PROCESS FOR PREPARING CHIRAL COMPOUNDS BACKGROUND OF THE INVENTION

The present invention is directed to a 2-deoxyribose-5-phosphate aldolase (DERA) chemoenzymatic process for making chiral compounds.

The use of DERA (deoxyribose aldolase) family of aldolases in chemoenzymatic processes has been described. See US Pat. No. 5,795,749, WO 03/006656, WO 2004/027075, WO 2005/012246; Gijsen, H. J. M., et al. JACS, 1994, 116, 8422-8423; Gijsen, H. J. M., et al., JACS, 1995, 117, 7585-7591 ; Greenberg, W. A., et al., PNAS, 2004, 101 , 5788-5793, US Pat No. 6,964,863 and Biotechonol J, 101 , pgs 537-548 (2006). However, some of the processes provided poor overall yield as well as a mixture of products. In addition, the processes were limited to specific substrates. Accordingly, there exists a need in the art for a chemoenzymatic process that is effective and efficient for alternative substrates.

SUMMARY OF THE INVENTION

The present invention relates to a process comprising the step of reacting acetaldehyde with an N-protected aminoaldehyde substrate selected from the group consisting of 3-phthalimidopropionaldehyde, N-formyl-3-aminopropionaldehyde, 3- succinimido-propionaldehyde or N-diBoc-3-aminopropionaldehyde under aldolase-catalyzed aldol condensation conditions to form the corresponding lactol.

The present invention also relates to a process wherein said aldolase is a 2- deoxyribose-5-phosphate aldolase (DERA) aldolase.

The present invention also relates to a process wherein said aldolase is DERA 04 comprising a nucleotide sequence of SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO: 17;

DERA 06 comprising a nucleotide sequence of SEQ ID NO: 3 or an amino acid sequence of SEQ ID NO: 18;

DERA 101 comprising a nucleotide sequence of SEQ ID NO: 8 or an amino acid sequence of SEQ ID NO: 23;

DERA 102 comprising a nucleotide sequence of SEQ ID NO: 9 or an amino acid sequence of SEQ ID NO: 24;

DERA 103 comprising a nucleotide sequence of SEQ ID NO: 10 or an amino acid sequence of SEQ ID NO: 25;

DERA 104 comprising a nucleotide sequence of SEQ ID NO: 11 or an amino acid sequence of SEQ ID NO: 26;

DERA 105 comprising a nucleotide sequence of SEQ ID NO: 12 or an amino acid sequence of SEQ ID NO: 27;

DERA 106 comprising a nucleotide sequence of SEQ ID NO: 13 or an amino acid sequence of SEQ ID NO: 28;

DERA 107 comprising a nucleotide sequence of SEQ ID NO 14 or an amino acid sequence of SEQ ID NO 29,

DERA 108 comprising a nucleotide sequence of SEQ ID NO 15 or an amino acid sequence of SEQ ID NO 30, or an aldolase having an ammo acid sequence identity of at least about 20% thereof

More specifically, the present invention also relates to a process wherein said aldolase is DERA 04 comprising a nucleotide sequence of SEQ ID NO 2 or an amino acid sequence of SEQ ID NO 17, DERA 06 comprising a nucleotide sequence of SEQ ID NO 3 or an amino acid sequence of SEQ ID NO 18 or DERA 102 comprising a nucleotide sequence of SEQ ID NO 9 or an amino acid sequence of SEQ ID NO 24

More specifically, the present invention also relates to a process wherein said aldolase is DERA 04 comprising a nucleotide sequence of SEQ ID NO 2 or an amino acid sequence of SEQ ID NO 17

More specifically, the present invention also relates to a process wherein said aldolase is DERA 102 compπsing a nucleotide sequence of SEQ ID NO 9 or an amino acid sequence of SEQ ID NO 24

The present invention also relates to a process wherein said N-protected aminoaldehyde substrate is 3-phthalιmιdopropιonaldehyde

The present invention also relates to a process wherein said N-protected aminoaldehyde substrate is N-formyl-3-amιnopropιonaldehyde or 3-succιnιmιdo- propionaldehyde

The present invention also relates to a process wherein said N-protected aminoaldehyde substrate is N-dιBoc-3-amιnopropιonaldehyde

The present invention relates to a process comprising the step of

(a) reacting an aldehyde with an N-protected aminoaldehyde substrate selected from the group consisting of 3-phthalιmιdopropιonaldehyde, N-formyl-3-amιnopropιonaldehyde, 3- succinimido-propionaldehyde or N-dιBoc-3-amιnopropιonaldehyde under aldolase-catalyzed aldol condensation conditions to form the corresponding lactol,

(b) oxidizing the lactol so formed to yield the corresponding lactone,

(c) reacting the lactone so formed with isopropyl alcohol and acetone under acidic catalysis to yield the corresponding isopropyl acetonide ester,

(d) treating the isopropyl acetonide ester so formed with a base to yield the corresponding amino acetonide isopropyl ester

The present invention relates to a process comprising the step of

(a) reacting an aldehyde with an N-protected aminoaldehyde substrate selected from the group consisting of 3-phthalιmιdopropιonaldehyde, N-formyl-3-amιnopropιonaldehyde, 3- succimmido-propionaldehyde or N-dιBoc-3-amιnopropιonaldehyde under aldolase-catalyzed aldol condensation conditions to form the corresponding lactol,

(b) oxidizing the lactol so formed to yield the corresponding lactone,

(c) reacting the lactone so formed with cyclopentanone to yield the corresponding cyclopentylidene phthalimido isopropyl ester, and

(d) treating the cyclopentylidene phthalimido isopropyl ester so formed with base to yield the corresponding amino cyclopentylidene isopropyl ester

The present invention relates to a process comprising the steps of

(a) reacting an aldehyde with an N-protected aminoaldehyde substrate selected from the group consisting of 3-phthalιmιdopropιonaldehyde, N-formyl-3-amιπopropιonaldehyde, 3- succinimido-propionaldehyde or N-dιBoc-3-amιnopropιonaldehyde under aldolase-catalyzed aldol condensation conditions to form the corresponding lactol,

(b) dehydrogenating the lactol so formed under catalytic dehydrogenation conditions to yield the corresponding heptanoic acid,

(c) treating said 3,5-dιhydroxyheptanoιc acid so formed with dicyclohexylamine to form the corresponding salt,

