)

(IX)

The compound of formula (IX) may exist as either the (E)- or (Z)-geometric isomer, or as a mixture of the two geometric isomers.

In a first embodiment A2E1 , the invention provides a compound of formula (IX) wherein: n is 1 and M ⁺ is selected from Li ⁺ , Na ⁺ , K ⁺ , Rb ⁺ , NH ₄ ⁺ , ((Ci -C ₃ )alkyl)NH ₃ ⁺ , ((Ci - C ₃ )alkyl) ₂ NH ₂ ⁺ , ((Ci -C ₃ )alkyl) ₃ NH ⁺ and ((Ci -C ₃ )alkyl) ₄ N ⁺ ; or

n is 2 and M ²⁺ is selected from Mg ²⁺ , Ca ²⁺ and Zn ²⁺ .

In a further embodiment A2E2, the invention provides a compound according to embodiment A2E1 wherein n is 1 and M ⁺ is selected from NH ₄ ⁺ and ((Ci -C ₃ )alkyl)NH ₃ ⁺ . In a further embodiment A2E3, the invention provides a compound according to embodiment A2E1 wherein n is 1 and M ⁺ is selected from Li ⁺ , Na ⁺ and K ⁺ .

In another aspect A3, the invention provides a compound of formula (VII).

(VII)

In a first embodiment A3E1 , the invention provides a compound of formula (VII) wherein -X- represents a single bond, -CH ₂ -, -0-; -NH-,-N((Ci -C ₃ )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-5/-/-furan-2-one;

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

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

1 ,4-Jb/s-(4-(2-methylpropenyl)-5/-/-furan-2-on-5-yl)piperazin e.

In another aspect A4, the invention provides a compound of formula (VIII).

(VIII) In a first embodiment A4E1 , the invention provides a compound of formula (VIII) wherein -Y- represents a single bond, -CH ₂ - -0-; -NH-, -N((Ci -C ₃ )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-Jb/s-(4-methyl-1 ,3-pentadien-1 -yl)piperazine. In another aspect A5, the invention provides a process for the manufacture of the compound of formula (VI ^A ) comprising the step of treating a compound of formula (VII) with water in the presence of an acid catalyst.

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

(a) preparing a compound of formula (VII)

(VII)

wherein -X- represents a single bond, -CH ₂ - -0-, -NH-, -N((Ci -C 3)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 hydrolysis step (b).

In a further embodiment A5E3, the invention provides a process according to embodiment A5E1 wherein hydrolysis step (b) is carried out directly following step (a) such that the compound of formula (VII) or (VII ^B ) is not isolated prior to hydrolysis step

(b).

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

(VIII)

wherein -Y- represents a single bond, -CH ₂ - -0-; -NH- -N((Ci -C ₃ )alkyl)-, -N(benzyl)-, or

with glyoxylic acid or its hydrate. In another aspect A6, the invention provides a process for the manufacture of a compound of formula (VI) wherein R is other than hydrogen, R ¹ -C(O)-, and R ² -SO ₂ - comprising the step of treating a compound of formula (VI ^A ) with an alcohol R-OH in the presence of an acid catalyst.

In an embodiment A6E1 , the invention provides a process for the manufacture of a compound of formula (VI) wherein R is other than hydrogen, R ¹ -C(O)-, and R ² -SO ₂ - comprising the steps of:

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

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

In another aspect A7, the invention provides a process for the manufacture of a compound of formula (VI) wherein R is other than hydrogen, R ¹ -C(O)-, and R ² -SO2- comprising the step of treating acompound of formula (VII) with an alcohol R-OH in the presence of stoichiometric acid.

In an embodiment A7E1 , the invention provides a process for the manufacture of a compound of formula (VI) wherein R is other than hydrogen, R ¹ -C(O)-, and R ² -SO2- comprising the steps of:

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

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

In another aspect A8, the invention provides a process for the manufacture of a compound of formula (VI) wherein R is R ¹ -C(O)-, comprising the step of treating a compound of formula (VI ^A ) with an acid chloride R ¹ -C(O)-CI or acid anhydride (R ¹ - C(O)) ₂ O.

In an embodiment A8E1 , the invention provides a process for the manufacture of a compound of formula (VI) wherein R is R ¹ -C(O)-, comprising the steps of:

(a) preparing a compound of formula (VI ^A ) using a process according to any of embodiments A5E1 , A5E2, A5E3 and A5E4 as defined above; and (b) treating the compound of formula (VI ^A ) with an acid chloride R ¹ -C(0)-Cl or acid anhydride (R ¹ -C(0)) ₂ 0.

In another aspect A9, the invention provides a process for the manufacture of a compound of formula (VI) wherein R is R ² -S0 ₂ - comprising the step of treating a compound of formula (VI ^A ) with a sulfonyl chloride R ² -S0 ₂ -Cl.

In an embodiment A9E1 , the invention provides a process for the manufacture of a compound of formula (VI) wherein R is R ² -S0 ₂ -, comprising the steps of:

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

(b) treating the compound of formula (VI ^A ) with a sulfonyl chloride R ² -S0 ₂ -Cl.

In another aspect A10, the invention provides a process for the manufacture of an enamine derivative of 4-methyl-2-pentenal.

In a first embodiment A10E1 , the invention provides a process for the manufacture of an enamine derivative of 4-methyl-2-pentenal comprising reacting acetaldehyde with isobutyraldehyde in the presence of a suitable amine.

In a further embodiment A10E2, the invention provides a process according to embodiment A10E1 wherein the suitable amine is a secondary amine.

In a further embodiment A10E3, the invention provides a process according to embodiment A10E2 wherein the secondary amine is selected from: ((Ci -C ₄ )alkyl) ₂ NH, pyrrolidine, piperidine, morpholine, piperazine, N-methylpiperazine, N-ethylpiperazine and N-benzylpiperazine.

In a further embodiment A10E4, the 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 of embodiments A10E1 , A10E2, A10E3 and A10E4 wherein the reaction is performed in the presence of an acid catalyst. In a further embodiment A10E6, the invention provides a process according to any of embodiments A10E1 , A10E2, A10E3, A10E4 and A10E5 wherein the isobutyraldehyde is combined with the suitable amine before addition of the acetaldehyde.

In a further embodiment A10E6, the invention provides a process according to any of embodiments A10E1 , A10E2, A10E3, A10E4 and A10E5 wherein the isobutyraldehyde and acetaldehyde are added to the reaction vessel simultaneously.

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

In a first embodiment A11 E1 , the invention provides a process for the manufacture of 3- aminomethyl-5-methylhexanoic acid (II) or a pharmaceutically acceptable salt thereof, comprising the steps:

(a) preparing 5-hydroxy-4-(2-methyl-1 -propenyl)-5/-/-2-furanone (VI ^A )

(VI ^A )

(b) converting said 5-hydroxy-4-(2-methyl-1 -propenyl)-5/-/-2-furanone (VI ^A ) into 5 hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanone (l ^A )

(i ^A )

and (c) converting said 5-hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanone (l ^A ) into 3-aminomethyl-5-methylhexanoic acid (II).

In a further embodiment A11 E2, the invention provides a process according to embodiment A11 E1 wherein the 3-aminomethyl-5-methylhexanoic acid (II) is (S)-3- aminomethyl-5-methylhexanoic acid ((S)-ll)

((S)-M)

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

In a further embodiment A11 E3, the invention provides a process according to embodiment A11 E1 or A11 E2 wherein step (a) comprises a process according to embodiments A5E1 , A5E2, A5E3 or A5E4 as described above. In a further embodiment A11 E4, the invention provides a process according to embodiment A11 E1 , A11 E2 or A11 E3 wherein step (b) comprises the steps:

(b1 ) treating the 5-hydroxy-4-(2-methyl-1 -propenyl)-5/-/-2-furanone (VI ^A ) with a metal oxide, hydroxide, carbonate or bicarbonate, ammonia, a mono- di- or tri-(C i - C3)alkylamine, or a tetra-(Ci -C 3)alkylammonium hydroxide to form a salt of formula (IX)

(IX)

wherein:

n is 1 and M ⁺ is selected from Li ⁺ , Na ⁺ , K ⁺ , Rb ⁺ , NH ₄ ⁺ , ((Ci -C ₃ )alkyl)NH ₃ ⁺ , ((d - C ₃ )alkyl) ₂ NH ₂ ⁺ , ((Ci -C ₃ )alkyl) ₃ NH ⁺ and ((Ci -C ₃ )alkyl) ₄ N ⁺ ; or

n is 2 and M ²⁺ is selected from Mg ²⁺ , Ca ²⁺ and Zn ²⁺ ;

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

(X) ; and

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

In a further embodiment A11 E5, the invention provides a process according to embodiment A11 E1 , A11 E2 or A11 E3 wherein step (b) comprises the steps:

(b1 ) converting the 5-hydroxy-4-(2-methyl-1 -propenyl)-5/-/-2-furanone (VI ^A ) to a compound of formula (VI) as defined in embodiment A1 E3, wherein R is a chiral (C 5-C i 5)hydrocarbon group;

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

(XI)

wherein R ^* is a chiral (Cs-Ci5)hydrocarbon group; and

(b3) treating the compound of formula (XI) with an acid to give ((S)-I ^A ). In another aspect A12, the invention provides a further process for the manufacture of 3-aminomethyl-5-methylhexanoic acid (II).

In a first embodiment A12E1 , the invention provides a process for the manufacture of 3- aminomethyl-5-methylhexanoic acid (II) or a pharmaceutically acceptable salt thereof, comprising the steps:

(a) preparing 5-hydroxy-4-(2-methyl-1 -propenyl)-5/-/-2-furanone (VI ^A )

(VI ^A )

(b) treating the 5-hydroxy-4-(2-methyl-1 -propenyl)-5/-/-2-furanone (VI ^A ) with ammonia or a mon -(C-i -C3)alkylamine to form a salt of formula (IX ^A )

(ix ^A )

wherein n is 1 and M ⁺ is selected from NH ₄ ⁺ and ((Ci -C ₃ )alkyl)NH ₃ ⁺

(c) hydrogenating th alt of formula (IX ^A ) to tain a salt of formula (X ^A )

(X ^A ) and

(d) treating the salt of formula (X ) with a transaminase or an amine oxidase/imine reductase enzyme to provide 3-aminomethyl-5-methylhexanoic acid (II).

In a further embodiment A12E2, the invention provides a process according to embodiment A12E1 wherein the 3-aminomethyl-5-methylhexanoic acid (II) is (S)-3- aminomethyl-5-methylhexanoic acid ((S)-ll)

((S)-ll)

wherein said (S)-3-aminomethyl-5-methylhexanoic acid has an enantiomeric excess of at least 80%. In a further embodiment A12E3, the invention provides a process according to embodiment A12E1 or A12E2 wherein step (a) comprises a process according to embodiments A5E1 , A5E2, A5E3 or A5E4 as described above.

In another aspect A13, the invention provides a further process for the manufacture of 5-hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanone (l ^A ).

In a first embodiment A13E1 , the invention provides a further process for the manufacture of 5-hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanone (l ^A ) which comprises the steps of:

(a) obtaining 3-isobutylidene-2-oxopentanedioic acid (XII ^A ) or its cyclised isomer (XII ^B )

(XII ^A ) (XII ^B ) , and

(b) sequentially or simultaneously reducing the carbon-carbon double bond and decarboxylating the a-keto acid functional group.

In a further embodiment A13E2, the invention provides a process according to embodiment A13E1 wherein the carbon-carbon double bond is reduced to provide 3- isobutyl-2-oxopentanedioic acid (XV) or its cyclised isomer (XV ^A )

(XV) (XV ^A )

before the decarboxylation of the a-keto acid functional group.

In a further embodiment A13E3, the invention provides a process according to embodiment A13E1 wherein the a-keto acid functional group is decarboxylated to provide 3-formyl- -methyl-3-pentenoic acid (XVI or its cyclised isomer (XVI ^A )

(XVI) (XVI ^A )

before the reduction of the carbon-carbon double bond. In a further embodiment A13E4, the invention provides a process according to embodiment A13E1 wherein the a-keto acid functional group is decarboxylated and the carbon-carbon double bond is reduced simultaneously.

In a further embodiment A13E5, the invention provides a process according to embodiments A13E1 , A13E2, A13E3 or A13E4 wherein the decarboxylation is carried out in the presence of a decarboxylase enzyme.

In a further embodiment A13E6, the invention provides a process according to embodiments A13E1 , A13E2, A13E3, A13E4 or A13E5 wherein the reduction of the carbon-carbon double bond is carried out in the presence of an enoate reductase enzyme.

In another aspect A14, the invention provides a compound selected from:

3-isobutylidene-2-oxopentanedioic acid;

3-isobutyl-2-oxopentanedioic acid; and 3-formyl-5-methyl-3-pentenoic acid,

or a salt, (Ci -C ₆ )alkyl ester or cyclised isomer thereof.

In another aspect A15, the invention provides a process for the manufacture of (S)-3- aminomethyl-5-methylhexanoic -ll)

((S)-N)

or a pharmaceutically acceptable salt thereof, comprising the steps:

(a) manufacturing 5-hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanone (l ^A ) using a process according to any one of embodiments A13E1 , A13E2, A13E3, A13E4, A13E5 or A13E6 as defined above, and

(b) converting said 5-hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanone into (S)- 3-aminomethyl-5-methylhexanoic acid.

In another aspect A16, the invention provides a process for converting (R)-3- aminomethyl-5-methylhexanoic acid into (S)-3-aminomethyl-5-methylhexanoic acid comprising treating the (R)-3-aminomethyl-5-methylhexanoic acid with a transaminase enzyme or an amine oxidase/imine reductase enzyme.

In another aspect A17, the invention provides a process 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 enzyme or an amine oxidase/imine reductase enzyme.

In another aspect A18, the invention provides a transaminase enzyme that is useful for the conversion of 5-hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanone (l ^A ) into Pregabalin.

In a first embodiment A18E1 , the invention provides a transaminase enzyme having an amino acid sequence that has at least 95% homology to the amino acid sequence MNKPQSWEARAETYSLYGFTDMPSLHX ²⁷ RGTWVTHGEGPYX ⁴ VDVX ⁴⁵ GRRYLDAN SGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGR VFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRX ⁴⁷ NAYHGVTAVSASMTGX ⁶³ P X ⁶⁵ NSVFGLPLPGFVHLX ⁸⁰ CPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAG FFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPD AIISSKNLTAGFFPVGAVILGPELX ³⁰⁴ KRLETAIEAIEEFPHGFTAX ³²⁴ GHPVGCAIALKAID WMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGN LSVSX ⁴⁰ RIANTCX ⁴⁰⁸ DLGLICX ^{4 5} X ^{4 6} X ^{4 7} GQSVILX ⁴²⁴ PPFILTEAQMDEMFDKLEKALD KVFAEVA (SEQ ID NO. 1 ) wherein

X ²⁷ is selected from glutamine (Q) and glutamic acid (E);

X ⁴¹ is selected from isoleucine (I) and valine (V);

X ⁴⁵ is selected from asparigine (N) and histidine (H);

_X 147 is selected from asparigine (N) and glutamine (Q);

_X 163 is selected from leucine (L) and methionine (M);

_X 165 is selected from tyrosine (Y) and histidine (H);

_X 180 is selected from threonine (T); glycine (G) and serine

_X 304 is selected from alanine (A) and serine (S);

_X 324 is selected from glycine (G) and serine (S);

_X 401 is selected from lysine (K) and glutamic acid (E);

χ408 is selected from threonine (T) and glutamine (Q);

χ415 is selected from serine (S) and alanine (A);

_X 416 is selected from proline (P) and alanine (A);

_X 417 is selected from leucine (L) and methionine (M); and

_X 424 is selected from cysteine (C) and serine (S).

In a further embodiment A18E2, the invention provides a transaminase enzyme according to embodiment A18E1 having the amino acid sequence of SEQ ID NO. 1.