(d) reacting the salt so formed with triisopropyl orthoformate and acetone under acidic catalysis to yield the corresponding isopropyl acetonide ester, and

(e) treating the isopropyl acetonide ester so formed with base to yield the corresponding ammo dicyclohexylamine isopropyl ester

The present invention relates to a process comprising the steps of

(a) reacting an aldehyde with an N-protected aminoaldehyde substrate selected from the group consisting of 3-phthalιmιdopropιonaldehyde, N-formyl-3-amιnopropιonaldehyde, 3- succinimido-propionaldehyde or N-dιBoc-3-amιnopropιonaldehyde under aldolase-catalyzed aldol condensation conditions to form the corresponding lactol,

(b) oxidizing the lactol so formed to yield the corresponding 3,5-dιhydroxyheptanoιc acid,

(c) treating said 3,5-dιhydroxyheptanoιc acid with dicyclohexylamine to form the corresponding salt, and

(d) reacting the salt so formed with tπisopropyl orthoformate to yield the corresponding isopropyl acetonide ester, and

(e) treating the isopropyl acetonide ester so formed with base to yield the corresponding amino acetonide isopropyl ester

The present invention relates to a process comprising the step of reacting an aldehyde with an aminoaldehyde substrate or an N-protected aminoaldehyde substrate under DERA 101 , DERA 102, DERA 103, DERA 104, DERA 105, DERA 106, DERA 107 or DERA 108 aldolase-catalyzed aldol condensation conditions to form the corresponding lactol

The present invention also relates to a process wherein said aminoaldehyde or said N-protected aminoaldehyde is N-Boc-3-amιnopropιonaldehyde, 3-amιnopropιonaldehyde, aminoacetaldehyde, N-CBz-3-amιnopropιonaldehyde, N-acetyl-3-amιnopropιonaldehyde, N- Fmoc-3-amιnopropιonaldehyde, or N-Fmoc-aminoacetaldehyde

More specifically, the present invention also relates to a process wherein said N- protected aminoaldehyde is N-Boc-3-amιnopropιoπaldehyde

More specifically, the present invention also relates to a process wherein said aminoaldehyde or said N-protected aminoaldehyde is N-CBz-3-amιnopropιonaldehyde or N- Fmoc-3-amιnopropιonaldehyde

More specifically, the present invention also relates to a process wherein said aminoaldehyde or said N-protected aminoaldehyde is N-CBz-3-amιnopropιonaldehyde

The present invention also relates to a process wherein said aldolase is DERA 102

The present invention relates to a process compnsing the step of reacting an aldehyde with an aminoaldehyde substrate or an N-protected aminoaldehyde substrate under DERA 101, DERA 102, DERA 103, DERA 104, DERA 105, DERA 106, DERA 107 or DERA 108 aldolase-catalyzed aldol condensation conditions to form the corresponding lactol, and oxidizing the lactol so formed to yield the corresponding lactone

The present invention relates to a process comprising the steps of

(a) reacting an aldehyde with an aminoaldehyde substrate or an N-protected aminoaldehyde substrate under DERA 101 , DERA 102, DERA 103, DERA 104, DERA 105, DERA 106, DERA 107 or DERA 108 aldolase-catalyzed aldol condensation conditions to form the corresponding lactol,

(b) dehydrogenating the lactol so formed under catalytic dehydrogenation conditions to yield the corresponding 3,5-dιhydroxyheptanoιc acid,

(c) treating said 3,5-dιhydroxyheptaπoιc acid so formed with dicyclohexylamine to form the corresponding salt, and

(d) reacting the salt so formed with triisopropyl orthoformate to yield the corresponding isopropyl acetoπide ester

The present invention relates to a process comprising the steps of

(a) reacting an aldehyde with an aminoaldehyde substrate or an N-protected aminoaldehyde substrate under DERA 101 , DERA 102, DERA 103, DERA 104, DERA 105, DERA 106, DERA 107 or DERA 108 aldolase-catalyzed aldol condensation conditions to form the corresponding lactol,

(b) oxidizing the lactol so formed to yield the corresponding 3,5-dιhydroxyheptaπoιc acid,

(c) treating said 3,5-dιhydroxyheptanoιc acid with dicyclohexylamine to form the corresponding salt, and

(d) reacting the salt so formed with triisopropyl orthoformate to yield the corresponding isopropyl acetonide ester

The present invention relates to a process comprising the step of reacting an aldehyde with an aminoaldehyde substrate compound of the general formula (I)

wherein n = 1 , 2, 3 or 4.

R' is hydrogen or an N-protecting group;

R" is hydrogen or an N-protecting group; or R' and R" taken together with nitrogen to which they are attached form a 5- or 6-membered heterocyclic moiety, under DERA 101 , DERA 102, DERA 103, DERA 104, DERA 105, DERA 106, DERA 107 or DERA 108 aldolase-catalyzed aldol condensation conditions to form the corresponding lactol.

The present invention also relates to the compound 2-[2-(4,6-Dihydroxy-tetrahydro- pyran-2-yl]-isoindole-1 ,3-dione.

More specifically, the present invention also relates to a compound of the formula

More specifically, the present invention also relates to a compound of the formula

The present invention also relates to the compound of the formula

The present invention also relates to the compound of the formula

The present invention also relates to the compound of the formula

The present invention also relates to the compound of the formula

The present invention also relates to the compound of the formula

The present invention also relates to the compound of the formula

The present invention also relates to the compound of the formula

The present invention also relates to the compound of the formula

The present invention relates to a crystalline form of 4-fluoro-alpha-[2-methyl-1- oxopropyl]-gamma-oxo-N, beta-diphenylbenzenebutanamide chacterized as having powder X-ray diffraction peaks of about 9 0, 12 7, 20 2, 22 6, and 25 2 degrees two-theta

The present invention relates to a crystalline form of (2R-trans)-5-(4-fluorophenyl)-2- (1-methylethyl)-N,4-dιphenyl-1-[2-(tetrahydro-4-hydroxy-6-o xo-2H-pyran-2-yl)ethyl]-1H- pyrrole-3-carboxamιde chacteπzed as having powder X-ray diffraction peaks of about 6 3, 12 7, 16 8, 21 1 and 25 5 degrees two-theta

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an experimental powder X-ray diffraction pattern for 4-fluoro-alpha-[2- methyl-1-oxopropyl]-gamma-oxo-N, beta-diphenylbenzenebutanamide The scale of the abscissa is degrees two-theta The ordinate is the intensity of the counts