In a further embodiment A18E3, the invention provides a transaminase enzyme according to embodiment A18E1 or A18E2 wherein:

X ²⁷ is glutamic acid (E);

X ¹⁴⁷ is glutamine (Q);

X ¹⁶⁵ is histidine (H); X ³⁰⁴ is serine (S);

X ³²⁴ is glycine (G);

X ⁴⁰¹ is lysine (K);

X ⁴⁰⁸ is glutamine (Q);

X ⁴¹⁶ is alanine (A);

X ⁴¹⁷ is methionine (M); and

X ⁴²⁴ is serine (S).

In a further embodiment A18E4, the invention provides a transaminase according to embodiment A18E2 having an amino acid sequence selected from:

MNKPQSWEARAETYSLYGFTDMPSLHQRGTVWTHGEGPYIVDVNGRRYLDANSGL YNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFY TNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAVSASMTGLPYNSVFG LPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGA GGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLT A GFFPVGAVILGPELAKRLETAIEAIEEFPHGFTASGHPVGCAIALKAIDWMNEGLAENV RRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSERIANTCT DLGLICSPMGQSVILCPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 2);

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYIVDVNGRRYLDANSGLY NMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYT NSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAVSASMTGLPYNSVFGL PLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAG GVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTA G FFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDWMNEGLAENVR RLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQD LGLICSALGQSVILCPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 3); MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYVVDVNGRRYLDANSGL YNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFY TNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFG LPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGA GGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLT A GFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDWMNEGLAENV RRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQ DLGLICSALGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 4);

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYIVDVNGRRYLDANSGLY NMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFYT NSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGMPHNSVFG LPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGA GGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLT A GFFPVGAVILGPALSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDWMNEGLAENV RRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQ DLGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 5);

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYVVDVNGRRYLDANSGL YNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFY TNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFG LPLPGFVHLGCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGA GGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLT A GFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDWMNEGLAENV RRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQ DLGLICAAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 6); and

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYIVDVHGRRYLDANSGLY N M VAG F D H KG L I D AAKAQ YE RF P G YH S F FG RM S D QTVM LS E KLVE VS P F D S G RVF YT NSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGL PLPGFVHLSCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAG GVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTA G FFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDWMNEGLAENVR RLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQD LGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 7).

In another aspect A19, the invention provides a process for the manufacture of (S)-3- aminomethyl-5-methylhexanoic acid ((S)-ll)

((S)-N)

or a pharmaceutically acceptable salt thereof, comprising the step of treating 5-hydroxy- 4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanone (l ^A ) and an amine with a transaminase enzyme according to any one of embodiments A18E1 , A18E2, A18E3 and A18E4.

Detailed Description of Preferred Embodiments

The term "alkyl" means a straight-chain or branched-chain saturated aliphatic hydrocarbon radical containing the specified number of carbon atoms. Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, fe/f-butyl, neopentyl and n-hexyl.

The term "alkoxy" means a group made up of an alkyl group as defined above connected to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy and isopropoxy.

The term "alkoxy-alkyl" means a straight-chain or branched-chain saturated aliphatic hydrocarbon radical in which an alkoxy group is substituted for an alkyl hydrogen atom. An example of an alkoxy-alkyl group is 2-methoxyethyl. The term "haloalkyl" means 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 replacing halogen atoms 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" means a phenyl or naphthyl group.

The term "aryl-alkyl" means a straight-chain or branched-chain saturated aliphatic hydrocarbon radical in which an aryl group is substituted for an alkyl hydrogen atom. An example of an aryl-alkyl group is benzyl. The term "cycloalkyl" means a saturated monocyclic or polycyclic carbocyclic ring containing the specified 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 reference to an alkyl or aryl group means that a hydrogen atom of the alkyl or aryl group may be replaced by one of the groups listed. The substitution may be made at any position within the alkyl or aryl group. When the optional substitution is with "one or more groups" then any number of hydrogen atoms of the alkyl or aryl group, up to a maximum equal to the number of hydrogens present in the alkyl or aryl group, may be replaced, and each replacement is independent of the others.

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

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

where "er" is the ratio of the more abundant enantiomer to the less abundant enantiomer. 5-Hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanone (l ^A ) is a convenient intermediate in the manufacture of 3-aminomethyl-5-methylhexanoic acid (II). Treatment of racemic (l ^A ) with ammonia in the presence of a chemical reducing agent provides racemic 3-aminomethyl-5-methylhexanoic acid ((R/S)-ll) (see WO2008/127646A2). The reaction is presumed to involve the ring-opened isomer (l ^B ).

(|A) (|B) (II)

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

The reaction of (l ^A ) with a suitable amine dehydrogenase/ imine reductase in the presence of ammonia can also be a suitable route to pregabalin. A co-factor such as NADH or NADPH may be needed in a stoichiometric amount, or a second oxidoreductase, such as formate dehydrogenase, may be included to recycle the co- factor.

(|A)

Where the dihydrofuranone (l ^A ) has a defined stereochemistry at the C-4 position, this stereochemistry may be preserved during the reductive amination reaction. Since Pregabalin has the (S)-stereochemistry it may be preferred to have this stereochemistry already present in the dihydrofuranone.

((S)-I ^A ) ((S)-ll)

Alternatively, the dihydrofuranone (l ^A ) may be used as in racemic form. The desired stereoisomer of the product may then be obtained either by carrying out the reductive amination reaction under conditions that allow for the stereoselective formation of a single enantiomer, such as by carrying out the transformation in the presence of a transaminase enzyme, or by subjecting the product to a separate resolution step, such as by crystallization with a chiral acid or base. The dihydrofuranone (l ^A ) may be conveniently prepared in racemic or enantiomerically enriched form using the methods set out below.

In a first method, the dihydrofuranone (l ^A ) is prepared by reduction of 5-hydroxy-4-(2- methyl-1 -propenyl)-5H-2-furanone (VI ^A ).

(VI ^A )

The reduction may conveniently be 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. Heterogeneous catalysts include finely divided metals and substrate-supported metals and metal oxides, where the substrate may be carbon, silica, alumina 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, then the product is the racemic dihydrofuranone (l ^A ). The use of a chiral catalyst may provide the dihydrofuranone (l ^A ) in an enantioselective manner. The selectivity of the hydrogenation reaction, and the overall yield, maybe improved when the reaction is carried out in an alkaline medium. Without being bound by theory, it is thought that in the presence of a base the furanone exists predominantly as the ring-opened salt (IX).

(IX)

Any suitable base may be used provided that it does not interfere with the hydrogenation process, such as by poisoning the catalyst. Examples of suitable bases include alkali (such as Li, Na, K and Rb) and alkaline earth metal (such as 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 may be used to prepare ammonium salts. Tetra-alkylammonium hydroxide may also be used, leading to the formation of tetra-alkylammonium salts.

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

(X)

The dihydrofuranone (l ^A ) is recovered, after the hydrogenation reaction, by treatment of this salt with a suitable acid. Alternatively, the salt may be converted directly to 3- aminomethyl-5-methylhexanoic acid (II) by treatment with a transaminase or amine oxidase/imine reductase enzyme. In this case it may be preferable to use the ammonium salt (M ⁺ = NH ₄ ⁺ ) or a primary alkylammonium salt (M ⁺ = alkyl-NH ₃ ⁺ ) since the ammonium or alkylammonium ion provides the co-substrate for the enzyme. The use of the isopropylammonium salt in combination with a transaminase enzyme is a preferred example.

Furanones of formula (VI) wherein R is other than hydrogen may also be reduced by hydrogenation. Where R is an alkyl, haloalkyl, alkoxyalkyi alkenyl, cycloalkyi, cycloalkyl-alkyl, aryl or aryl-alkyl group, these furanones may be prepared from the compound of formula (VI ^A ) by reaction with an alcohol R-OH in the presence of an acid catalyst. Where R is R ¹ -C(O)- the furanones may be prepared from the compound of formula (VI ^A ) by reaction with an acid anhydride (R ¹ -C(O)) ₂ O or acid chloride R ¹ -C(O)- Cl, optionally in the presence of a base, for example a tertiary amine. Where R is R ² - SO ₂ - the furanones may be prepared from the compound of formula (VI ^A ) by reaction with a sulfonyl chloride R ² -SO ₂ -CI, optionally in the presence of a base, for example a tertiary amine.

Following the hydrogenation step the dihydrofuranone of formula (l ^A ) can be generated from the reduction product by treatment with acid (where R is an alkyl, haloalkyl, alkoxyalkyi alkenyl, cycloalkyi, cycloalkyl-alkyl, aryl or aryl-alkyl group) or base (where R is R ¹ -C(O)- or R ² -SO ₂ -).

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

For example, the furanone (VI ^A ) is reacted with a chiral alcohol R ^* -OH to afford a chiral ether derivative (VI ^B ).

(VI ^A ) (V| )

Suitable chiral alcohols may include a-aryl alcohols such as 1 -phenylethanol and 1 - naphthylethanol, as well as terpene alcohols such as menthol and borneol. Hydrogenation of derivative (VI ^B ) may proceed in an enantioselective manner, and treatment of the resulting product with a suitable acid and in the presence of water then provides the dihydrofuranone (l ^A ) in chiral form.

(VI ^B ) ((S)-I ^A )

The furanone (VI ^A ) may be prepared from a compound of formula (VII)

(VM)

wherein -X- represents a single bond, -CH ₂ -, -O-, -NH-, -N((Ci-C ₃ )alkyl)-, -N(benzyl)- or

by treating the compound of formula (VII) with water in the presence of an acid catalyst. Suitable acids include mineral acids such as sulphuric acid. Alternatively, the compound of formula (VII) can be treated with an alcohol R-OH to provide directly a compound of formula (VI) wherein R is an alkyl, haloalkyl, alkoxyalkyl alkenyl, cycloalkyl, cycloalkyl-alkyl, aryl or aryl-alkyl group.

The compounds of formula (VII) can be prepared by the reaction of a dienam formula (VIII).

(VIII)

wherein -Y- represents a single bond, -CH ₂ - -O-; -NH-, -N((Ci -C ₃ )alkyl)-, -N(benzyl)-, or

with glyoxylic acid or its hydrate.

(VII)

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

The compounds of formula (VIII) wherein -Y- represents a single bond or -CH ₂ - have been prepared by the reaction of 4-methyl-2-pentenal with pyrrolidine or piperidine (Kienzle, F. et al., Helv. Chim. Acta 1985, 68(5), 1 133-39). Other compounds of formula (VIII) may be prepared analogously.

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

H ^<

-CHO

H ₃ C (VIII)

Y = single bond, CH ₂ , O, N(alkyl), N(benzyl)

If piperazine is used as the amine then a Jb/s-dienamine is obtained.

The compound of formula (VIII) wherein Y is NH may be obtained by using mono- protected piperazine as the amine, followed by a deprotection step.

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

This mode of reaction of isobutyraldehyde and acetaldehyde differs from the direct base catalysed reaction (eg using potassium carbonate as base: UK patent GB834100) which only gives the adduct 2,2-dimethyl-3-hydroxybutanal where isobutyraldehyde acts as a nucleophile .

90%

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

The method of the present invention is more economical and more amenable to scale- up than the literature methods of effecting this 'umpolung' of normal acetaldehyde reactivity, in which acetaldehyde is converted to an O-silylated enol derivative and coupled with the isobutyraldehyde under Mukiyama aldol conditions, In the present invention, both coupling partners are activated simultaneously - electronic and steric effects directing the observed reactivity pattern. The other 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-aminofuranones (VII).

These dienamine derivatives of formula (VIII) may be isolated and purified, or alternatively they can be treated directly with glyoxylic acid (or its hydrate), which provides furanone derivative (VII) directly. The furanone derivatives (VII) may be isolated and purified. Treatment with aqueous acid then provides furanone (VI ^A ).

Overall, the transformations set out above provide a short route for the manufacture of pregabalin using inexpensive and safe starting materials.

In an alternative embodiment, the dihydrofuranone (I) is prepared from 3-isobutylidene- 2-oxopentanedioic acid (XII), which is readily obtained by the condensation of isobutyraldehyde with 2-oxopentanedioic acid (a-ketoglutaric acid) (XIV).

Conversion of diacid (XII) to dihydrofuranone (I) requires a decarboxylation step and a reduction step. These two process steps may be carried out separately, in which case either 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 carried out first, the intermediate (XV) will be produced. When the decarboxylation step is carried out first the intermediate (XVI) will be produced.

(XV) (XVI) The reduction step may be carried out chemically, such as by hydrogenation, but is preferably achieved using an enzyme-mediated reduction, such as by treating with an enoate reductase enzyme. The decarboxylation step is preferably performed by treating the compound with a decarboxylase enzyme.

Where enzyme-mediated transformations are contemplated, the enzyme may be an isolated enzyme, including an enzyme immobilized on a carrier, it 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. Cells may include those which express the desired enzyme naturally, and cells that have been manipulated so as to express the desired enzyme.

The enzyme-mediated reductive amination of the compound of formula (l ^A ) is reversible, and so treatment of 3-aminomethyl-5-methylhexanoic acid (II) with a transaminase enzyme or an amine oxidase/imine reductase enzyme can lead to the formation of the dihydrofuranone (l ^A ). The ring-opened isomer of this is compound (l ^B ), which is epimerizable. In view of this, it is possible to convert ((R)-ll) into ((S)-ll), or to increase the optical purity of a mixture of ((R)-ll) and ((S)-ll) 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 Doublet

DIW De-ionised water

dd Doublet of doublets

eq or eq. Equivalent

e.e. or ee Enantiomeric excess

ES ⁺ Positive mode electrospray ionization

EtOAc Ethyl Acetate

EtOH Ethanol

GC Gas chromatography

GC/MS Gas chromatography / Mass spectroscopy

HOAc Acetic acid

HPLC High Performance Liquid Chromatography

Hr or h Hour

¹ H NMR Proton Nuclear Magnetic Resonance Spectroscopy

L Litre

LCMS Liquid chromatography / Mass spectroscopy

m Multiplet

mbar Millibar

MeOH Methanol

min Minute

mL Millilitre

mmol Millimole

mol Mole

Mp Melting point

MTBE Methyl tert-butyl Ether

NAD ⁺ Nicotinamide adenine dinucleotide (oxidised)

NADP ⁺ Nicotinamide adenine dinucleotide phosphate (oxidised)

NADH Nicotinamide adenine dinucleotide (reduced) NADPH Nicotinamide adenine dinucleotide phosphate (reduced)

PLP Pyridoxal phosphate

ppm Parts per million

pTsOH Para-toluenesulfonic acid

q Quartet

qNMR Quantitative nuclear magnetic resonance spectroscopy

R _f Retention factor

RT Room temperature

s Singlet

t Triplet

ThDP Thiamine diphosphate

TLC Thin layer chromatography

TsOH Toluenesulfonic acid (= pTsOH)

UPLC Ultra Performance Liquid Chromatography

XRD X-ray diffraction crystallography

δ Chemical shift

Commercial chemicals were used as received unless stated otherwise. Thin layer chromatography was performed on pre-coated plastic plates (Merck silica 60F254), and visualised using UV light and KMn0 ₄ dip. Proton ( ¹ H) and carbon ( ¹³ C) NMR spectra were recorded on a Varian INOVA 300 MHz spectrometer. Chemical shifts are quoted relative to tetramethylsilane and referenced to residual solvent peaks as appropriate. Unless otherwise indicated, chiral HPLC analysis was performed using an Agilent 1200 HPLC system and data was processed using the Chemstation software or with a Varian semiprep/analytical HPLC using Galaxie software.

Example 1

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

A 50% solution of glyoxylic acid in water (29.6g, 0.2mol) was added to a biphasic stirred mixture of morpholine (17.8g, 0.2mol) and heptanes (75ml_), which had been pre-cooled to 0-10°C. The temperature was kept less than 10°C. The mixture was warmed to 20°C and 4-methyl-2-pentenal (19.6g, 0.2mol) was added. The mixture was stirred at 45°C for 20h. A large quantity of solid was formed. Water (100ml_) was added at ambient temperature and the mixture stirred for 2h. The mixture was extracted with cyclopentyl methyl ether (100ml_) and the organic solution washed twice with water (100ml_) and concentrated to leave 30.4g of crude product. This solid was purified by recrystallisation from methanol (100ml_) to afford 17.7g (40%) of pure 4-(2-methyl-1 -propenyl)-5- morpholino-5/-/-2-furanone.