Figure 2 is the differential scanning caloπmetry (DSC) thermogram for 4-fluoro-alpha- [2-methyl-1-oxopropyl]-gamma-oxo-N, beta-diphenylbenzenebutanamide

Figure 3 is the infrared (FTIR) spectrum for 4-fluoro-alpha-[2-methyl-1-oxopropyl]- gamma-oxo-N, beta-diphenylbenzenebutanamide

Figure 4 is the Raman spectrum for 4-fluoro-alpha-[2-methyl-1-oxopropyl]-gamma- oxo-N, beta-diphenylbenzenebutanamide

Figure 5 is an experimental powder X-ray diffraction pattern for (2R-trans)-5-(4- fluorophenyl)-2-(1-methylethyl)-N,4-dιphenyl-1-[2-(tetrahyd ro-4-hydroxy-6-oxo-2H-pyran-2- yl)ethyl]-1 H-pyrrole-3-carboxamιde The scale of the abscissa is degrees two-theta The ordinate is the intensity of the counts

Figure 6 is the differential scanning calorimetry (DSC) thermogram for (2R-trans)-5- (4-fluorophenyl)-2-(1-methylethyl)-N,4-dιphenyl-1-[2-(tetra hydro-4-hydroxy-6-oxo-2H-pyran-2- yl)ethyl]-1 H-pyrrole-3-carboxamιde

Figure 7 is the infrared (FTIR) spectrum for (2R-trans)-5-(4-fluorophenyl)-2-(1- methylethyl)-N,4-dιphenyl-1-[2-(tetrahydro-4-hydroxy-6-oxo- 2H-pyran-2-yl)ethyl]-1 H-pyrrole-3- carboxamide

Figure 8 is the Raman spectrum for (2R-trans)-5-(4-fluorophenyl)-2-(1-methylethyl)- N,4-dιphenyl-1-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl )ethyl]-1H-pyrrole-3- carboxamide

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

Unless indicated otherwise, the following terms are defined as follows:

The article "a" or "an" as used herein refers to both the singular and plural form of the object to which it refers.

The term "aldolase-catalyzed aldol condensation conditions" as used herein refers to any aldol condensation conditions known in the art that can be catalyzed by an aldolase, as described herein.

The aldehyde for use in the present invention may be any aldehyde that will undergo an aldol condensation with a substrate, as described herein, in the presence of an aldolase, as described herein. An example of suitable aldehyde is, but is not limited to, acetaldehyde.

A substrate for use in the present invention may be any aminoaldehyde or N- protected aminoaldehyde. Such an aminoaldehyde or N-protected aminoaldehyde will react with an aldehyde under aldolase-catalyzed aldol condensation conditions, each as described herein.

Suitable N-protecting groups for the aminoaldehyde include, but are not limited to, phthalimido, N-formyl, succinimdo, di-butoxycarbonyl (di-Boc), benzyloxycarbonyl (CBz), butoxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl (Fmoc), benzyl, and dibenzyl.

Examples of a suitable aminoaldehyde substrate include, but are not limited to: propionaldehyde acetaldehyde N-CBz-3-Aminopropionaidehyde

ldehyde /V-Boc-3- Aminopropionaldehyde

N-Fmoc-3-Aminopropioπaldehyde N-Fmoc-acetaldehyde

S-Succinimido-propionaldehyde _and λ/-dιBoc-3-Amιnopropιonaldehyde

In one embodiment of the invention, the aminoaldehyde substrate is 3- phthahmidopropionaldehyde, N-formyl-3-amιnopropιonaldehyde, N-Boc-3- aminopropionaldehyde, 3-succιnιmιdo-propιonaldehyde or N-dιBoc-3-amιnopropιonaldehyde In another embodiment of the invention, the aminoaldehyde substrate is N-CBz-3- aminopropionaldehyde or N-Fmoc-3-amιnopropιoπaldehyde In another embodiment of the invention, the aminoaldehyde substrate is 3-amιno-propιonaldehyde In another embodiment of the invention, the aminoaldehyde substrate is amino-acetaldehyde In another embodiment of the invention, the aminoaldehyde substrate is N-CBz-3-amιnopropιonaldehyde (commercially available from Aldπch) In another embodiment of the invention, the aminoaldehyde substrate is N-acetyl-3-amιnopropιonaldehyde In another embodiment of the invention, the aminoaldehyde substrate is N-Fmoc-3-amιnopropιonaldehyde

Both N-Fmoc-aminoaldehydes were obtained via standard Dess-Martin oxidation of the corresponding N-Fmoc aminoalcohol

The N-acetyl-3-amιnopropιonaldehyde was obtained from 3-amιno-1-propanol by a two step procedure N-acetylation of the 3-amιno-1-propanol by methyl actetate followed by Dess-Martin oxidation to give the desired product with the correct ESI-MS [M+H] ^* 11625 and [M+Na] ⁺ 138 20

An aldolase for use in the present invention may be any enzyme that has aldolase activity towards an aminoaldehyde substrate, N-protected aminoaldehyde substrate, or pyrrole aldehyde substrate, each as descπbed herein In one embodiment of the invention, the aldolase is a 2-deoxyrιbose-5-phosphate aldolase (DERA) Examples of a suitable DERA aldolase include, but are not limited to

DERA 03 (£ coli) (commercially available from Sigma Aldrich, St Louis, MO),

DERA 04 (William A Greenberg, er a/ , PNAS, (2004), VoI 101 ,, No 16, pp 5788- 5793 or a modified version thereof),

DERA 06 (GenBank Accession NP_294929 or a modified version thereof),

DERA 08 (GenBank Accession NP_465519 or a modified version thereof),

DERA 11 (GenBank Accession NP_439273),

DERA 12 (GenBank Accession NP_229359),

DERA 15 (Haruhiko Sakuraba, et al , Journal of Biological Chemistry (2003), VoI 278, No 12, pp 10799-10806),

DERA 101 (GenBank Accession NP_906068 1 or a modified version thereof),

DERA 102 (GenBank Accession NP_813976 1 or a modified version thereof),

DERA 103 (GenBank Accession NP_01130044 1 or a modified version thereof),

DERA 104 (GenBank Accession YP_924715 1 or a modified version thereof),

DERA 105 (GenBank Accession YP_148352 1 or a modified version thereof),

DERA 106 (GenBank Accession NP_471437 1 or a modified version thereof),

DERA 107 (GenBank Accession NP_242218 1 or a modified version thereof), and

DERA 108 (GenBank Accession ZP_00875069 1 or a modified version thereof)

In one embodiment of the invention, the aldolase is an aldolase having an amino acid sequence identity of at least about 20% thereof, preferably, at least 70% thereof, to a DERA aldolase described herein In one embodiment of the invention, the DERA aldolase is DERA 04, DERA 06 or DERA 102 In one embodiment of the invention, the DERA aldolase is DERA 102

According to the invention, DERA 03, DERA 04, DERA 06, DERA 08, DERA 11, DERA 12, DERA 15, DERA 101 , DERA 102, DERA 103, DERA 104, DERA 105, DERA 106, DERA 107 and DERA 108 are identified by their nucleotide sequences and amino acid sequences set forth in Examples 1-30

More specifically, DERA 03 is an aldolase having a nucleotide sequence of SEQ ID NO 1 and an ammo acid sequence of SEQ ID NO 16

DERA 04 is an aldolase having a nucleotide sequence of SEQ ID NO 2 and an amino acid sequence of SEQ ID NO 17

DERA 06 is an aldolase having a nucleotide sequence of SEQ ID NO 3 and an amino acid sequence of SEQ ID NO 18

DERA 08 is an aldolase having a nucleotide sequence of SEQ ID NO 4 and an amino acid sequence of SEQ ID NO 19

DERA 11 is an aldolase having a nucleotide sequence of SEQ ID NO 5 and an amino acid sequence of SEQ ID NO 20

DERA 12 is an aldolase having a nucleotide sequence of SEQ ID NO 6 and an amino acid sequence of SEQ ID NO 21

DERA 15 is an aldolase having a nucleotide sequence of SEQ ID NO 7 and an amino acid sequence of SEQ ID NO 22

DERA 101 is an aldolase having a nucleotide sequence of SEQ ID NO 8 and an amino acid sequence of SEQ ID NO 23

DERA 102 is an aldolase having a nucleotide sequence of SEQ ID NO 9 and an amino acid sequence of SEQ ID NO 24

DERA 103 is an aldolase having a nucleotide sequence of SEQ ID NO 10 and an amino acid sequence of SEQ ID NO 25

DERA 104 is an aldolase having a nucleotide sequence of SEQ ID NO 11 and an amino acid sequence of SEQ ID NO 26

DERA 105 is an aldolase having a nucleotide sequence of SEQ ID NO 12 and an amino acid sequence of SEQ ID NO 27

DERA 106 is an aldolase having a nucleotide sequence of SEQ ID NO 13 and an amino acid sequence of SEQ ID NO 28

DERA 107 is an aldolase having a nucleotide sequence of SEQ ID NO 14 and an amino acid sequence of SEQ ID NO 29

DERA 108 is an aldolase having a nucleotide sequence of SEQ ID NO 15 and an amino acid sequence of SEQ ID NO 30

The DERA aldolases described herein can be prepared by any means known in the art, including but not limited to Standard protocols for protein expression in recombinant E coll (Sambrook and Russell, Molecular Cloning A Laboratory Manual, 3 ^rd Ed , Cold Spring Harbor, NY 2001) As would be understood by one of skill in the art, modified versions of known DERA aldolases may be necessary or may result depending on cloning conditions and are encompassed by the present invention

The following Schemes illustrate the present invention

Preparation A

In Preparation A, 3-phthalιmιdopropιonaldehyde is prepared by reacting phthalimide with acrolein in the presence of benzyltrimethyl ammonium hydroxide (Triton-B) The reaction is stirred at a temperature between about 53 ⁰ C to about 67 5 ⁰ C, preferably about 60 ⁰ C, for a time penod between about 30 minutes to about 3 hours, preferably about 90 minutes

In Preparation B, N-formyl-3-amιnopropιonaldehyde is prepared by reacting ethyl formate with 1-amιno-3,3-dιmethoxypropane and treating the amide so formed with acid

Preparation C

In Preparation C, N-Boc-3-amιnopropιonaldehyde is prepared by reacting 1-amιno- 3,3-dιmethoxypropane with BOC anhydride and treating the amide so formed with acid

Preparation D

In Preparation D, N-dι-Boc-3-amιnopropιonaldehyde is prepared by reacting 1-amιno- 3,3-dιmethoxypropane with BOC anhydrdride in the presence of 4-dι(methylamιno)pyπdιne and treating the amide so formed with acid

Preparation E

3-sucαnιmιdoproDionaldehvde

Acrolein is added to a solution of succinimide in the presence of catalytic sodium ethoxide and a polar protic solvent, such as ethanol The reaction mixture is stirred at a temperature between about 10 ⁰ C to about 40 ⁰ C, preferably about 20-30 ⁰ C, for a time peπod between about 20 hours to about 60 hours, preferably about 48 hours

Scheme 1

Scheme 1 describes in general a process encompassed by the present invention As set forth in Scheme 1 , a DEFRA aldolase catalyzes two sequential aldol condensation reactions between 3-phthalιmιdopropιonaldehyde and 2 mol of acetaldehyde in the presence of other suitable solvents such as methyl tert-butyl ether (MTBE) and water to yield the protected desired amino-lactol (A) Suitable DERA aldolases include, but are not limited to,

DERA 04, DERA 06, DERA 101 , DERA 102, DERA 104, DERA 105, DERA 106, DERA107 and DERA 108, preferably DERA 04 and DERA 102 The acetaldehyde is added to the mixture of 3-phthalιmιdopropιonaldehyde and DERA aldolase over a time period between about 7 hours to about 12 hours, preferably about 10 hours The mixture so formed is further stirred at a temperature between about 15 ⁰ C to about 30 ⁰ C, preferably about 22 ⁰ C, for a time period between about 20 hours to about 60 hours, preferably about 48 hours

The amino-lactol (A) can undergo catalytic (e g platinum on carbon or palladiumon carbon) dehydrogenation to form carboxyhc acid (C), which can then undergo lactonization to form (B)

Any catalytic dehydrogenation means known in the art to convert (A) to (C) are encompassed by the present invention Examples of suitable catalysts include, but are not limited to, Pt/C, Pd/C, Pt/Bi/C, Pd/Bi/C and any other dehydrogenation catalysts In one embodiment of the invention, the catalytic dehydrogenation is performed at about pH 7 to about pH 10 using air or oxygen as terminal oxidant