GC/MS: m/z = 223

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

Example 2

Isobutyraldehyde (46.90g, 0.65mol, 1 .43eq.) was stirred in acetonitrile (300ml_). Morpholine (56.63g, 0.65mol, 1 .43eq.) followed by pTsOH (8.63g, 0.1 equiv) were slowly added at room temperature to the isobutyraldehyde solution. A solution of acetaldehyde (20g, 0.454mol) in acetonitrile (100ml_) was added drop-wise over 1 hr with internal temperature monitoring at 50°C. After complete addition the mixture was stirred for 30m in at 50°C and then cooled to room temperature before evaporation of the solvent in vacuo to give an orange oil (123.3g). Assay of the crude by quantitative NMR using benzyl benzoate as the internal standard was 40% which give 65% yield of the desired dienamine.

GC/MS: m/z = 167

¹ H NMR: δ 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-5H-2-furanone from crude 4- (4-meth -1 ,3-pentadien-1 -vDmorpholine

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

GC/MS: m/z = 223

NMR: as example 1 .

Example 4

Preparation of 4-(2-methyl-1 -propenyl)-5-morpholino-5H-2-furanone in one pot from isobutyraldehvde and acetaldehvde.

Isobutyraldehvde (102.90g, 1 .43mol) was stirred in acetonitrile (600ml_). Morpholine (124.3g, 1 .43mol) followed by pTsOH (19.0g, 0.1 equiv) were slowly added at room temperature to the isobutyraldehyde solution. A solution of acetaldehyde (44.05g, LOmol) in acetonitrile (150ml_) was added drop-wise over 1 hr with internal temperature monitoring at 50°C. After complete addition the mixture was stirred for 30m in at 50°C and then cooled to <10°C. Glyoxylic acid (50wt%, 21 1 .3g, 1 .43mol) was added and the resultant biphasic mixture stirred at 50°C for 18h. The reaction mixture was cooled to room temperature and the solvent removed by rotary evaporation. The residue was partitioned between ethyl acetate (1 L) and saturated sodium carbonate solution (1 L). The aqueous was extracted with ethyl acetate and combined organics washed with brine and evaporated to a thick oil which solidified on standing (166g). The crude product was triturated with MTBE at room temperature. The product is collected by filtration and washed with MTBE to give pure 4-(2-methyl-1 -propenyl)-5-morpholino-5/-/- 2-furanone (84g, 37% yield) identical to the material prepared in Example 1 . Analysis of the crude solid (166g) indicated ca 50% yield.

Example 5

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

A solution of piperazine (43. Og, 0.5mol) and 4-toluenesulfonic acid monohydrate (4.4g, 0.023mol) in acetonitrile (600ml_) was prepared. This solution was heated to 50°C and isobutyraldehyde (100ml_, 1 .10mol) added over 10 minutes. The solution went red- orange in colour and a transient white precipitate occurred. A solution of acetaldehyde (30.8g, 0.70mol) in acetonitrile (30 ml_) was then added via syringe pump over 3h at 50°C. A suspension was formed which was stirred at 50°C for 0.5h. The solvent was removed and the residual solid isolated from methanol (500m L) at -5°C. It was filtered and washed with chilled methanol and dried to afford 52.9g (60%) of 1 ,4-bis-(4-methyl- 1 ,3-pentadien-1 -yl)piperazine.

GC/MS: m/z = 246.

¹ H NMR: δ 6.00 (d, 2H); 5.70 (d, 2H); 5.28 (dd, 2H); 2.92 (s, 8H); 1 .73 (s,6H); 1 .68 (s, 6H). ¹³ C NMR (CDCI3): 140.6 (C), 126.3 (CH), 123.4 (CH), 100.2 (CH), 48.2 (CH ₂ ), 25.8 (CH ₃ ), 18.1 (CH ₃ ). Example 6

Preparation of 5,5'-(piperazine-1 ,4-di yl)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 charged to methanol (300ml_). The temperature was adjusted to 34°C and a 50% solution of glyoxylic acid in water (44.9g, 0.302 mol) was added rapidly (over 5 minutes). The resulting suspension was stirred at 45°C for 15h; cooled to 0-10°C 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 extra 5% could be obtained from the mother-liquor by concentration.

1H NMR: δ 5.96 (d, 2H), 5.87 (d, 2H), 5.56 (m, 2H), 2.71 (s, 8H), 2.07-1 .90 (m, 12H). ¹³ C NMR (CDCI3): 172.3 (C), 159.2 (C), 150.9 (C), 1 16.6 (CH), 1 15.4 (CH), 99.2 (CH), 46.7 (CH ₂ ), 28.2 (CH ₃ ), 21 .4 (CH ₃ ).

This reaction can also be conducted in isopropanol, acetonitrile-water, toluene-water or in heptane water with similar yields.

Example 7

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

Tosic acid (6.5g, 0.03mol) was charged to a solution of piperazine (59g, 0.69 mol) in acetonitrile (240ml_). The reaction was heated to 50°C and agitated until dissolution of solids was observed, before addition of isobutyraldehyde (138ml_, 1.51 mol). The reaction was held at 50°C before a solution of acetaldehyde (60ml_; 1.07mol) in acetonitrile (30m L) was charged to the vessel over 3hrs via syringe pump. On complete addition, the reaction was stirred for a further 30 minutes before a 50% w/w solution of glyoxylic acid in water (148g, 1 .0 mol) was added over 5min to the reaction mixture followed by water (50ml). The reaction was then heated to 70°C for 2h, then to 50°C overnight. The reaction was then cooled to 5°C and held for 30 minutes. 5,5'- (Piperazine-1 ,4-diyl)bis(4-(2-methylprop-1 -en-1 -yl)furan-2(5H)-one) precipitated and was isolated by filtration and washed with acetonitrile (2 x 100ml_) to give the desired product (126 g, 93.5% assay, 66% yield from acetaldehyde).

Example 8

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

Isobutyraldehyde (79g, 100ml_, 1 .1 mol) was charged to a 3-necked round bottom flask and argon was flushed through the system. Piperazine (43g, 0.5 mol) and TsOH-H ₂ O (4.4g; 0.023 mol) were divided into 4 equal portions containing piperazine (10.75 g) and TsOH-H ₂ O (1 .1 g). Addition of the first portion of piperazine (10.75g) resulted in exotherm from 24°C to 35°C. This was followed by the first portion of TsOH (1 .1 g). The reaction was agitated until all the piperazine had dissolved (t=35°C), after which the second portion of piperazine (10.75g) was added, followed by TsOH (1 .1 g) (t=41 °C). Stirring was continued until all of piperazine had dissolved (t=41 °C), then the third portion of piperazine (10.75g) was added, followed by TsOH (1 .1 g). After the 3 ^rd charge a clear solution was generated and then the fourth portion of piperazine (10.75g) & TsOH-H ₂ 0 were added, followed by TsOH (1 .1 g) (t=52°C). On complete addition the reaction was stirred at 50°C for 30 min.

Another flask (50ml_) was charged with acetaldehyde (30.8g, 39ml_, 0.7mol) and placed in an ice-water bath (0-2°C). The acetaldehyde flask was connected to the 3-necked flask by cannula via septa. A stream of argon was then passed through the acetaldehyde flask at the rate which ensures that the addition of the acetaldehyde was complete within 3 h. After all acetaldehyde was added, the reaction was stirred for 30 min at 50°C, then cooled to 40°C and 50% glyoxylic acid (104g, 78ml_) was added dropwise over 45 min at a rate to keep temperature below 50°C. Water (100ml_) was then added and the reaction was heated at 70°C for 5 h. The reaction was cooled to 4°C in an ice-water bath. The precipitated product was filtered and filter cake was washed with cold water (100 mL). After drying in vacuo at 50°C 88.9g (71 %) of desired product was obtained. Precipitate contains 78.5% of the title compound (HPLC assay).

Example 9

Alternative preparation of 5,5'-(piperazine-1 ,4-diyl)bis(4-(2-methylprop-1 -en-1 - yl)furan-2(5H)-one) with simultaneous addition of both aldehydes

Piperazine (236.6g) was charged to a clean 3L dry vessel fitted with thermometer, addition funnel (100ml_) and reflux condenser. pTsOH (25.3g) was charged to vessel followed by acetonitrile (550m L). The agitator was started and the vessel was inerted with nitrogen. Isobutyraldehyde (150ml_, 27% of the total charge) was charged to the agitated piperazine / pTsOH slurry and a temperature rise to ca 43°C was observed. The white suspension was then heated to 50°C (+/- 5°C). A pre-mixed chilled solution of isobutyraldehyde (400m L, 73% of total charge) and chilled acetaldehyde (260m L) were charged to a clean, dry 1 L vessel and this mixture was maintained in an ice bath. The isobutyraldehyde / acetaldehyde mixture was charged to the contents of the 3L vessel in 100ml_ aliquots over 5-6h at 50°C. The contents of the reaction flask changed from a white suspension to a wine red solution and finally an orange suspension during the addition of the acetaldehyde. After complete addition the suspension was agitated at 50°C for ca. 0.5 to 1 .0h.

Aqueous glyoxylic acid (50% wt/wt) solution was charged to the suspension over 10-15 min and the temperature rose to ca 75°C. 5,5'-(Piperazine-1 ,4-diyl)bis(4-(2-methylprop- 1 -en-1 -yl)furan-2(5/-/)-one) was observed to crystallise from solution. The suspension was stirred at 70°C (+/ 5°C) for 6h, then cooled with stirring to ambient. The batch was then cooled to -5 to 0°C for and held for 3-4h before the suspension was filtered and the cake washed with chilled methanol (1 x 500ml_) then 2 x 500ml_ of methanol at ambient temperature. The washed product was dried in a vacuum oven at 40-50°C to constant weight to afford 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

Preparation of 5-hvdroxy-4-(2-methylprop-1 -en-1 -yl)furan-2(5H)-one from 4-(2- methyl-1 -propenyl)-5-morpholino-2(5H)-furanone or 5,5'-(piperazine-1 ,4-diyl)bis(4- (2-methylprop-1 -en-1 -yl)furan-2(5H)-one).

A 10% w/w aqueous sulphuric acid solution (1 10g) was charged to 4-(2-methyl-1 - propenyl)-5-morpholino-2(5/-/)-furanone (20. Og, 0.896mol) and the mixture was stirred at reflux for 4h or until TLC (100:3 CPME: HOAc) indicated reaction completion. The mixture remained a suspension at all times. It was then cooled to 5°C, 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(5/-/)-one (12.9g, 93%).

Alternatively, a 10% w/w aqueous solution of sulphuric acid (412g) was charged to a vessel containing 5,5'-(piperazine-1 ,4-diyl)bis(4-(2-methylprop-1 -en-1 -yl)furan-2(5/-/)- one) (60g, 0.167mol). The contents were agitated and then heated to reflux. The reaction was held at reflux until the starting material was consumed, which was determined by dissolution. On completion of reaction the batch was cooled to 35°C at which point the target compound began to crystallise. The slurry was further cooled to 0-5°C and then transferred to a filter. The product was filtered, washed with water (2 x 100ml_) and then dried under vacuo at <50°C, to give 5-hydroxy-4-(2-methylprop-1 -en- 1 -yl)furan-2(5H)-one (42.0g, 81 %).

GC/MS: m/z = 154

¹ H NMR (CDCI3): δ 6.10 (s, 1 H); 5.92 (s, 1 H); 5.88 (s, 1 H); 5.35 (bs, 1 H, O-H); 1 .98 (s,3H); 1 .93 (s, 3H). ¹³ C NMR (CDCI ₃ ): 172.8 (C), 161 .4 (C), 152.3 (C), 1 15.3 (CH), 1 14.9 (CH), 99.5 (CH), 28.2 (CH ₃ ), 21 .4 (CH ₃ ).

Example 11

Preparation of 5-hvdroxy-4-(2-methylprop-1 -en-1 -yl)furan-2(5H)-one in one pot

toluene

A mixture of piperazine (43. Og, 0.5 mol) and isobutyraldehyde (200ml_) were refluxed under Dean-Stark until the theoretical amount of water (18ml_) collected. The excess isobutyraldehyde was distilled off to leave a crystalline mass of 1 ,4 bis(2-methylpropen- 1 -yl)piperazine. This was dissolved in acetonitrile (1 .2L) and extra isobutyraldehyde (100 ml_, LOmol) added. Extra piperazine (4.4g) was added followed by p- toluenesulfonic acid (4.4g, 23.2mmol). The temperature was adjusted to 40°C and a solution of acetaldehyde (44g, LOmol) in acetonitrile (40ml_) was added over 4h. The reaction mixture was stirred at 40°C for 1 h and at ambient temperature for 4h. The acetonitrile was distilled off and the residue suspended in toluene (400ml_). A mixture of 50% glyoxylic acid (148g) and water (150ml_) was added over 0.5h. The mixture was then stirred at 45°C for 15h. There was a solid precipitate. Dilute sulphuric acid (10%, 1 L) was added and the mixture refluxed for 3h. A biphasic mixture resulted. The toluene phase was separated. It contained ca 50% yield (based on acetaldehyde) of 5- hydroxy-4-(2-methylpropen-1 -yl)-2-furanone by analysis Example 12

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

4-(2-Methyl-1 -propenyl)-5-morpholino-2(5/-/)-furanone (7.70g, 34.5mmol) was stirred in MeOH (50ml_) with H ₂ SO ₄ (4mL, 2.2eq.) at reflux for 3h until disappearance of the starting material by GC. The reaction mixture was cooled to room temperature. The solvent was then evaporated in vacuo and the residue was diluted with EtOAc (50ml_). The organic phase was washed with H ₂ O (3x 50ml_). Solvent was then 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 can be used without purification.

Alternatively, 5,5'-(piperazine-1 ,4-diyl)bis(4-(2-methylprop-1 -en-1 -yl)furan-2(5/-/)-one) (130g, 363mmol) and MeOH (690ml_) were charged in 2L round bottom flask with mechanical agitation to form a suspension. Concentrated H ₂ SO ₄ (43ml_, 762mmol) was added dropwise with stirring at 20-25°C - a slight exotherm was observed, and nature of the solids present changed from dispersed and easily mixed to a thicker slurry. The reaction mixture was refluxed for 5h. After 0.5h complete dissolution (orange liquid) was observed. After 5h LCMS showed ~99% of the desired product. The mixture was cooled to ambient and left for crystallization of piperazine sulfate. The sediments were filtered, and filter cake was washed with ice cold MeOH (400ml_). The filtrate was concentrated in vacuo (40°C bath temperature), and the residue was partitioned between water (50ml_) and MTBE (300ml_). Water layer was separated and additionally extracted (2x300ml_ of MTBE). Combined organic extracts were washed with saturated aq. NaHCO ₃ (200ml_). MTBE layer was dried over Na ₂ SO ₄ with stirring 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%). Distilled at 0.2-0.3 mbar vacuum with bp 100-105°C. GC/MS: m/z = 168; ¹ H NMR (400 MHz, CDCI3) δ 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). ¹³ C NMR (CDCI3): 171.4 (C), 159.2 (C), 151.9 (C), 115.8 (CH), 115.2 (CH), 104.4 (CH), 56.1 (CH ₃ ), 28.2 (CH ₃ ), 21.4 (CH ₃ ).

The following compounds were prepared from 4-(2-methyl-1-propenyl)-5-morpholino- 2(5/-/)-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-M ethyl butoxy)-4-(2-methyl rop-1 -en-1 -yl)furan-2(5H)-one

Distilled at 0.4 mbar vacuum with bp 139-140°C; GC/MS: m/z = 224; ¹ H-NMR (400 MHz, CDCI3) δ 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-M ethyl prop-1 -en-1 -yl)-5-pentyloxyfuran-2(5H)-one

Distilled at 0.08 mbar vacuum with bp 123-134°C; GC/MS: m/z = 224; ¹ H-NMR

(400 MHz, CDCI3) δ 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-methylprop-1 -en-1 -yl)furan-2(5H)-one

Distilled at 0.05 mbar vacuum with bp 136-146°C; GC/MS: m/z = 210; ¹ H-NMR (400 MHz, CDCI3) δ 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=7.4 Hz) Example 13

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

A solution of 5-methoxy-4-(2-methylprop-1 -en-1 -yl)furan-2(5H)-one (100 g, see example

12) in MTBE (700ml_), 5g (5 wt%) of 10% Pd/C was charged to a hydrogenation vessel. 1 atm of hydrogen gas was introduced and pressure was kept constant during the reaction. After 7h at 20°C, the reaction was filtered through a celite pad (060mm, H=30mm) and rinsed with MTBE (3x50ml_). The filtrate was washed with 1 M NaHC0 ₃ solution (200ml_), water, brine and dried on Na ₂ S0 ₄ . After solvent removal 5-methoxy- 4-(2-methylpropyl)-dihydrofuran-2(3H)-one was obtained as colourless liquid (98g, 96%).

GC/MS: m/z = 172

¹ H NMR (400 MHz, CDCI ₃ ) δ 5.25 (d, J = 5.0 Hz, 0.53 H, major isomer), 5.04 (d, J = 2.4 Hz, 0.26H, minor isomer), 3.48 (s, 0.92H, minor isomer), 3.46 (s, 1 .66H, major isomer), 2.78 (dd, J = 17.7, 8.6 Hz, 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, 1 1 .8 Hz, 0.63H, major isomer), 2.15 (dd, J = 17.7, 4.6 Hz, 0.33H, minor isomer), 1 .67 - 1 .31 (m, 2.63H both isomers), 1 .28 - 1 .19 (m, 0.33H, minor isomer), 0.95 - 0.86 (m, 6H both isomers). ¹³ C NMR (CDCI ₃ ): 177.7 (C, major isomer), 176.04 (C, minor isomer), 1 10.1 (CH, minor isomer), 106.1 (CH, major isomer), 57.0 (CH ₃ , minor isomer), 56.6 (CH ₃ , major isomer), 41 .2 (CH ₂ , minor isomer), 39.1 (CH ₂ , minor isomer), 38.3 (CH ₂ , major isomer), 37.1 (CH ₂ , major isomer), 33.9 (CH, minor isomer), 32.8 (CH, major isomer), 26.0 (CH, major isomer), 25.8 (CH, minor isomer), 23.0 (CH ₃ , major isomer), 22.9 (minor isomer), 22.7 (major isomer), 22.6 (minor isomer).

Using the same general method 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)-dihvdrofuran-2(3H)-one GC/MS: m/z = 228; ¹ H-NMR (400 MHz, CDCI ₃ ) δ 5.35 (0.86H, d, J=5.0 Hz) 5.13 (0.14H, d, J=2.7 Hz) 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-pentyloxydihydrofuran-2(3H)-one

GC/MS: m/z = 228; ¹ H-NMR (400 MHz, CDCI ₃ ) δ 5.35 (0.77H, d, J=5.0 Hz) 5.13 (0.23H, d, J=2.6 Hz,) 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, 11.8 Hz) 2.15 (0.27H, dd, J=17.7, 5.0 Hz) 1.67-1.45 (4H, m) 1.41 -1.25 (5H, m) 0.96-0.86 (9H, m)

Example 13C

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

GC/MS: m/z = 214; ¹ H-NMR (400 MHz, CDCI ₃ ) δ 5.35 (0.8H, d, J=5.0 Hz) 5.13 (0.2H, d, J=2.6 Hz,) 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, 11.7 Hz) 2.15 (0.24H, dd, J=17.6, 5.0 Hz) 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-Menthyloxy)-4-(2-methyl-1-propenyl)-2(5H)- fu ran one

A mixture of 5-hydroxy-4-(2-methyl-1-propenyl)-2-furanone (59.5g, 0.386 mol), L- menthol (89.5g, 1.5 equivalents) and methanesulfonic acid (1.5g) was stirred at 70-80°C under vacuum (to remove water) for 100h. The liquid mass was poured (hot) into acetonitrile (450ml_) and the title compound isolated by cooling, filtration and washing. The yield was 76.2g (67%). Suitable crystals forXRD were grown by slow evaporation of an acetone solution. The configuration at the acetal carbon was (R). Hydrogenation of 5-(L-menthyloxy)-4-(2-methyl-1 -propenyl)-2-furanone in ethyl acetate using the conditions of Example 13, gave the saturated derivative in quantitative yield. By ¹ H NMR, the compound was a 1 : 1 mixture of diastereomers.

Example 15

Preparati -acetoxy-4-(2-methyl rop-1 -en-1 -yl)furan-2(5H)-one

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

m/z: 196 Example 16

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

The material of example 34 was hydrogenated under similar conditions to those outlined in Example 13 to give a >90% yield of 5-acetoxy-4-(2-methylpropyl)-3,4-dihydro-2(5/-/)- furanone as a 1 :1 mixture of diastereomers. The product is accompanied by ca. 5% of 4-(2-methylpropyl)-3,4-dihydrofuran-2(5/-/)-one. The product can be hydrolysed as in Example 30 to give yields of 5-hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanone/ 3-formyl-5-methylhexanoic acid.

Example 17

Preparation of 5-hvdroxy-4-(2-methylpropyl)dihydrofuran-2(3H)-one by hydrogenation of 5-hydroxy-4-(2-methylprop-1 -en-1 -yl)furan-2(5H)-one in alkaline solution

5-Hydroxy-4-(2-methylpropen-1 -yl)-2-furanone (3.35g, 21 .7mmol) and water (20ml_) were charged to a hydrogenator. Potassium hydroxide (1 .21 g, 1 eq) was then charged to the vessel and the contents were heated to 40°C until dissolution had occurred. 10% Pd/carbon catalyst (0.67g) was charged to the vessel and the reactor contents were then hydrogenated at 40°C and 5barg hydrogen pressure. On completion of reaction the reaction was cooled and the catalyst removed by filtration. The pH was adjusted to pH 2 by addition of 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-methylprop-1 -en-1 -yl)furan-2(5H)-one in neutral solution,

5-Hydroxy-4-(2-methylpropen-1 -yl)-2-furanone was hydrogenated in 6ml_ of water per gram starting material with 10% w/w of Pd/C catalyst. (22h, 40°C, 10 bar). On filtration, an oily phase separated from the water. This was a 1 : 1 mixture of 5-hydroxy-4-(2- methylpropyl)dihydrofuran-2(3H)-one (l ^A ) and 4-(2-methylpropyl)-dihydrofuran-2-one. Compound (l ^A ) can easily be separated pure by an acid base extraction. This reaction was repeated in organic solvents with 2-propanol giving relatively pure (l ^A ). Example 19

Preparation of 5-hvdroxy-4-(2-methylpropyl)dihydrofuran-2(3H)-one from 5,5'- (piperazine-1 ,4-diyl)bis(4-(2-methylprop-1 -en-1 -yl)furan-2(5H)-one) in one pot

5,5'-(Piperazine-1 ,4-diyl)bis(4-(2-methylprop-1 -en-1 -yl)furan-2(5/-/)-one) (50.0g, 0.14mol) was charged to water (300ml_) containing sulphuric acid (16.0g, 0.163mol). Isopropyl acetate (200m L) was added. 50% water wet 5% palladium on carbon (3.0g) was added and the mixture was hydrogenated (5bar hydrogen) at ambient temperature for 15h. The reaction mixture was filtered from catalyst, washed with isopropyl acetate (300ml_) - this was also used to wash out the vessel. The organic phase was separated, washed with water (100ml_) and concentrated on a rotary evaporator to afford 38.8g of clear oil. This was a mixture of desired compound (l ^A ) and the over- reduced lactone (4-(2-methylpropyl)-dihydrofuran-2-one). It was readily purified by extraction with aqueous potassium carbonate solution and wash with toluene. Acidification of the potassium carbonate extract with formic acid gave the desired product (l ^A ) (25.6 g, 58%).

¹ H NMR (D ₂ 0 with added K ₂ C0 ₃ ): δ 9.5 (1 H), 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-oxopentanedioic acid mono-potass

A 2L round bottom was charged 300g a-ketoglutaric acid and 450ml_ of ice water. With stirring and cooling 314g of potassium hydroxide in 450m L water was charged keeping the pot temperature less than 25°C. Reactor was placed under nitrogen and isobutyraldehyde was added at 0.2mL/min over 50hrs. The resulting two phase yellow solution was separated and washed with 100ml_ MTBE. The lower aqueous phase pH was adjusted to 3.2-3.4 and was concentrated to 1/3 volume at 65°C under vacuum. After cooling to <5°C the resulting solids were collected and washed with several small volumes of water to give after drying 230g of a white solid containing 60% 3- isobutylidene-2-oxopentanedioic acid mono-potassium salt and remainder KCI.

1H NMR (D ₂ 0 at pH 8-10, 400MHz) δ 6.4 (d, 1 H), 3.3 (s, 2H), 2.7 (m, 1 H), 0.96 (d, 6H). ¹³ C NMR (D ₂ 0 at pH 8-10, 400MHz) δ 23 (2CH ₃ ), 31 (CH), 35 (CH ₂ ), 133 (C), 164 (CH), 170 (C), 176 (C), 182 (C).

Example 21

Preparation of 3-(2-methylpropyl)-2-oxopentanedioic acid mono-potassium salt

An aqueous solution of 3-isobutylidene-2-oxopentanedioic acid mono-potassium salt as generated in example 20 was adjusted to pH 6-8 and 5% Pd/C was added and the solution was hydrogenated at 10bar H ₂ 25°C for 10hours. The catalyst was removed by filtration and the pH of the solution adjusted to pH 3.8. The mixture was cooled to <5°C and filtered and washed with a small amount of cold water. The resulting white crystals were dried at 40°C under vacuum to give the title compound, 286g, 58% yield over 2 steps.

¹ H NMR (D ₂ 0 at pH 8-10, 400MHz) δ 3.5 (m, 1 H), 2.5 (dd, 1 H), 2.3 (dd, 1 H), 1 .6-1 .5 (m, 2H) 1 .3-1 .2 (m, 1 H) 0.9 (d, 6H). ¹³ C NMR (D ₂ 0 at pH 8-10, 400MHz) δ 21 .7 (CH ₃ ), 22.2 (CH ₃ ), 25.5 (CH), 38.4 (CH ₂ ), 39.3 (CH ₂ ), 43.4 (CH), 167.8 (C), 170.6 (C), 180.7 (C).

Example 22

Expression of Decarboxylase Enzymes in E. coli

PubMed was used to search the literature for decarboxylase enzymes with a broad spectrum of activity. The KEGG (Kyoto Encyclopedia of Genes and Genomes) program was used to search for microbial decarboxylases with activity on compounds somewhat similar in structure to 3-(2-methylpropyl)-2-oxopentanedioic acid or 3-isobutylidene-2- oxopentanedioic acid. Seven classes of decarboxylases were chosen for investigation based on reports of activity on compounds with some structural similarity. The decarboxylase classes were glutamate decarboxylase, diaminopimelate decarboxylase, indolepyruvate decarboxylase, branched-chain a-keto acid decarboxylase, aromatic-L- amino-acid decarboxylase, lysine decarboxylases and benzoylformate decarboxylase. Forty one sequences of genes for proven, or putative decarboxylase enzymes were selected. The genes were codon optimized for expression in E. coli, synthesized by GeneArt (Germany), DNA2.0 (Menlo Park, CA USA), or Blue Heron Biotechnology (Bothell, WA USA), cloned into expression vector pSTRC52 (Pfizer Inc., USA), and put into the expression strain E. coli BDG62 (Pfizer Inc., USA) (Table 1 below). Two genes were amplified from genomic DNA from the appropriate organisms by PCR and cloned into the same expression vector and E. coli strain. Four decarboxylase enzymes were purchased from Sigma and tested for activity on 3-(2-methylpropyl)-2-oxopentanedioic acid.

Each E. coli strain containing the cloned decarboxylase gene was grown overnight in LB broth with the appropriate antibiotic. A small amount (50μΙ_ - 100μΙ_) of the overnight seed culture was used to inoculate 4.0ml_ of Terrific Broth medium with appropriate antibiotics in 20mm diameter culture tubes. The cultures were grown in a shaking incubator at 32°C and 300rpm. After 5h of growth IPTG was added to 0.4mM final concentration to induce the enzyme expression. The cultures were returned to the incubator and grown for an additional 19h.

Example 23

Decarboxylation of 3-(2-methylpropyl)-2-oxopentanedioic acid potassium salt and 3-isobutylidene-2-oxopentanedioic acid potassium salt with Decarboxylase

Enzymes

Recombinant decarboxylase enzymes were tested for decarboxylation of 3-(2- methylpropyl)-2-oxopentanedioic acid (XV) and 3-isobutylidene-2-oxopentanedioic acid (XII) using E. coli cells prepared as described in Example 22. Reactions (1 ml_) were carried out at 37°C in potassium phosphate buffer (100mM, pH 6.4) with decarboxylase (60mg wet cells), 50mM 3-(2-methylpropyl)-2-oxopentanedioic acid or 3-isobutylidene- 2-oxopentanedioic acid, ThDP (0.1 mM), and MgS0 ₄ (2.5mM). Decarboxylation of 3-(2- methylpropyl)-2-oxopentanedioic acid and 3-isobutylidene-2-oxopentanedioic acid was determined by a UPLC assay. An aliquot (0.1 mL) of the reaction was treated with 0.5ml_ of potassium phosphate buffer (100mM, adjusted to pH 2.2 with phosphoric acid) and 0.3ml_ of a solution of 2,4-dinitrophenylhydrazine (20mM in 1 M HCI:acetonitrile, 3:1 , v/v) for 30min at 50°C. The derivatized samples were diluted with 0.5ml_ of acetonitrile, filtered and analyzed by UPLC on a Agilent Eclipse Plus C18 column (100mm x 3.0mm, 1 .8pm) eluted with 0.1 % trifluoroacetic acid in watenacetonitrile (55:45, v/v) at 1 .1 ml_/min. The column was maintained at 40°C and the effluent was monitored at 360nm and ES ⁺ mass spectroscopy. Positive results were indicated by the presence of a peak for 3-formyl-5-methylhexanoic acid from 3-(2-methylpropyl)-2-oxopentanedioic acid or 3-formyl-5-methylhex-3-enoic acid from 3-isobutylidene-2-oxopentanedioic acid. Results of the analyses are shown in Table 1.