Any lactonization means known in the art to convert carboxyhc acid (C) to lactone (B) are encompassed by the present invention including, but not limited to, the use of acid catalysts such as, but not limited to, hydrochloπc acid, sulfuric acid, methanesulfonic acid (MSA), p-toluenesulfonic acid (TSA) and any other lactonization acids known in the art More specifically, the 7-(1 ,3-Dιoxo-1 ,3-dιhydro-ιsoιndo-2-yl)-3,5-dιhydroxy-heptanoιc acid (C) is converted to the corresponding 2-[2-(4-Hydroxy-6-oxo-tetrahydro-pyran-2-yl]-ιsoιndole-1,3 - dione (B) by treating (C) with anhydrous hydrochloric acid in the presence of ethyl acetate The reaction is stirred at room temperature for a time period between about 1 hour to about 4 hours, preferably about 2-3 hours

Alternatively, oxidation of the lactol (A) to lactone (B) or carboxyhc acid (C) can be performed by use of any oxidation means known in the art that will achieve the desired transformation More specifically, 2-[2-(4,6-dιhydroxy-tetrahydro-pyran-2-yl]-ιsoιndole-1 ,3- dione (A) is converted to the corresponding 2-[2-(4-hydroxy-6-oxo-tetrahydro-pyran-2-yl]- ιsoιndole-1 ,3-dιone (B) by oxidizing (A) in the presence of an oxidizing agent , such as sodium chlorite The reaction is stirred at a temperature between about 10 ⁰ C to about 30 ⁰ C, preferably about 23 ⁰ C, for a time period between about 2 hours to about 6 hours, preferably about 4 hours The 2-[2-(4,6-dιhydroxy-tetrahydro-pyran-2-yl]-ιsoιndole-1 ,3-dιone (A) can also be converted to the corresponding 7-(1 ,3-dιoxo-1 ,3-dιhydro-ιsoιndo-2-yl)-3,5-dιhydroxy- heptanoic acid (C) by oxidizing (A) in the presence of an oxidizing agent , such as sodium chlorite, a phosphate buffer, a polar aprotic solvent, such as dimethyl sulfoxide, and an alcohol, such as isopropanol The reaction is maintained at room temperature and a pH between about 5 to about 6 for a time period between about 2 hours to about 6 hours, preferably about 4 hours

The 7-(1 ,3-dιoxo-1 ,3-dιhydro-ιsoιndo-2-yl)-3,5-dιhydroxy-heptanoιc acιd (C) is converted to the corresponding dicyclohexyl amine (DCA) salt (D) by treating (C) with dicyclohexyl amine in the presence of ethyl acetate The DCA salt (D) is then converted to

the phthalimido acetonide isopropyl ester (E) by reacting (D) with DCM, tπisopropyl orthoformate in the presence of acetone and methanesulfonic acid

The phthalimido acetonide isopropyl ester (E) may also be prepared by reacting 2-[2- (4-hydroxy-6-oxo-tetrahydro-pyran-2-yl]-ιsoιndole-1 ,3-dιone (B) with isopropyl alcohol in the presence of acetone and methanesulfonic acid (MSA) The reaction mixture is stirred at room temperature at a pH between about 1 to about 2, preferably about 1 5, for a time period between about 20 hours to about 28 hours, preferably about 24 hours.

The phthalimido acetonide isopropyl ester (E) is deprotected to give the corresponding amino acetonide isopropyl ester (F) by treating (E) with a base, such as primary amine, i e an alkylamine, diamine such as ethylene diamine or an hydroxylamme, in the presence of a polar protic solvent, such as methanol The reaction mixture is stirred at room temperature for a time penod between about 30 minutes to about 4 hours, preferably about 2 hours.

The amino acetonide isopropyl ester (F) can be further reacted with 4-fluoro-alpha-[2- methyl-1-oxopropyl]-gamma-oxo-N, beta-diphenylbenzenebutanamide of formula Il

to give the corresponding pyrrole πng containing acetonide isopropyl ester of formula III below

According to the invention, as would be understood by one of skill in the art, the stereoselectivity of the enzymatic step can be confirmed via chemical preparation of racemic standards and the development of the related chiral chromatographic methods

The PXRD pattern for 4-fluoro-alpha-[2-methyl-1-oxopropyl]-gamma-oxo-N, beta- diphenylbenzenebutanamide is shown in Fig 1

The main peaks (greater than 13% relative intensity) are given in Table 1 4-fluoro- alpha-[2-methyl-1-oxopropyl]-gamma-oxo-N, beta-diphenylbenzenebutanamide displays characteristic diffraction peaks at 9 0, 12 7, 20 2, 22 6 and 25 2 degrees two theta + 0 1

degree. The DSC thermogram is shown in Fig. 2. 4-fluoro-alpha-[2-methyl-1-oxopropyl]- gamma-oxo-N, beta-diphenylbenzenebutanamide shows a sharp endothermic peak at 213° C + 2°C. The FT-IR spectrum is illustrated in Fig. 3. The FT-IR peak table is given in Table 2. 4- fluoro-alpha-[2-methyl-1-oxopropyl]-gamma-oxo-N, beta-diphenylbenzenebutanamide displays characteristic peaks at 696, 1492, 1327, 843, 1151 cm ^"1 (in this order). The FT- Raman spectrum is illustrated in Fig. 4. The FT-Raman peak table is given in Table 3. 4- fluoro-alpha-[2-methyl-1-oxopropyl]-gamma-oxo-N, beta-diphenylbenzenebutanamide displays characteristic peaks at 1004, 115, 87, 877, 1601 cm ^"1 .