Table 1

Enzyme Reaction on Reaction on

Source Organism

Name (XV) (XII)

Branched-chain α-Keto Acid decarboxylase

Pa bkd Photorhabdus asymbiotica - -

Sv bkdA Streptomyces virginiae - -

LI kdcA Lactococcus lactis + +

Indolepyruvate decarboxylases

Ss ipd Staphylococcus saprophyticus - +

Dd ipd Desulfovibrio desulfuricans + +

Bm ipd Bacillus megaterium - -

Dv ipd Desulfovibrio vulgaris - -

Aa ipd Aromatoleum aromaticum - -

Ab ipd Azospirillum brasilense - -

Bs ipd Brady rhizobi urn sp. BTAil - -

NC ipd NC10 bacterium - -

Rr ipd Rhodospirillum rubrum - -

Aromatic-L-amino-acid decarboxylases

Ab dcd Acinetobacter baumannii - -

Bp dcd Bacillus pumilus - -

Cg dcd Chryseobacterium gleum - -

As dcd Anaeromyxobacter sp. Fw109-5 - -

Bs dcd Burkholderia sp. 383 + -

Cs dcd Cyanothece sp. PCC 8801 - -

Lysine decarboxylases

Cr lysA Candidatus Carsonella ruddii - -

Dt lysA Dictyoglomus thermophilum - -

Sa lysA Sphingopyxis alaskensis - -

Benzoylformate decarboxylases

Rp bfd Rhodopseudomonas palustris + +

Sc bfd Streptomyces coelicolor + +

Rc bfd Ricinus communis - -

Pp bfd Pseudomonas putida + +

Ms bfd Mycobacterium smegmatis - -

As bfd Arthrobacter sp. FB24 + + Enzyme Reaction on Reaction on

Source Organism

Name (XV) (XII)

Pb bfd Pseudomonas brassicacearum + +

Ct bfd Comamonas testosterone + +

Dd bfd Desulfovibrio desulfuricans + +

Rj bfd Rhodococcus jostii + +

Pf bfd Pseudomonas fluorescens + +

Purified Enzymes (purchased from Sigma- Aldrich)

L-Histidine

Lactobacillus 30a - NT

Decarboxylase

L-Lysine

Bacterium cadaveris - NT

Decarboxylase

L-Tyrosine

Streptococcus faecalis - NT

Decarboxylase

Pyruvate

Saccharomyces cerevisiae - NT

Decarboxylase

a -: No detection of appropriate product

b NT: Not tested

c +: Detection of appropriate product Example 24

Preparation of 3-Formyl-5-methylhex-3-enoic acid

In a 250ml_ round bottom flask 80g of cell concentrate comprising E. Coli expressing Pseudomonas putida benzoylformate decarboxylase (see example 23) was charged. To this was added a pH 6.2 adjusted solution of 3-isobutylidene-2-oxopentanedioic acid mono-potassium salt (10g, see example 20) in 50ml_ water, with 0.5g of magnesium sulfate and 0.5g of thiamine pyrophosphate. The resulting pH 6.2 slurry was heated to 50°C and stirred for 145hrs keeping the pH adjusted between 6.2 and 7.2 with concentrated HCI. The reaction mixture was cooled to RT and centrifuged. The aqueous decants were washed with 50ml_ MTBE with minimum agitation. The pH of the aqueous phase was adjusted to 4 and filtered through celite. The filtrate was extracted with minimum agitation three times with 50ml_ MTBE. The MTBE was dried over anhydrous sodium sulfate and concentrated to a thick red oil. This oil was extracted with several portions of hot hexanes; the combined hexanes were cooled to <0° and the resulting crystals were isolated and dried in air to give 3-formyl-5- methylhex-3-enoic acid as a white solid (0.6g).

¹ H NMR (D ₂ 0 at pH 8-10, 400MHz) δ 9.2 (s, 1 H), 6.6 (d, 1 H), 3.1 (s, 2H), 2.7 (m, 1 H), 1 (d, 6H) Example 25

Expression of Enoate Reductase Homologues in E. coli

The DNA sequence corresponding to the gene for Lycopersicon escuientum (tomato) 12-oxophytodienoate reductase 1 (OPR1 ) was retrieved from the Genbank database (accession number AC Q9XG54) and was synthesized by GeneArt (Germany). The sequence was codon optimized for expression in E. coli, and was subcloned into an E. coli expression plasmid pSTRC18 (Pfizer Inc. , USA). The protein sequence is shown below. The OPR1 expression construct was transformed into BL21 (DE3) E. coli (Stratagene, Agilent Technologies, Santa Clara, CA, USA) as directed and overnight cultures were incubated in LB + streptomycin media. The LB culture was used to inoculate expression cultures (LB, M9Y, or TB), which were incubated at 37°C (210rpm) After the culture reached a suitable biomass concentration (OD 1 at /4600), IPTG was added (1 mM) and cultures were incubated for another 20 h (30°C, 210 rpm). The cells were harvested by centrifugation (4000 x g, 30 min, 4°C) and stored at -20 °C.

The BLASTP program was used to search the NCBI non-redundant protein sequences database for gene sequences related to 12-Oxophytodienoate reductase (OPR1 ) from Lycopersicon escuientum. Thirty eight sequences for related genes were selected, codon optimized for expression in E.coli, and subcloned into the pET28b(+) E. coli expression plasmid (Novagen, EMD Chemicals, Gibbstown, NJ, USA). The OPR1 related expression constructs were transformed into BL21 (DE3) E. coli (Stratagene, Agilent Technologies, Santa Clara, CA, USA) as directed and overnight cultures were grown in LB + kanamycin media. The LB cultures were used to inoculate expression cultures grown in Overnight Express Instant TB Medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). Cultures were incubated for 20h at 30°C, and the cells were harvested by centrifugation (4000 x g, 30m in, 4°C) and stored at -20 °C. Lycopersicon esculentum (tomato) 12-Oxophytodienoate Reductase 1 protein sequence:

MENKWEEKQ VDKIPLMSPC KMGKFELCHR WLAPLTRQR SYGYIPQPHA ILHYSQRSTN GGLLIGEATV ISETGIGYKD VPGIWTKEQV EAWKPIVDAV HAKGGIFFCQ IWHVGRVSNK DFQPNGEDPI SCTDRGLTPQ IRSNGIDIAH FTRPRRLTTD EIPQIVNEFR VAARNAIEAG FDGVEIHGAH GYLIDQFMKD QVNDRSDKYG GSLENRCRFA LEIVEAVANE IGSDRVGIRI SPFAHYNEAG DTNPTALGLY MVESLNKYDL AYCHWEPRM KTAWEKIECT ESLVPMRKAY KGTFIVAGGY DREDGNRALI EDRADLVAYG RLFISNPDLP KRFELNAPLN KYNRDTFYTS DPIVGYTDYP FLETMT (SEQ ID NO. 8)

Lycopersicon esculentum (tomato) 12-Oxophytodienoate Reductase 1 codon optimized sequence:

ATGGAAAACAAAGTTGTGGAAGAAAAACAGGTTGATAAAATCCCGCTGATGAGCCC GTGTAAAATGGGTAAATTCGAGCTGTGTCATCGCGTTGTACTGGCACCGCTGACTC GTCAGCGTTCTTATGGTTACATTCCGCAGCCGCACGCAATCCTGCATTACTCTCAG CGCAGCACCAACGGTGGCCTGCTGATCGGTGAAGCAACCGTGATCAGCGAAACT GGCATCGGTTACAAAGATGTGCCGGGTATCTGGACGAAAGAGCAGGTTGAGGCCT GGAAACCGATCGTCGACGCGGTGCATGCCAAAGGTGGTATTTTCTTTTGTCAGATC TGGCACGTTGGTCGTGTATCCAACAAAGATTTTCAGCCGAACGGCGAAGATCCGA TTTCCTGTACTGACCGCGGCCTGACCCCGCAGATCCGTTCCAACGGCATTGACAT TGCCCACTTCACCCGTCCACGTCGCCTGACTACTGACGAGATTCCGCAGATCGTG AACGAGTTCCGCGTTGCAGCGCGTAATGCTATTGAAGCGGGTTTCGATGGCGTCG AG ATTC ATG GTG C C C AC G GTTAC CTG ATC G AC C AATTC ATG AAAG AC C AAGTTAAC GACCGCAGCGATAAGTATGGCGGTTCTCTGGAGAACCGTTGTCGCTTCGCGCTGG AAATCGTTGAAGCAGTAGCCAACGAGATTGGCTCCGACCGTGTTGGTATCCGTATC TCTCCATTCGCACACTACAACGAAGCGGGCGACACTAACCCGACCGCACTGGGCC TGTATATGGTGGAGAGCCTGAATAAATACGACCTGGCGTATTGTCACGTGGTCGA GCCGCGCATGAAAACCGCCTGGGAAAAGATTGAGTGCACCGAAAGCCTGGTGCC GATGCGTAAAGCCTACAAAGGCACCTTCATCGTAGCTGGTGGCTACGACCGTGAA GACGGTAACCGCGCTCTGATCGAAGACCGTGCCGACCTGGTTGCGTACGGTCGT CTGTTCATCAGCAACCCAGACCTGCCGAAGCGTTTTGAACTGAACGCTCCGCTGA AC AAATAC AAC C GTG AC ACTTTCTAC ACTTC C G AC C C G ATC GTTG GTTAC AC C GAT TACCCGTTTCTGGAAACTATGACTTAATAA (SEQ ID NO. 9) Example 26

Reduction of (E)-3-formyl-5-methylhex-3-enoic acid with Recombinant

Reductases

Recombinant enoate reductases were tested for reduction of (E)-3-formyl-5-methylhex- 3-enoic acid using E. coli cells prepared as described in Example 25. Reactions (0.5ml_) were carried out at 30°C in potassium phosphate buffer (100mM, pH 7.0) with E. coli cells (100mg wet cells/mL), NADPH (10mM), NADH (10mM) and (E)-3-formyl-5- methylhex-2-enoic acid (10mM). After 16 h, acetonitrile (0.5ml) was added to each reaction and the resulting mixtures were centrifuged (2000rpm x 5min). Aliquots (0.1 mL) of the resulting supernatants were treated with 0.1 mL of potassium phosphate buffer (100mM, adjusted to pH 2.2 with phosphoric acid) and 0.225mL of a solution of 2,4-dinitrophenylhydrazine (20mM in 1 M HCI:acetonitrile, 3: 1 , v/v) for 30min at 50°C. The derivatized samples were diluted with 0.225mL of acetonitrile and analyzed by HPLC on a Phenomenex Lux 5μ Amylose-2 column (250mm x 4.6mm id) eluted with 0.1 % trifluoroacetic acid in water: acetonitrile (65:35, v/v) at 2mL/min. The column was maintained at 50°C and the effluent was monitored at 360nm. Results of HPLC analyses are shown in Table 2. Table 2

Accession %

Entry Enzyme Name /Source

Number Conversion ¹ fumarate reductase / Shewanella

5 Q07WU7 3.4

frigidimarina

Pentaerythritol tetranitrate

6 Q6JL81 85.6 reductase / Enterobacter cloacae

Unnamed enzyme / Arabidopsis

7 BAH57049 3.7

thaliana

Allyl alcohol dehydrogenase /

8 BAA89423 5.0

Nicotiana tabacum

Artemisinic aldehyde delta-1 1 (13)

9 1WLY_A 37.1 reductase / Artemisia annua

2-haloacrylate reductase /

10 NP_390263 4.2

Burkholderia sp. WS

NADPH dehydrogenase / Bacillus

1 1 YP_390263 82.8 subtilis subsp. subtilis str. 168

NADH:flavin

oxidoreductase/NADH oxidase /

12 YP_001664021 54.9

Thermoanaerobacter

pseudoethanolicus

Unnamed enzyme / Clostridium

13 CAA71086 6.0

tyrobutyricum

Unnamed enzyme / Moorella

14 Q2RGT7 4.8

thermoautotrophica oxophytodienoate reductase

15 Q9XG54 31 .4 (OPR1 ) / Lycopersicon esculentum

Oxophytodienoate reductase

16 Q9FEW9 15.7 (OPR3) / Lycopersicon esculentum

N-ethylmaleimide reductase /

17 Q3Z206 100.0 Shigella sonnei Accession %

Entry Enzyme Name /Source

Number Conversion ¹

12-oxo-phytodienoic acid

18 Q49HE0 100.0 reductase / Zea mays

12-oxo-phytodienoic acid

19 Q49HE4 100.0 reductase / Zea mays

20 Unnamed enzyme / Vitis vinifera A5BF80 7.5

Unnamed enzyme / Populus

21 B9MWG6 100.0 trichocarpa

12-oxophytodienoate reductase /

22 Q8GYB8 100.0 Arabidopsis thaliana

12-oxophytodienoate reductase /

23 B9SK95 5.4

Castor bean

NADH:flavin

24 oxidoreductase/NADH oxidase / D0YIM0 100.0 Klebsiella variicola

Unnamed enzyme / Citrobacter

25 A8AH31 100.0 koseri

N-ethylmaleimide reductase /

26 C1 M473 100.0 Citrobacter sp.

N-ethylmaleimide reductase /

27 D2THI8 100.0 Citrobacter rodentium

N-ethylmaleimide reductase /

28 Q5PH09 100.0 Salmonella paratyphi

N-ethylmaleimide reductase /

29 C9Y3L1 100.0 Cronobacter turicensis

Unnamed enzyme / Providencia

30 B2Q290 100.0 stuartii

NADPH dehydrogenase / Yarrowia

31 Q6CI57 9.1 lipolytica Accession %

Entry Enzyme Name /Source

Number Conversion ¹

N-ethylmaleimide reductase /

32 Q88I29 17.4 Pseudomonas putida

12-oxophytodienoate reductase /

33 150864790 7.8

Pichia stipitis

NAPDH dehydrogenase / Pichia

34 126131638 12.6 stipitis

Unnamed enzyme / Pichia

35 146393506 21 .6 guilliermondii unnamed enzyme / Candida

36 50293551 36.5 glabrata

Unnamed enzyme /

37 50405397 9.1 Debaryomyces hansenii

Unnamed enzyme/ Geobacillus

38 Q5KXG9 100.0 kaustophilus HTA426

Oxophytodienoate reductase

39 (OPR2) / Lycopersicon Q9XG54 14.0 esculentium

¹ %Conversion of (E)-3-formyl-5-methylhex-2-enoic acid to 3-formyl-5- methylhexanoic acid

Example 27

Reduction of (E)-3-formyl-5-methylhex-3-enoic acid with Recombinant

Reductases and Formate Dehydrogenase

Recombinant enoate reductases were evaluated for reduction of (E)-3-formyl-5- methylhex-3-enoic acid with NAD ⁺ and formate dehydrogenase. Enoate reductases were expressed in E. coli cells as described in Example 25. Formate dehydrogenase was expressed in E. coli cells as follows: The pET26b formate dehydrogenase expression construct was transformed into BL21 (DE3) E. coli (Stratagene, Agilent Technologies, Santa Clara, CA, USA) as directed and overnight cultures were grown in LB + kanamycin media. The LB cultures were used to inoculate expression cultures grown in Overnight Express Instant TB Medium + kanamycin (Novagen, EMD Chemicals, Gibbstown, NJ, USA). Cultures were incubated for 20 h at 30°C, and the cells were harvested by centrifugation (4000 x g, 30 min, 4°C) and stored at -20 °C. A variant (D74M) of oxophytodienoate reductase 3 (Accession number Q9FEW9) was expressed in E coli cells as follows: QuikChange Site-directed Mutagenesis kit from Stratagene (La Jolla, CA,USA) was used to create oxophytodienoate reductase 3 variant D74M as directed. Primers were ordered from Integrated DNA Technologies (Coralville, IA,USA). The pSTRC18 oxophytodienoate reductase 3 (OPR3) expression construct was transformed into BL21 (DE3) E coli (Stratagene, Agilent Technologies, Santa Clara, CA, USA) as directed and overnight cultures were grown in expansion broth + streptomycin (Zymo Research, Irvine, CA, USA). The LB cultures were used to inoculate expression cultures grown in Overexpression broth + streptomycin (Zymo Research, Irvine, CA, USA). Cultures were incubated for 20h at 23°C, and the cells were harvested by centrifugation (4000 x g, 30m in, 4°C) and stored at -20°C.

Reactions (0.5mL) were carried out at 30°C in potassium phosphate buffer (100mM, pH 7.0) with enoate reductases (40mg wet cells/mL), formate dehydrogenase (80mg wet cells/ml_), NAD ⁺ (0.02mM), ammonium formate (30mM) and (E)-3-formyl-5-methylhex- 3-enoic acid (20mM). After 24 h, reaction mixtures were acidified with 0.025ml_ of 4N HCI and extracted with 1 ml_ of ethyl acetate. Aliquots (0.5ml_) of the ethyl acetate extracts (0.5ml_) were dried over anhydrous sodium sulfate and treated with methanol (0.02ml_) and (trimethylsilyl)diazomethane (0.01 mL of a 2M solution in diethyl ether) to derivatize carboxylic acid moieties to their corresponding methyl esters. The derivatized samples were analyzed by GC on a Chiraldex™ G-TA column (30M x 0.25mm column, column temperature: 135°C isothermal, injector temperature: 200°C, carrier gas: helium, flow rate approximately 1 ml_/min) to give the results shown in Table 3.