Table 1 : Main PXRD Peaks for 4-fluoro-alpha-[2-methyl-1-oxopropyl]-gamma-oxo-N, beta-diphenylbenzenebutanamide

Table 2: FT-IR Peaks for 4-fluoro-alpha-[2-methyl-1-oxopropyl]-gamma-oxo-N, beta- diphenylbenzenebutanamide

Experimental error is ± 2 cm ^'1 (w: weak, m: medium, s: strong)

Table 3 FT-Raman Peaks for 4-fluoro-alpha-[2-methyl-1-oxopropyl]-gamma-oxo-N, beta-diphenylbenzenebutanamide Ex erimental error is ± 2 cm ^'1 , w: weak m: medium s: stron vs: ver stron

The PXRD pattern for (2R-trans)-5-(4-fluorophenyl)-2-(1-methylethyl)-N,4-dιpheny l-1- [2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1 H-pyrrole-3-carboxamιde is shown in Fig 5 The main peaks (greater than 12% relative intensity) are given in Table 4 (2R-trans)- 5-(4-fluorophenyl)-2-(1-methylethyl)-N,4-dιphenyl-1-[2-(tet rahydro-4-hydroxy-6-oxo-2H-pyran-

2-yl)ethyl]-1H-pyrrole-3-carboxamide displays characteristic diffraction peaks at 6.3, 12.7, 16.8, 21.1 and 25.5 degrees two theta + 0.1 degree. The DSC thermogram is shown in Fig. 6. (2R-trans)-5-(4-fluorophenyl)-2-(1-methylethyl)-N,4-diphenyl -1-[2-(tetrahydro-4-hydroxy-6- oxo-2H-pyran-2-yl)ethyl]-1H-pyrrole-3-carboxamide shows a sharp endothermic peak at 166°C + 2°C. The FT-IR spectrum is illustrated in Fig. 7. The FT-IR peak table is given in Table 5. (2R-trans)-5-(4-fluorophenyl)-2-(1-methylethyl)-N,4-diphenyl -1-[2-(tetrahydro-4- hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1H-pyrrole-3-carboxamide displays characteristic peaks at 851 , 1220, 1047, 757, 1153 cm ^"1 (in this order). The FT-Raman spectrum is illustrated in Fig. 8. The FT-Raman peak table is given in Table 6 (2R-trans)-5-(4-fluorophenyl)-2-(1- methylethyl)-N,4-diphenyl-1-[2-(tetrahydro-4-hydroxy-6-oxo-2 H-pyran-2-yl)ethyl]-1H-pyrrole-3- carboxamide displays characteristic peaks at 1531 , 997, 114, 99, 1605 cm ^'1 .

Table 4: Main PXRD Peaks for (2R-trans)-5-(4-fluorophenyl)-2-(1-methylethyl)-N,4- diphenyl-1-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethy l]-1H-pyrrole-3-carboxamide

Table 5: FT-IR Peaks for (2R-trans)-5-(4-fluorophenyl)-2-(1-methylethyl)-N,4- diphenyl-1-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethy l]-1H-pyrrole-3-carboxamide Experimental error is ± 2 cm ^'1 . (w:weak, m: medium, s: strong)

Table 6: FT-Raman Peaks for (2R-trans)-5-(4-fluorophenyl)-2-(1-methylethyl)-N,4- diphenyl-1-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethy l]-1 H-pyrrole-3-carboxamide Experimental error is ± 2 cm ^"1 . (w: weak, m: medium, s: strong, vs: very strong)

Scheme 2

As set forth in Scheme 2, the cyclopentylidene phthalimido isopropyl ester (G) may be prepared by reacting 2-[2-(4-hydroxy-6-oxo-tetrahydro-pyran-2-yl]-isoindole-1 ,3-dione (B) with cyclopentanone and isopropyl alcohol in the presence of magnesium sulfate and methanesulfonic acid (MSA). The reaction mixture is stirred at room temperature at a pH between about 1 to about 2, preferably about 1.5, for a time period between about 20 hours to about 28 hours, preferably about 24 hours.

The cyclopentylidene phthalimido isopropyl ester (G) is deprotected to give the corresponding amino cyclopentylidene isopropyl ester (H) by treating (G) with a base, such as primary amine, i.e. an alkylamine, diamine such as ethylene diamine or an hydroxyamine, in the presence of a polar protic solvent, such as methanol. The reaction mixture is stirred at room temperature for a time period between about 30 minutes to about 4 hours, preferably about 2 hours.

The amino cyclopentylidene isopropyl ester (H) so formed can be further reacted with 4-fluoro-alpha-[2-methyl-1-oxopropyl]-gamma-oxo-N, beta-diphenylbenzenebutanamide of formula Il

to give the corresponding pyrrole ring containing cyclopentylidene isopropyl ester of formula IV below

Scheme 3

oxidation

Cat dehydrogenation or oxidation

OH OH

OH

Scheme 3 describes in general a process encompassed by the present invention. As set forth in Scheme 3, a DERA aldolase catalyzes two sequential aldol condensation reactions between an N-protected aminopropionaldehyde substrate (i.e. R1 = protecting group) selected from the group consisting of N-formyl-3-amιnopropionaldehyde, 3- succimmido-propionaldehyde, N-diBoc-3-aminopropionaldehyde, N-Boc-3- aminopropionaldehyde, aminoacetaldehyde, N-CBz-3-aminopropionaldehyde, N-acetyl-3- aminopropionaldehyde, N-Fmoc-3-aminopropιonaldehyde or N-Fmoc-aminoacetaldehyde, and 2 mol of acetaldehyde in the presence of a suitable co-solvent such as methyl tert-butyl ether (MTBE) and water to yield the protected desired amino-lactol (I). Suitable DERA

aldolases include, but are not limited to, DERA 04, DERA 06, DERA 101, DERA 102, DERA 104, DERA 105, DERA 106, DERA107 and DERA 108, preferably DERA 04 and DERA 102 The acetaldehyde is added to a mixture of the N-protected aminoaldehyde and DERA aldolase over a time period between about 7 hours to about 12 hours, preferably about 10 hours The mixture so formed is further stirred at a temperature between about 15 ⁰ C to about 30 ⁰ C, preferably about 22 ⁰ C, for a time period between about 20 hours to about 60 hours, preferably about 48 hours

The amino-lactol (I) can undergo catalytic (e g Pt/C, Pd/C) dehydrogenation to form carboxylic acid (K), which can then undergo lactonization to form (J)

Any catalytic dehydrogenation means known in the art to convert (I) to (K) are encompassed by the present invention Examples of suitable catalysts include, but are not limited to, Pt/C, Pd/C, Pt/Bi/C, Pd/Bi/C and any other dehydrogenation catalysts In one embodiment of the invention, the catalytic dehydrogenation is performed at about pH 7 to about pH 10 using air or oxygen as terminal oxidant

Any lactonization means known in the art to convert carboxylic acid (K) to lactone (J) are encompassed by the present invention including, but not limited to, the use of aαd catalysts such as, but not limited to, hydrochloπc acid, sulfuric acid, methanesulfonic acid (MSA), p-toluenesulfonic acid (TSA) and any other lactonization acids known in the art

Alternatively, oxidation of the lactol (I) to lactone (J) or carboxylic acid (K) can be performed by use of any oxidation means known in the art that will achieve the desired transformation

Scheme 4

R= H CBz Boc Fmoc benzyl or dώenzyl

As set forth in Scheme 4, a DERA aldolase catalyzes an aldol condensation reaction between an aminoaldehyde or an N-protected aminoaldehyde and 2 mol of acetaldehyde to give the desired amino-lactol (M)

The following non-limiting examples illustrate the invention

Example 1 2-r2-(4.6-Dιhvdroxy-tetrahvdro-pyran-2-yll-ιsoιndole-1.3- dιone

To a suspension of 3-phthalιmιdo-propιonaldehyde (10 0 grams, 49 2 mmol) in 20 mL of tert-butyl methyl ether (MTBE) was added a solution of DERA 04 lysate (52 0 mL, 10,400 units, prepared from 13 0 grams of wet cells of DERA 04 in phosphate buffer, pH 7 0, 0 01M)

and phosphate buffer (102 mL, pH 7 0, 0 01 M) with vigorous stirring at 22°C Acetaldehyde (4 8 grams, 108 2 mmol, Aldrich) dissolved in water (10 mL) was continuously added into the reaction mixture by a programmed pump for 10 hours The pH of the reaction mixture was kept 70 by titration with 1 0 N sodium hydroxide The reaction mixture was further stirred at 22 ⁰ C for 10 hours and the conversion was monitored by high pressure liquid chromatography (HPLC) After 20 hours, about 95% of the starting material was consumed and 50-55% of the desired lactol was produced based on high pressure liquid chromatography analysis, and the resulting reaction mixture was used directly in the subsequent oxidation step LC-ESIMS of lactol m/z [M+H] ⁺ 292 3

Example 2 2-f2-(4-Hvdroxy-6-oxo-tetrahvdro-pyran-2-yll-isoindole-1.3-d ione

To a suspension of crude lactol (200 mL, prepared according to Example 1) was added dimethyl sulfoxide (10 mL) with stirring Then a solution of sodium chlorite (1 5 eq , 8 3 grams, Aldrich) in water (18 mL) was added dropwise over 30 minutes The temperature was controlled in the range of 20-25 ⁰ C The pH of the reaction mixture should be kept above 4 0 After 4 hours, acetone (200 mL) was added The reaction mixture was stirred at 0-5 ⁰ C for 1 hour and then filtered through a celite pad (10 grams) in a buchel funnel The filtered cake was washed with acetone (50 mL twice) The combined acetone filtrate was concentrated to remove acetone and tert-butyl methyl ether (MTBE) under vacuum The remaining aqueous solution was adjusted to pH of approximately 4 0 and extracted with ethyl acetate (100 mL three times) The combined ethyl acetate solution was dried over magnesium sulfate and concentrated to about 100 mL in vacuum, which was treated with dry hydrochloric acid (0 6 mL, 4M in dioxane) in presence of magnesium sulfate (2 grams) and stired at room temperature for 4 hours Then the reaction mixture was washed with saturated sodium bicarbonate/bnne and dπed over sodium sulfate The solution of ethyl acetate was concentrated to 50 mL to which was then added 50 mL of heptane The formed solid was filtered and washed with heptane (20 mL), and dried in oven to afford lactone as a white solid (40%-45% for three steps, 95% chemical purity, ee >99%, de >86%) LC-ESIMS [M+Na] ⁺ m/z 312 0 ¹ H NMR (CDCI ₃ , 400 MHz) 6 7 82 (m, 2H), 7 68 (m, 2H), 4 78 (m, 1H), 441 (m, 1 H), 3 84 (m, 2H), 2 65 (m, 2H), 1 94-2 14 (m, 3H), 1 81 (m, 1H) ¹³ C NMR (CDCI ₃ , 100 MHz) δ 170 15, 168 61 (2), 134 32 (2), 132 20 (2), 123 58 (2), 73 82 (2), 62 85, 38 63, 35 70, 34 47, 34 40

Example 3

2-[2-(4.6-Dιhvdroxy-tetrahvdro-pyran-2-yll-ιsoιndole-1 .3-dιone

To a suspension of £ co// cells containing DERA 102 (4 grams wet cells suspended in 190 mL of phosphate buffer, pH 7 0, 0 01 M) was added a mixture of 3-phthalιmιdo- propionaldehyde (2 0 grams, 9 8 mmol) and acetaldehyde (0 96 grams, 21 8 mmol, Aldπch) in dimethyl sulfoxide (15 mL) by a programmed pump over 10 hours The reaction mixture was further stirred at 22 ⁰ C for 14 hours The progress of the reaction was monitored by high pressure liquid chromatography (HPLC) After 24 hours, the reaction mixture was extracted with ethyl acetate (100 mL twice) After the separation of two layers by centπfugation, the organic layer was dried and evaporated to give the crude lactol (1 6 grams, 45-50%) as a solid, which was directly submitted to next oxidation step LC-ESIMS of lactol m/z [M+H] ⁺ 292 3

Example 4 7-(1.3-Dioxo-i .3-dihvdro-isoindo-2-vO-3.5-dihvdroxv-heptanoic acid

To a mixture of crude lactol (1 6 grams, prepared according to Example 3) in isopropanol (4 8 mL) and dimethyl sulfoxide (1 0 mL) and 26 mL of phosphate buffer (pH 6 0, 0 01 M) was added a solution of sodium chlorite (0 9 grams, Aldπch) in water (2 mL) at room temperature The pH of the reaction mixture was kept between 5 0 and 6 0 After 4 hours, the reaction mixture was neutralized to pH 7 0 withi N sodium hydroxide and extracted with ethyl acetate (30 mL) After removal of the organic layer, the aqueous layer was acidified to pH 4 0 with 1 N hydrochlonc acid and extracted with ethyl acetate (30 mL three times) The combined organic layer containing crude acid was treated with dicyclohexylamine (1 5 mL) to afford the corresponding dicyclohexylamine salt (1 5 grams, approximately 90% purity) at cold temperature (5-1O ⁰ C) LC-ESIMS m/z [M+Na] ⁺ 330 0 ¹ H NMR (CDCI ₃ , 400 MHz) ό 7 59 (m, 4H), 388 (m, 1 H), 3 58 (m, 1 H), 3 56 (m, 2H), 3 03 (m, 2H), 2 07-2 19 (m, 2H), 1 40-1 82 (m, 14H), 0 80-1 20 (m, 10H) ¹³ C NMR (CDCI ₃ , 100 MHz) δ 180 22, 170 82, 134 65 (2), 131 52 (2), 123 32 (2), 6736, 6731, 5323 (2), 4487, 43 14, 34 82, 34 57, 29 14 (4), 2464 (2), 24 04 (4)