Table 3

Accession %

Entry Enzyme Name /Source

Number Conversion ¹

Cronobacter turicensis DSM 18703

Unnamed enzyme / Geobacillus

10 Q5KXG9 21 .7

kaustophilus HTA426

12-oxophytodienoate reductase

1 1 Q9XG54 39.8

(OPR1 ) / Lycopersicon esculentum

¹ %Conversion of (E)-3-formyl-5-methylhex-2-enoic acid to 3-formyl-5- methylhexanoic acid (all reactions gave approximately 1 : 1 mixtures of (S)- and

(R)-enantiomers

Example 28

Reduction of (E)-3-formyl-5-methylhex-3-enoic acid with Recombinant

Reductases

Recombinant enoate reductases were evaluated for reduction of (E)-3-formyl-5- methylhex-3-enoic acid with NADP ⁺ and Lactobacillus brevis alcohol dehydrogenase (X- zyme). Enoate reductases were expressed in E. coli cells as described in Example 25. A variant (D74M) of oxophytodienoate reductase 3 (OPR3) was prepared as described in Example 27. Reactions (0.5ml_) were carried out at 30°C in potassium phosphate buffer (100mM, pH 7.0) with enoate reductases (40mg wet cells/mL), Lactobacillus brevis alcohol dehydrogenase (32U/ml_), NADP ⁺ (0.02mM), 2-propanol (3 vol%) and (E)-3-formyl-5-methylhex-2-enoic acid (20mM). After 24 h, reaction mixtures were acidified with 0.025ml_ of 4N HCI and extracted with 1 ml_ of ethyl acetate. Aliquots (0.5ml_) of the ethyl acetate extracts (0.5ml_) were dried over anhydrous sodium sulfate and treated with methanol (0.02ml_) and (trimethylsilyl)diazomethane (0.01 mL of a 2M solution in diethyl ether) to derivatize carboxylic acid moieties to their corresponding methyl esters. The derivatized samples were analyzed by GC on a Chiraldex™ G-TA column (30M x 0.25mm column, column temperature: 135°C isothermal, injector temperature: 200°C, carrier gas: helium, flow rate approximately 1 ml_/min) to give the results shown in Table 4.

Table 4

Accession %

Entry Enzyme Name /Source

Number Conversion ¹

12-oxophytodienoate reductase

1 1 Q9XG54 34.3

(OPR1 ) / Lycopersicon esculentum

¹ %Conversion of (E)-3-formyl-5-methylhex-2-enoic acid to 3-formyl-5- methylhexanoic acid (all reactions gave approximately 1 : 1 mixtures of (S)- and (R)-enantiomers

Example 29

Preparation of 3-formyl-5-methylhexanoic acid

A reaction vessel was charged with 7.5ml_ potassium phosphate buffer (0.1 M, pH 7.0), 8.4mg NADP ⁺ , 0.2ml_ Lactobacillus brevis alcohol dehydrogenase (32U/ml_, X-zyme), 0.3ml_ 2-propanol, pentaerythritol tetranitrate reductase (2ml_ of a 200mg/ml_ suspension of E. coli cells in potassium phosphate buffer), and 156mg of (E)-3-formyl-5- methylhex-3-enoic acid, and agitated at 40°C. After 6.75h, the reaction mixture was centrifuged and the supernatant was adjusted to pH 2 with 4N HCI and extracted with ethyl acetate (2 x 10ml_). The ethyl acetate extract was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to give 141 mg of colorless oil (89% yield).

¹ H NMR (D ₂ 0 at pH 8 - 10) δ 2.53 - 2.43 (m, 2 H), 2.34 - 2.28 (m, 1 H), 1.53 - 1 .42 (m, 1 H), 1 .41 - 1 .34 (m, 1 H), 1 .20 - 1.12 (m, 1 H), 0.76 (d, 3H), 0.74 (d, 3H).

Example 30

Biotransformation of 3-formyl-5-methylhexanoic acid to pregabalin with recombinant co-transaminases.

Transamination of 3-formyl-5-methylhexanoic acid to form Pregabalin was evaluated with various recombinant transaminases. Recombinant co-transaminases from Vibrio fluvialis, Rhodobacter sphaeroides, and Paracoccus denitrificans were expressed in E. coli as follows: The pET28b co-transaminase expression constructs were transformed into BL21 (DE3) E. coli (Stratagene, Agilent Technologies, Santa Clara, CA, USA) as directed and overnight cultures were grown in LB + kanamycin media. The LB cultures were used to inoculate expression cultures grown in Overnight Express Instant TB + kanamycin Medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). Cultures were incubated for 20 h at 30°C, and the cells were harvested by centrifugation (4000 x g, 30 min, 4°C) and stored at -20°C.

Reactions (0.5mL) were carried out at 30°C in potassium phosphate buffer (100mM, pH 7.0), pyridoxal phosphate (2mM), isopropylamine (150mM), 3-formyl-5-methylhexanoic acid (50mM) and co-transaminase (40mg wet cells/mL) from Vibrio fiuviaiis, Rhodobacter sphaeroides, or Paracoccus denitrificans. After 24h, reaction samples (0.1 mL) were diluted with 0.4mL acetonitrile:water (1 : 1 , v/v). Aliquots (0.05mL) of the diluted reaction samples were treated with saturated aqueous sodium bicarbonate (0.01 mL) and Marfey's reagent (/V-a-(2,4-dinitro-5-fluorophenyl)alaninamide, 0.2mL of 5g/L solution in acetonitrile) at 40°C. After 1 h, the derivatization reactions were quenched with 0.01 mL 1 N aqueous hydrochloric acid and diluted with 0.23mL of acetonitirle. The derivatized reaction samples were analyzed by UPLC (column: BEH C18, 50mm x 2.1 mm id, gradient elution: 70% A:30% B to 55% A:45% B in 5 min (A = 1 % triethylamine (pH 3 with phosphoric acid); B = acetonitrile), flow rate: 0.8mL/min, column temperature: 30°C, detection: 210-400nm) to give the results shown in Table 5. Table 5

Example 31

Biotransformation of 3-formyl-5-methylhexanoic acid to pregabalin with recombinant co-transaminases.

Recombinant variants of Vibrio fiuviaiis were tested for the reductive amination of 3- formyl-5-methylhexanoic acid to form Pregabalin. Variants of V. fiuviaiis co-transaminase (Accession number AEA39183) were expressed in E. coli as follows: QuikChange Site- directed Mutagenesis kits from Stratagene (La Jolla, CA, USA) was used to create V. fiuviaiis aminotransferase variants as directed. Primers were ordered from Integrated DNA Technologies (Coralville, IA, USA). The pET28b co-transaminase expression constructs were transformed into BL21 (DE3) E. coli (Stratagene, Agilent Technologies, Santa Clara, CA, USA) as directed and overnight cultures were grown in LB + kanamycin media. The LB cultures were used to inoculate expression cultures grown in Overnight Express Instant TB + kanamycin Medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). Cultures were incubated for 20h at 30°C, and the cells were harvested by centrifugation (4000 x g, 30m in, 4°C) and stored at -20°C.

Reactions (0.5mL) were carried out at 30°C in potassium phosphate buffer (100mM, pH 7.0) with pyridoxal phosphate (2mM), isopropylamine (300mM), 3-formyl-5- methylhexanoic acid (100mM) and V. fiuviaiis co-transaminase wild-type or variants (40mg wet cells/mL). After 28h, reaction samples (0.1 mL) were diluted with 0.4m L acetonitrile:water (1 : 1 , v/v). Aliquots (0.05mL) of the diluted reaction samples were treated with saturated aqueous sodium bicarbonate (0.01 mL) and Marfey's reagent (N- a-(2,4-dinitro-5-fluorophenyl)alaninamide, 0.2mL of 5g/L solution in acetonitrile) at 40°C. After 1 h, the derivatization reactions were quenched with 0.01 mL 1 N aqueous hydrochloric acid and diluted 0.23mL of acetonitrile. The derivatized reaction samples were analyzed by UPLC (column: BEH C18, 50mm x 2.1 mm id, gradient elution: 70% A:30% B to 55% A:45% B in 5min (A = 1 % triethylamine (pH 3 with phosphoric acid); B = acetonitrile), flow rate: 0.8mL/min, column temperature: 30°C, detection: 210-400nm) to give the results shown in Table 6. Table 6

V. fluvialis ω-transaminase %

Entry % ee

variant Conversion

33 Vfat888 ² 95.0 95.5

: Entry 25 thru 33 - reactions were performed using 4( DOmM of 3-1 Ormyl-5 methylhexanoic acid , 3mM PLP, 800mM IPM at 45°C.

2: Vfat 888:

DNA SEQUENCE

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTAC

GGCTTCACTGATATGCCATCTCTGCACCAGCGTGGTACCGTGGTTGTCACC

CACGGCGAGGGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGA CGCAAACTCCGGCCTGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCT GATCGACGCAGCAAAGGCCCAGTACGAACGCTTCCCGGGTTACCATAGCTT CTTCGGTCGTATGTCTGATCAAACTGTTATGCTGAGCGAGAAACTGGTAGA GGTGTCTCCATTCGACAGCGGTCGCGTGTTCTATACTAACTCCGGCTCCGA GGCTAACGATACTATGGTGAAAATGCTGTGGTTTCTGCACGCCGCAGAGGG CAAGCCGCAAAAACGCAAAATCCTGACTCGTAACAACGCATACCACGGTGT AACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGTACAACTCTGTATTCGG CCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTGGCGTTA CGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCG AGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTC TTTGCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAG GTTACTTCCAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTA TCTCTGATGAAGTTATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTG CGTTACCTATGACTTCACCCCGGATGCGATCATCTCCAGCAAAAATCTGACC GCCGGTTTCTTTCCGGTTGGTGCTGTGATTCTGGGTCCGGAACTGGCGAAA CGCCTGGAAACGGCGATCGAAGCTATCGAAGAGTTCCCGCACGGCTTTAC GGCCAGCGGTCACCCGGTGGGTTGCGCTATCGCTCTGAAAGCAATCGATG TTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCCGCCTGGCACCGCGT TTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACATCGGTGAATAT CGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAAGCATCT AAAACCCCATTCGATGGTAATCTGTCTGTGAGCGAGCGTATCGCTAACACCT GTACCGACCTGGGCCTGATCTGTAGCCCGATGGGTCAGTCCGTTATCCTGT GCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAAC TGGAGAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA (SEQ ID NO. 10)

AMINO ACID SEQUENCE

MNKPQSWEARAETYSLYGFTDMPSLHQRGTVWTHGEGPYIVDVNGRRYLDA NSGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVS PFDSGRVFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAV SASMTGLPYNSVFGLPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETI QREGADTIAGFFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRT GNTWGCVTYDFTPDAIISSKNLTAGFFPVGAVILGPELAKRLETAIEAIEEFPHGF TASGHPVGCAIALKAIDWMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIG FMWALEAVKDKASKTPFDGNLSVSERIANTCTDLGLICSPMGQSVILCPPFILTE AQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 2)

Example 32

Enzymatic reduction of (E)-3-formyl-5-methylhex-2-enoic acid to 3-formyl-5- methylhexanoic acid and in situ conversion to pregabalin with co-transaminase

Reduction of (E)-3-formyl-5-methylhex-3-enoic acid to 3-formyl-5-methylhexanoic acid with pentaerythntol tetranitrate reductase and in situ conversion to pregabalin was evaluated with various co-transaminases. Recombinant co-transaminases from Vibrio fluvialis, Rhodobacter sphaeroides, and Paracoccus denitrificans were expressed in E. coli as follows: The pET28b co-transaminase expression constructs were transformed into BL21 (DE3) E. coli (Stratagene, Agilent Technologies, Santa Clara, CA, USA) as directed and overnight cultures were grown in LB + kanamycin media. The LB cultures were used to inoculate expression cultures grown in Overnight Express Instant TB + kanamycin Medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). Cultures were incubated for 20h at 30°C, and the cells were harvested by centrifugation (4000 x g, 30min, 4°C) and stored at -20°C.

Reactions (0.5mL) were carried out at 30°C in potassium phosphate buffer (100mM, pH 7.0) with pentaerythntol tetranitrate reductase (40mg wet cells/mL), Lactobacillus brevis alcohol dehydrogenase (32U/ml_), NADP ⁺ (0.02mM), 2-propanol (3 vol%), pyridoxal phosphate (2 mM), isopropylamine (100mM), (E)-3-formyl-5-methylhex-2-enoic acid (20mM) and co-transaminase (40mg wet cells/mL) from Vibrio fiuviaiis, Rhodobacter sphaeroides, or Paracoccus denitrificans. After 43 h, reaction samples (0.1 ml_) were diluted with 0.1 mL acetonitrile:water (1 : 1 , v/v). Aliquots (0.1 ml_) of the diluted reaction samples were treated with saturated aqueous sodium bicarbonate (0.01 mL) and Marfey's reagent (/V-a-(2,4-dinitro-5-fluorophenyl)alaninamide, 0.4mL of 5g/L solution in acetonitrile) at 40°C. After 1 h, the derivatization reactions were quenched with 0.1 mL 1 N aqueous hydrochloric acid. The derivatized reaction samples were analyzed by UPLC (column: BEH C18, 50mm x 2.1 mm id, gradient elution: 70% A: 30% B to 55% A:45% B in 5min (A = 1 % triethylamine (pH 3 with phosphoric acid); B = acetonitrile), flow rate: 0.8mL/min, column temperature: 30°C, detection: 210-400nm) to give the results shown in Table 7. Table 7

Example 33

Enzymatic reduction of (E)-3-formyl-5-methylhex-2-enoic acid to 3-formyl-5- methylhexanoic acid and in-situ conversion to pregabalin with V. fiuviaiis co-transaminase

Reduction of (E)-3-formyl-5-methylhex-3-enoic acid to 3-formyl-5-methylhexanoic acid with pentaerythntol tetranitrate reductase and in-situ conversion to pregabalin was evaluated with variants of V. fluvialis aminotransferase. Variants of V. fluvialis co- transaminase (Accession number AEA39183) were expressed in E. coli as follows: QuikChange Site-directed Mutagenesis kits from Stratagene (La Jolla, CA, USA) was used to create V. fluvialis aminotransferase variants as directed. Primers were ordered from Integrated DNA Technologies (Coralville, IA, USA). The pET28b co-transaminase expression constructs were transformed into BL21 (DE3) E. coli (Stratagene, Agilent Technologies, Santa Clara, CA, USA) as directed and overnight cultures were grown in LB + kanamycin media. The LB cultures were used to inoculate expression cultures grown in Overnight Express Instant TB + kanamycin Medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). Cultures were incubated for 20h at 30°C, and the cells were harvested by centrifugation (4000 x g, 30m in, 4°C) and stored at -20°C.

Reactions (0.5mL) were carried out at 30°C in potassium phosphate buffer (100mM, pH 7.0) with pentaerythntol tetranitrate reductase (40mg wet cells/mL), Lactobacillus brevis alcohol dehydrogenase (32U/mL), NADP ⁺ (0.1 mM), 2-propanol (3 vol%), pyridoxal phosphate (2mM), isopropylamine (300mM), (E)-3-formyl-5-methylhex-2-enoic acid (100mM) and V. fluvialis co-transaminase wild-type or variants (40mg wet cells/mL). After 48h, reaction samples (0.02mL) were diluted with 0.18mL acetonitrile:water (1 : 1 , v/v). Aliquots (0.1 mL) of the diluted reaction samples were treated with saturated aqueous sodium bicarbonate (0.01 mL) and Marfey's reagent (/V-a-(2,4-dinitro-5- fluorophenyl)alaninamide, 0.4mL of 5 g/L solution in acetonitrile) at 40°C. After 1 h, the derivatization reactions were quenched with 0.1 mL 1 N aqueous hydrochloric acid. The derivatized reaction samples were analyzed by UPLC (column: BEH C18, 50mm x 2.1 mm id, gradient elution: 70% A:30% B to 55% A:45% B in 5 min (A = 1 % triethylamine (pH 3 with phosphoric acid); B = acetonitrile), flow rate: 0.8mL/min, column temperature: 30°C, detection: 210-400nm) to give the results shown in Table 8.

Table 8

V. fiuviaiis transaminase %Yield %ee

Entry

Variant Pregabalin (S)-Pregabalin

4 N166V 38.1 73.1

5 C424A 76.5 70.6

6 L100M 83.7 71 .4

7 S283A 68.1 71

8 L417M 68.7 71 .8

9 Wild-type 62.7 61 .5

Example 34

Alternative variants of Vibrio fluvalis ω-transaminase

The following further recombinant variants of Vibrio fiuviaiis co-transaminase were expressed in E. coli as follows: The pET28b co-transaminase expression constructs were transformed into BL21 (DE3) E. coli (Stratagene, Agilent Technologies, Santa Clara, CA, USA) as directed and overnight cultures were grown in LB + kanamycin media. The LB cultures were used to inoculate expression cultures grown in Overnight Express Instant TB + kanamycin Medium (Novagen, EMD Chemicals, Gibbstown, NJ, USA). Cultures were incubated for 20 h at 30°C, and the cells were harvested by centrifugation (4000 x g, 30 min, 4°C) and stored at -20°C.

Example 34a: Vfat906

DNA Sequence:

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTT CACTGATATGCCATCTCTGCACgAGCGTGGTACCGTGGTTGTCACCCACGGCGAG GGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCC TGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGC CCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAA CTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGT GTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGT TTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTaacAAC GCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGTACAACTC TGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTGG CGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCG AGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGC TGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCA GGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTA TCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACC CCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTG CTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTAT CGAAGAGTTCCCGCACGGCTTTACGGCCggcGGTCACCCGGTGGGTTGCGCTATC GCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCC GCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACAT CGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAA GCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCaaaCGTATCGCTAACACC TGTcagGACCTGGGCCTGATCTGTAGCgCGCTGGGTCAGTCCGTTATCCTGTGCCC G C C GTTC ATC CTG AC C GAG G C G C AAATGG ATG AG ATGTTTG AC AAACTG G AGAAG GCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA (SEQ ID NO. 1 1 )

Amino acid sequence:

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYIVDVNGRRYLDANSGLY N M VAG F D H KG L I D AAKAQ YE RF P G YH S F FG RM S D QTVM LS E KLVE VS P F D S G RVF YT NSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAVSASMTGLPYNSVFGL PLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAG GVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTA G FFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVR RLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQD LGLICSALGQSVILCPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 3)

Example 34b: Vfat999

DNA Sequence:

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTT CACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAG GGCCCATACGTGGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGC CTGTAC AATATG GTTG C C G G CTTC G AC C AC AAG G GTCTG ATC GAC G C AG C AAAG G CCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAA ACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCG TGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGG TTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAA ACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAA CTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATT GGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCG CGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTT GCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTC CAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGT TATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCA CCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGT GCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTA TCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTA TCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCG CCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAAC ATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACA AAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAAC ACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGCTGGGTCAGTCCGTTATCCTGA GCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGA GAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA (SEQ ID NO. 12)

Amino acid sequence:

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYVVDVNGRRYLDANSGL YNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFY TNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFG LPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGA GGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLT A GFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENV RRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQ DLGLICSALGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 4)

Example 34c: Vfat1010

DNA Sequence:

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTT CACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAG GGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCC TGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGC CCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAA CTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGT GTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGT TTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAAA CGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTATGCCGCACAAC TCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTG GCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGC GAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTG CTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCC AGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTT ATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCAC CCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGT GCTGTGATTCTGGGTCCGGCACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTA TCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTA TCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCG CCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAAC ATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACA AAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAAC ACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGA GCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGA GAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA (SEQ ID NO. 13) Amino acid sequence:

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYIVDVNGRRYLDANSGLY N M VAG F D H KG L I D AAKAQ YE RF P G YH S F FG RM S D QTVM LS E KLVE VS P F D S G RVF YT NSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGMPHNSVFG LPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGA GGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLT A GFFPVGAVILGPALSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENV RRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQ DLGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 5) Example 34d: Vf at 1020

DNA Sequence:

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTT CACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAG GGCCCATACGTGGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGC CTGTAC AATATG GTTG C C G G CTTC G AC C AC AAG G GTCTG ATC GAC G C AG C AAAG G CCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAA ACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCG TGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGG TTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAA ACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAA CTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGGGTTGTCCGCACTATT GGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCG CGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTT GCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTC CAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGT TATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCA CCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGT GCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTA TCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTA TCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCG CCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAAC ATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACA AAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAAC ACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGA GCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGA GAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA (SEQ ID NO. 14)

Amino acid sequence:

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYVVDVNGRRYLDANSGL YNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFY TNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFG LPLPGFVHLGCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGA GGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLT A GFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENV RRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQ DLGLICAAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 6)

Example 34e: Vfat1030

DNA Sequence: ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTT CACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAG GGCCCATACATCGTGGACGTCCACGGTCGCCGTTACCTGGACGCAAACTCCGGC CTGTAC AATATG GTTG C C G G CTTC G AC C AC AAG G GTCTG ATC GAC G C AG C AAAG G CCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAA ACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCG TGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGG TTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAA ACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAA CTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGAGCTGTCCGCACTATT GGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCG CGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTT GCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTC CAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGT TATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCA CCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGT GCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTA TCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTA TCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCG CCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAAC ATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACA AAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAAC ACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGA GCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGA GAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA (SEQ ID NO. 15)

Amino acid sequence:

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYIVDVHGRRYLDANSGLY N M VAG F D H KG L I D AAKAQ YE RF P G YH S F FG RM S D QTVM LS E KLVE VS P F D S G RVF YT NSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGL PLPGFVHLSCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAG GVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTA G FFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVR RLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQD LGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA (SEQ ID NO. 7) Example 35

Biotransformation of 3-isobutylidene-2-oxopentanedioic acid to Pregabalin

The genes for the Pseudomonas putida benzoylformate decarboxylase, Enterobacter cloacae pentaerythritol tetranitrate reductase, Lactobacillus brevis alcohol dehydrogenase, and Vibrio fiuviaiis co-transaminase were cloned into expression vector pDSTRC52 (Pfizer Inc. , USA) and put into the expression strain E. coli BDG62 (Pfizer Inc. , USA). The culture was grown and enzyme production was induced as described in Example 22. Enzyme expression was determined by polyacrylamide gel electrophoresis using Novex gels and stains (Invitrogen Corporation Carlsbad, California).

Reactions (1 .0ml_) were carried out at 40°C in phosphate buffer (100mM, pH 6.4) with P. putida decarboxylase, V. fiuviaiis transaminase, L. brevis alcohol dehydrogenase, E. cloacae pentaerythritol tetranitrate reductase, and NADP (0.1 mM) in one cell. 200μΙ_ 1 M 3-isobutylidene-2-oxopentanedioic acid, 100μΙ_ 1 mM ThDp + 25mM MgS04, 40μΙ_ 50mM PLP, 30μΙ_ isopropanol, 250μΙ_ 2M isopropylamine. Adjust pH to 6.4 for 24 hours, then adjust pH to 6.8 for additional 24 hours. An aliquot (0.5ml_) of the reaction was treated with 1 M aqueous sodium bicarbonate (0.05ml_) and Marfey's reagent (Λ/-α-(2,4- dinitro-5-fluorophenyl)alaninamide, 0.5ml_ of 5g/L solution in acetonitrile) at 40°C. After 1 h, the derivatization reactions were quenched with 0.05ml_ 1 N aqueous hydrochloric acid. The derivatized reaction samples were filtered and analyzed by UPLC (column: Agilent Eclipse Plus C18 column (100mm x 3.0mm, 1 .8μΓη) eluted with 0.1 % trifluoroacetic acid in watenacetonitrile (60:40, v/v) at 1 .3ml_/min. The column was maintained at 30°C and the effluent was monitored at 340nm and ES ⁺ mass spectroscopy. The reaction yielded a small amount of Pregabalin of which 82% was the desired S-isomer. Example 36

Biotransformation of 3-(2-methyl ropyl )-2-oxopentanedioic acid to Pregabalin

The genes for the Pseudomonas putida benzoylformate decarboxylase and Vibrio fiuviaiis co-transaminase were cloned into expression vector pDSTRC52 (Pfizer Inc., USA) and put into the expression strain E. coli BDG62 (Pfizer Inc., USA). The culture was grown and enzyme production was induced as described in Example 22. Enzyme expression was determined by polyacrylamide gel electrophoresis using Novex gels and stains (Invitrogen Corporation Carlsbad, California).

Reactions (2.0ml_) were carried out at 40°C in phosphate buffer (100mM, pH 6.4) with 500μΙ_ of culture (42.5mg dry cell weight) from a 24 hour fermentation tanks with P. putida decarboxylase and V. fiuviaiis co-transaminase cloned in one plasmid, 400μΙ_ 0.5M 3-(2-methylpropyl)-2-oxopentanedioic acid, 200ul_ 10 x ThDP (final 0.1 mM) and MgS0 ₄ (final 2.5mM), 80μΙ_ 50mM PLP (final 2mM), 300μΙ_ 2M isopropylamine (final 0.3M). Adjust pH to 6.4 and incubate at 45°C for 24 hours, then adjust pH to 6.8 for additional 24 hours. An aliquot (0.5ml_) of the reaction was treated with 1 M aqueous sodium bicarbonate (0.05ml_) and Marfey's reagent (/V-a-(2,4-dinitro-5- fluorophenyl)alaninamide, 0.5ml_ of 5g/L solution in acetonitrile) at 40°C. After 1 h, the derivatization reactions were quenched with 0.05ml_ 1 N aqueous hydrochloric acid. The derivatized reaction samples were filtered and analyzed by UPLC (column: Agilent Eclipse Plus C18 column (100mm x 3.0mm, 1 .8μΓη) eluted with 0.1 % trifluoroacetic acid in watenacetonitrile (60:40, v/v) at 1 .3ml_/min. The column was maintained at 30°C and the effluent was monitored at 340nm and ES ⁺ mass spectroscopy. The reaction yielded 100μg/mL of Pregabalin of which 65% was the desired S-isomer. Example 37

Preparation of (S)-Pregabalin via hydrolysis of 5-methoxy-4-(2-methyl ropyl )- dihydrofuran-2(3H)-one and enzymatic transamination

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 via syringe over 5mins. The reaction was removed from the ice/water bath and stirred at rt for 90m ins. The pH was adjusted to 7.0 using formic acid. The reaction mixture was then used as feedstock in the subsequent transaminase reaction.

Transaminase enzyme solution (12.5g), PLP (35mg), DIW (15ml_) and 4.0M isopropylamine. HCI aq soln (7.5ml_, 30mmol) were charged to a 100ml_ flask and warmed to 45°C. The hydrolysis reaction was added in one portion followed by DIW (6 ml_, used as a vessel rinse). The pH of the reaction was adjusted to 7.25 using neat isopropylamine and the reaction was stirred at 45°C until reaction completion. The reaction mixture was then heated to an internal temperature of 55°C and the pH adjusted to 4.0 using 95% formic acid. Darco carbon (125mg) was added and the mixture was allowed to cool to room temperature before cooling on ice/water for 20 minutes. The mixture was then filtered through Whatman paper no. 3. The filtrate was concentrated to 1 /3 of its weight and then heated to 55°C. The pH of the solution was then adjusted to pH 7.5 using 50% KOH after which the solution was cooled to ambient and then to 0-5°C in an ice/water bath. Precipitation of product was observed in the cooldown. The slurry was filtered and washed with DIW/EtOH (10ml_, 1 : 1 , 0°C). The white precipitate was dried for 12 hours in a vacuum oven (45°C) to yield (S)- Pregabalin in 61 % yield, 98.6% w/w purity and 99. 8% ee (preferred S-isomer). Example 38

Preparation of (S)-Pregabalin via hydrolysis of 5-butoxy-4-(2-methyl ropyl )-

5-Butoxy-4-(2-methylpropyl)-dihydrofuran-2(3H)-one (3.21 g, 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 via syringe over 5mins. The reaction was removed from the ice/water bath and stirred at rt for 90m ins. The reaction pH was then adjusted to pH 7.0 using formic acid, and the reaction mixture was then used as feedstock in the subsequent transaminase reaction.

Transaminase enzyme solution (12.5g), PLP (35mg), DIW (15ml_) and 4.0M isopropylamine.HCI solution (7.5ml_, 30mmol) were charged to a 100ml_ flask and warmed to 45°C. The hydrolysis reaction (see above) was added in one portion followed by DIW (6ml_, used as a vessel rinse). The pH of the reaction was adjusted to 7.25 using neat isopropylamine and the reaction was stirred at 45°C.

The reaction mixture was heated to an internal temperature of 55°C and pH adjusted to 4.0 using 95% formic acid. Darco carbon (125mg) was added and the mixture was allowed to cool to room temperature before being cooled on ice/water for 20 minutes. The mixture was then filtered through Whatman paper no 3. The filtrate was concentrated to 1/3 of its weight then heated to 55°C. The pH of the solution was then adjusted to pH 7.5 using 50% KOH after which the solution was cooled to ambient and then to 0-5°C in an ice/water bath. Precipitation of product was observed in the cooldown. The slurry was filtered and washed with DIW/EtOH (10ml_, 1 : 1 , 0°C). The white precipitate was dried for 12 hours in a vacuum oven (45°C) to yield (S)- Pregabalin in 51 % yield, 98.4% w/w purity and 99.9% e.e preferred S-isomer. Example 39

Preparation of (S)-Pregabalin from 5-hvdroxy-4-(2-methylpropyl)-dihvdrofuran- 2(3H)-one via enzymatic transamination

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. Potassium carbonate (0.863g, 6.3mmol) was added portion wise over the course of 5mins. The reaction was removed from the ice/water bath and stirred at rt for 90m ins. The pH was adjusted to 7.0 using formic acid and the reaction mixture was then used as feedstock in the subsequent transaminase reaction.

Transaminase enzyme solution (10.4g), PLP (30mg), DIW (12.5ml_) and aq 4.0M isopropylamine. HCI aq soln (6.3ml_, 30mmol) were charged to a 100ml_ flask and warmed to 45°C. The hydrolysis reaction was added in one portion followed by DIW (5m L, used as a vessel rinse). The pH of the reaction was adjusted to 7.25 using neat isopropylamine and the reaction was stirred at 45°C. The reaction mixture was heated to an internal temperature of 55°C and adjusted to pH 4.0 using 95% formic acid. Darco carbon (125mg) was added and the mixture was allowed to cool to room temperature before being cooled on ice/water for 20 minutes. The mixture was then filtered through Whatman paper no 3. The filtrate was concentrated to 1 /3 of its weight then heated to 55°C. The pH of the solution was then adjusted to pH 7.5 using 50% KOH after which the solution was cooled to ambient and then to 0-5°C in an ice/water bath. Precipitation of product was observed in the cooldown. The slurry was filtered and washed with DIW/EtOH (10ml_, 1 : 1 , 0°C). The white precipitate was dried for 12 hours in a vacuum oven (45°C) to yield (S)-Pregabalin in 61 % yield, 98.3% w/w purity and 99.9% e.e of the preferred S-isomer. Example 40

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

A 0.01 M solution of 5-hydroxy-4-(2-methylprop-1 -en-1 -yl)furan-2(5H)-one in 30% aqueous ammonia solution was hydrogenated (10 bar, ambient temperature) for 48h in the presence of 10mol% Raney Nickel catalyst. The catalyst was filtered and the solution concentrated to leave a solid. The product was isolated by addition of hydrochloric acid to a methanol suspension and found to be pure 3-aminomethyl-5- methylhexanoic acid hydrochloride.

The use of palladium as a catalyst gave mixtures 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-hvdroxy-4-(2- methylpropyl)-3,4-dihvdro-5H-2-furanone using a transaminase

A solution of R-3-aminomethyl-5-methylhexanoic acid in D. I. water at pH 7.5/ 45 C is stirred with a transaminase enzyme lysate, or whole cell preparation containing pyridoxal phosphate (PLP) and acetone. The isopropylamine produced is removed via a nitrogen sweep. Analysis of the solution after 24h show 3-aminomethyl-5-methyl- hexanoic acid with a lower e.e. and the presence of compound 5-hydroxy-4-(2- methylpropyl)-3,4-dihydro-5/-/-2-furanone. The lowering of the chiral purity of the 5- hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanone is due to the selective conversion of (4S)-5-hydroxy-4-(2-methylpropyl)-3,4-dihydro-5/-/-2-furanon e to S- pregabalin. Example 42

De-racemisation of rac-pregabalin using a transaminase

A solution of racemic pregabalin in DIW at pH 7.5 is treated with a suitable transaminase lysate. PLP is added and the reaction progressed for 12h at 45°C with a nitrogen sweep. Then, isopropylamine is added and the reaction is continued for a further 12h. The product is isolated as in Example 38 to afford enantiomerically enriched pregabalin.

It is also possible to effect the transformations exemplified in Examples 41 and 42 using a suitable amine oxidase/ imine reductase enzyme system with a co-factor such as NADP.

SEQUENCE LISTING SEQ ID NO. 1

Generic Vfat aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHX ²⁷ RGTWVTHGEGPYX ⁴ VDVX ⁴⁵ GRRYLDAN SGLYNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGR VFYTNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRX ⁴⁷ NAYHGVTAVSASMTGX ⁶³ P X ⁶⁵ NSVFGLPLPGFVHLX ⁸⁰ CPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAG FFAEPVMGAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPD AIISSKNLTAGFFPVGAVILGPELX ³⁰⁴ KRLETAIEAIEEFPHGFTAX ³²⁴ GHPVGCAIALKAID WMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGN LSVSX ⁴⁰ RIANTCX ⁴⁰⁸ DLGLICX ^{4 5} X ^{4 6} X ^{4 7} GQSVILX ⁴²⁴ PPFILTEAQMDEMFDKLEKALD KVFAEVA

X ²⁷ is selected from glutamine (Q) and glutamic acid (E); X ⁴¹ is selected from isoleucine (I) and valine (V); X ⁴⁵ is selected from asparigine (N) and histidine (H); X ¹⁴⁷ is selected from asparigine (N) and glutamine (Q); X ¹⁶³ is selected from leucine (L) and methionine (M); X ¹⁶⁵ is selected from tyrosine (Y) and histidine (H); X ¹⁸⁰ is selected from threonine (T); glycine (G) and serine (S); X ³⁰⁴ is selected from alanine (A) and serine (S); X ³²⁴ is selected from glycine (G) and serine (S); X ⁴⁰¹ is selected from lysine (K) and glutamic acid (E); X ⁴⁰⁸ is selected from threonine (T) and glutamine (Q); X ⁴¹⁵ is selected from serine (S) and alanine (A); X ⁴¹⁶ is selected from proline (P) and alanine (A); X ⁴¹⁷ is selected from leucine (L) and methionine (M); and X ⁴²⁴ is selected from cysteine (C) and serine (S).

SEQ ID NO. 2

Vfat888 aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHQRGTVWTHGEGPYIVDVNGRRYLDANSGL YNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFY TNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAVSASMTGLPYNSVFG LPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGA GGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLT A GFFPVGAVILGPELAKRLETAIEAIEEFPHGFTASGHPVGCAIALKAIDWMNEGLAENV RRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSERIANTCT DLGLICSPMGQSVILCPPFILTEAQMDEMFDKLEKALDKVFAEVA

SEQ ID NO. 3

Vfat906 aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYIVDVNGRRYLDANSGLY N M VAG F D H KG L I D AAKAQ YE RF P G YH S F FG RM S D QTVM LS E KLVE VS P F D S G RVF YT NSGSEANDTMVKMLWFLHAAEGKPQKRKILTRNNAYHGVTAVSASMTGLPYNSVFGL PLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAG GVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTA G FFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDWMNEGLAENVR RLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQD LGLICSALGQSVILCPPFILTEAQMDEMFDKLEKALDKVFAEVA SEQ ID NO. 4

Vfat999 aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYVVDVNGRRYLDANSGL YNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFY TNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFG LPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGA GGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLT A GFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENV RRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQ DLGLICSALGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA

SEQ ID NO. 5

Vfat1010 aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYIVDVNGRRYLDANSGLY N M VAG F D H KG L I D AAKAQ YE RF P G YH S F FG RM S D QTVM LS E KLVE VS P F D S G RVF YT NSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGMPHNSVFG LPLPGFVHLTCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGA GGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLT A GFFPVGAVILGPALSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENV RRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQ DLGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA SEQ ID NO. 6

Vfatl 020 aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYVVDVNGRRYLDANSGL YNMVAGFDHKGLIDAAKAQYERFPGYHSFFGRMSDQTVMLSEKLVEVSPFDSGRVFY TNSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFG LPLPGFVHLGCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGA GGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLT A GFFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENV RRLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQ DLGLICAAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA

SEQ ID NO. 7

Vfatl 030 aa sequence

MNKPQSWEARAETYSLYGFTDMPSLHERGTVWTHGEGPYIVDVHGRRYLDANSGLY N M VAG F D H KG L I D AAKAQ YE RF P G YH S F FG RM S D QTVM LS E KLVE VS P F D S G RVF YT NSGSEANDTMVKMLWFLHAAEGKPQKRKILTRQNAYHGVTAVSASMTGLPHNSVFGL PLPGFVHLSCPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVMGAG GVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTA G FFPVGAVILGPELSKRLETAIEAIEEFPHGFTAGGHPVGCAIALKAIDVVMNEGLAENVR RLAPRFEERLKHIAERPNIGEYRGIGFMWALEAVKDKASKTPFDGNLSVSKRIANTCQD LGLICSAMGQSVILSPPFILTEAQMDEMFDKLEKALDKVFAEVA

SEQ ID NO. 8

Lycopersicon esculentum (tomato) 12-Oxophytodienoate Reductase 1 protein sequence:

MENKVVEEKQVDKIPLMSPCKMGKFELCHRWLAPLTRQRSYGYIPQPHAILHYSQRS TNGGLLIGEATVISETGIGYKDVPGIWTKEQVEAWKPIVDAVHAKGGIFFCQIWHVGRV SNKDFQPNGEDPISCTDRGLTPQIRSNGIDIAHFTRPRRLTTDEIPQIVNEFRVAARNAI EAGFDGVEIHGAHGYLIDQFMKDQVNDRSDKYGGSLENRCRFALEIVEAVANEIGSDR VGIRISPFAHYNEAGDTNPTALGLYMVESLNKYDLAYCHWEPRMKTAWEKIECTESLV PMRKAYKGTFIVAGGYDREDGNRALIEDRADLVAYGRLFISNPDLPKRFELNAPLNKYN RDTFYTSDPIVGYTDYPFLETMT SEQ ID NO. 9

Lycopersicon esculentum (tomato) 12-Oxophytodienoate Reductase 1 codon optimized sequence:

ATGGAAAACAAAGTTGTGGAAGAAAAACAGGTTGATAAAATCCCGCTGATGAGCCC GTGTAAAATGGGTAAATTCGAGCTGTGTCATCGCGTTGTACTGGCACCGCTGACTC GTCAGCGTTCTTATGGTTACATTCCGCAGCCGCACGCAATCCTGCATTACTCTCAG CGCAGCACCAACGGTGGCCTGCTGATCGGTGAAGCAACCGTGATCAGCGAAACT GGCATCGGTTACAAAGATGTGCCGGGTATCTGGACGAAAGAGCAGGTTGAGGCCT GGAAACCGATCGTCGACGCGGTGCATGCCAAAGGTGGTATTTTCTTTTGTCAGATC TGGCACGTTGGTCGTGTATCCAACAAAGATTTTCAGCCGAACGGCGAAGATCCGA TTTCCTGTACTGACCGCGGCCTGACCCCGCAGATCCGTTCCAACGGCATTGACAT TGCCCACTTCACCCGTCCACGTCGCCTGACTACTGACGAGATTCCGCAGATCGTG AACGAGTTCCGCGTTGCAGCGCGTAATGCTATTGAAGCGGGTTTCGATGGCGTCG AG ATTC ATG GTG C C C AC G GTTAC CTG ATC G AC C AATTC ATG AAAG AC C AAGTTAAC GACCGCAGCGATAAGTATGGCGGTTCTCTGGAGAACCGTTGTCGCTTCGCGCTGG AAATCGTTGAAGCAGTAGCCAACGAGATTGGCTCCGACCGTGTTGGTATCCGTATC TCTCCATTCGCACACTACAACGAAGCGGGCGACACTAACCCGACCGCACTGGGCC TGTATATGGTGGAGAGCCTGAATAAATACGACCTGGCGTATTGTCACGTGGTCGA GCCGCGCATGAAAACCGCCTGGGAAAAGATTGAGTGCACCGAAAGCCTGGTGCC GATGCGTAAAGCCTACAAAGGCACCTTCATCGTAGCTGGTGGCTACGACCGTGAA GACGGTAACCGCGCTCTGATCGAAGACCGTGCCGACCTGGTTGCGTACGGTCGT CTGTTCATCAGCAACCCAGACCTGCCGAAGCGTTTTGAACTGAACGCTCCGCTGA ACAAATACAACCGTGACACTTTCTACACTTCCGACCCGATCGTTGGTTACACCGAT TACCCGTTTCTGGAAACTATGACTTAATAA

SEQ ID NO. 10

Vfat 888 DNA SEQUENCE

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTT CACTGATATGCCATCTCTGCACCAGCGTGGTACCGTGGTTGTCACCCACGGCGAG GGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCC TGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGC CCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAA CTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGT GTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGT TTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTAACAA CGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGTACAACT CTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTG GCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGC GAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTG CTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCC AGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTT ATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCAC CCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGT GCTGTGATTCTGGGTCCGGAACTGGCGAAACGCCTGGAAACGGCGATCGAAGCTA TCGAAGAGTTCCCGCACGGCTTTACGGCCAGCGGTCACCCGGTGGGTTGCGCTAT CGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGC CGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACA TCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAA AGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCGAGCGTATCGCTAACA CCTGTACCGACCTGGGCCTGATCTGTAGCCCGATGGGTCAGTCCGTTATCCTGTG CCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGAG AAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA SEQ ID NO. 11

Vfat 906 DNA SEQUENCE

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTT

CACTGATATGCCATCTCTGCACgAGCGTGGTACCGTGGTTGTCACCCACGGCGAG

GGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCC TGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGC CCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAA CTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGT GTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGT TTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTaacAAC GCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGTACAACTC TGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTGG CGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGCG AGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTGC TGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCCA GGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTTA TCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCACC CCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGTG CTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTAT CGAAGAGTTCCCGCACGGCTTTACGGCCggcGGTCACCCGGTGGGTTGCGCTATC GCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCGCC GCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAACAT CGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACAAA GCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCaaaCGTATCGCTAACACC TGTcagGACCTGGGCCTGATCTGTAGCgCGCTGGGTCAGTCCGTTATCCTGTGCCC G C C GTTC ATC CTG AC C GAG G C G C AAATGG ATG AG ATGTTTG AC AAACTG G AGAAG GCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA

SEQ ID NO. 12

Vfat 999 DNA SEQUENCE

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTT CACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAG GGCCCATACGTGGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGC CTGTAC AATATG GTTG C C G G CTTC G AC C AC AAG G GTCTG ATC GAC G C AG C AAAG G CCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAA ACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCG TGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGG TTTCTG C AC G C C G C AG AG G G C AAG C C G CAAAAAC G C AAAATCCTG ACTC GTC AAA ACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAA CTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATT GGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCG CGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTT GCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTC CAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGT TATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCA CCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGT GCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTA TCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTA TCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCG CCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAAC ATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACA AAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAAC ACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGCTGGGTCAGTCCGTTATCCTGA GCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGA GAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA

SEQ ID NO. 13

Vfat 1010 DNA SEQUENCE

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTT CACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAG GGCCCATACATCGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGCC TGTACAATATGGTTGCCGGCTTCGACCACAAGGGTCTGATCGACGCAGCAAAGGC CCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAAA CTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCGT GTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGGT TTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAAA CGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTATGCCGCACAAC TCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGACCTGTCCGCACTATTG GCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCGC GAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTTG CTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTCC AGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGTT ATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCAC CCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGT GCTGTGATTCTGGGTCCGGCACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTA TCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTA TCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCG CCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAAC ATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACA AAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAAC ACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGA GCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGA GAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA SEQ ID NO. 14

Vfat 1020 DNA SEQUENCE

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTT CACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAG GGCCCATACGTGGTGGACGTCAACGGTCGCCGTTACCTGGACGCAAACTCCGGC CTGTAC AATATG GTTG C C G G CTTC G AC C AC AAG G GTCTG ATC GAC G C AG C AAAG G CCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAA ACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCG TGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGG TTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAA ACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAA CTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGGGTTGTCCGCACTATT GGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCG CGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTT GCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTC CAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGT TATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCA CCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGT GCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTA TCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTA TCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCG CCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAAC ATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACA AAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAAC ACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGA GCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGA GAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA SEQ ID NO. 15

Vfat 1030 DNA SEQUENCE

ATGAATAAACCACAAAGCTGGGAAGCGCGTGCTGAAACTTACTCTCTGTACGGCTT CACTGATATGCCATCTCTGCACGAGCGTGGTACCGTGGTTGTCACCCACGGCGAG GGCCCATACATCGTGGACGTCCACGGTCGCCGTTACCTGGACGCAAACTCCGGC CTGTAC AATATG GTTG C C G G CTTC GAC C AC AAG G GTCTG ATC GAC G C AG C AAAG G CCCAGTACGAACGCTTCCCGGGTTACCATAGCTTCTTCGGTCGTATGTCTGATCAA ACTGTTATGCTGAGCGAGAAACTGGTAGAGGTGTCTCCATTCGACAGCGGTCGCG TGTTCTATACTAACTCCGGCTCCGAGGCTAACGATACTATGGTGAAAATGCTGTGG TTTCTGCACGCCGCAGAGGGCAAGCCGCAAAAACGCAAAATCCTGACTCGTCAAA ACGCATACCACGGTGTAACTGCTGTTTCCGCTTCCATGACGGGTCTGCCGCACAA CTCTGTATTCGGCCTGCCGCTGCCGGGTTTCGTTCACCTGAGCTGTCCGCACTATT GGCGTTACGGCGAAGAAGGTGAAACCGAAGAGCAGTTTGTTGCTCGTCTGGCCCG CGAGCTGGAGGAAACTATCCAACGTGAAGGCGCGGACACGATTGCGGGCTTCTTT GCTGAGCCGGTCATGGGCGCGGGCGGCGTAATCCCGCCGGCGAAAGGTTACTTC CAGGCGATCCTGCCGATTCTGCGTAAGTACGACATCCCGGTTATCTCTGATGAAGT TATCTGCGGCTTTGGTCGTACCGGTAATACTTGGGGTTGCGTTACCTATGACTTCA CCCCGGATGCGATCATCTCCAGCAAAAATCTGACCGCCGGTTTCTTTCCGGTTGGT GCTGTGATTCTGGGTCCGGAACTGAGCAAACGCCTGGAAACGGCGATCGAAGCTA TCGAAGAGTTCCCGCACGGCTTTACGGCCGGCGGTCACCCGGTGGGTTGCGCTA TCGCTCTGAAAGCAATCGATGTTGTGATGAATGAGGGTCTGGCAGAGAACGTGCG CCGCCTGGCACCGCGTTTTGAGGAGCGTCTGAAACACATTGCCGAACGTCCGAAC ATCGGTGAATATCGTGGCATCGGTTTTATGTGGGCACTGGAGGCTGTGAAAGACA AAGCATCTAAAACCCCATTCGATGGTAATCTGTCTGTGAGCAAACGTATCGCTAAC ACCTGTCAGGACCTGGGCCTGATCTGTAGCGCGATGGGTCAGTCCGTTATCCTGA GCCCGCCGTTCATCCTGACCGAGGCGCAAATGGATGAGATGTTTGACAAACTGGA GAAGGCTCTGGACAAAGTCTTTGCGGAGGTGGCGTAA