Example 5

2-r2-(4-Hvdroxy-6-oxo-tetrahvdro-Dyran-2-vn-isoondole-1.3 -dione

The crude acid (1.0 grams, prepared according to Example 4) in ethyl acetate (20 mL) was treated with anhydrous hydrocholic acid in dioxane (4 M, 50 μL) and the reaction mixture was stirred at room temperature for 2-3 hours. The reaction mixture was washed with water (pH 7.0, 50 mL twice). The organic layer was dried over Na ₂ SO ₄ and evaporated to give the desired lactone as a white solid (0.94 grams, approximately 94% chemical purity, >99% eβ,

Example 6 Phthalimido acetonide isopropyl ester

Phthalimido lactone (5.0 grams, 17.3 mmol) was suspended in toluene (100 mL). IPA (6.6 mL, 86.0 mmol, 5 eq.), acetone (6.3 mL, 86.0 mmol, 5 eq.), magnesium sulfate (5.0 grams) and methanesulfonic acid (0.4 mL, 6.0 mmol, 0.35 eq.) were added. pH = 1.5 (required < 2). The mixture was stirred at room temperature for 24 hours. The reaction was quenched with triethylamine (0.9 mL, 6.5 mmol) and the mixture was filtered through a grade 4 sinter funnel, washing with toluene (20 mL). The filtrate was washed with sat. aq. NaHCθ ₃ (20 mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give a colourless oil, 6.88 grams, 100%.

Example 7 Amino acetonide isopropyl ester

Ethylene diamine cX, MeOH H ₂ N A

Phthalimido acetonide isopropyl ester (6 55 g, 16 8 mmol) was dissolved in methanol (65 mL, 10 volumes) Ethylene diamine (10 1 grams, 168 mmol, 10 eq ) was added dropwise and the solution was stirred at room temperature

HPLC analysis after 1 hour indicated no starting material After 2 hours the reaction mixture was concentrated in vacuo on a rotavap The residue was partitioned between toluene (65 mL, 10 volumes) and water (65 mL, 10 volumes) - agitated for 15 minutes then allowed to stand for 15 minutes The cloudy aqueous phase was re-extracted with toluene (65 mL) - agitated for 15 minutes then allowed to stand for 15 minutes The combined toluene extracts were washed with water (65 mL) - agitated for 15 minutes then allowed to stand for 15 minutes The toluene extracts were concentrated in vacuo to give an oil product, 2 85 grams, 65 0% yield

Example 8

Pyrrolyl acetonide isopropyl ester (AIE)

4-fluoro-alpha-[2-methyl-1 -oxopropyl]-gamma-oxo-N, beta- diphenylbenzenebutanamide (4 64 grams, 11 1 mmol, 1 03 eq ) was weighed into a one-neck 50 mL rbf Amino acetonide isopropyl ester (2 80 grams, 10 8 mmol) in tert-butyl methyl ether (MTBE, 11 mL) was added followed by a tetrahydrofuran flush (4 2 mL) Triethylamine (1 09 grams, 10 8 mmol, 1 eq ) was added and the slurry was heated to 5O ⁰ C Pivalic acid (1 10 grams, 10 8 mmol, 1 eq ) was added and the mixture was heated at reflux (67-68 ⁰ C) for 88 hours On cooling, the volatiles were removed in vacuo and the residue was taken up in isopropyl alcohol (IPA, 17 5 mL) and heated to 80 ⁰ C Further IPA (10 mL) was required to give a clear solution The solution was allowed to cool to room temperature - no crystallisation occurred The solution was seeded with authentic product and crystallisation occurred The slurry was cooled to O ⁰ C and held for 30 minutes The product was collected on a grade 2 sinter funnel and washed with isopropyl alcohol (ι e, IPA, 3 times with 10 mL) The product was dried in a vacuum oven at 40-50 ⁰ C for 18 hours to give a pale yellow solid (4 15 grams, 60 0 % yield)

Example 9

Cvclopentylidene -Phthahmido-isopropyl ester

Phthahmido lactone (5 0 grams, 17 3 mmol) was suspended in toluene (50 mL) IPA (6 6 mL, 86 0 mmol, 5 eq ), cyclopentanone (3 0 grams, 34 8 mmol, 2 eq ), magnesium sulfate (5 0 grams) and methanesulfonic aαd (0 4 mL, 6 0 mmol, 0 35 eq ) were added pH of 1 5 (less than pH of 2 required) The mixture was stirred at room temperature for 24 hours The reaction was quenched with triethylamine (0 9 mL, 6 5 mmol) and the mixture was filtered through a grade 4 sinter funnel, washing with toluene (20 mL) The filtrate was washed with sat aq NaHCO ₃ (20 mL), dried over magnesium sulfate, filtered and concentrated in vacuo to give a colourless oil, 7 18 grams, 100%

Example 10 Amino cvclopentvlidene isopropvl ester

Cyclopentylidene phthahmido isopropyl ester (10 0 grams, 24 1 mmol) was dissolved in methanol (50 mL, 5 volumes) Ethylene diamine (2 9 grams, 482 mmol, 2 eq ) was added dropwise and the solution was stirred at room temperature

High pressure liquid chromatography (HPLC) analysis after 1 hour indicated no starting mateπal After 2 hours the reaction mixture was concentrated in vacuo on a rotavap The residue was partitioned between toluene (100 mL, 10 volumes) and water (100 mL, 10 volumes) - agitated for 15 minutes then allowed to stand for 15 minutes The cloudy aqueous phase was re-extracted with toluene (65 mL) - agitated for 15 minutes then allowed to stand for 15 minutes The combined toluene extracts were washed with water (65 mL) - agitated for 15 minutes then allowed to stand for 15 minutes The toluene extracts were concentrated in vacuo to give the product as an oil, 645 grams, 94 0% yield It is important to ensure absence of ethylenediamine from the crude product as it leads to the formation of an impurity (bispyrrole) in the subsequent Paal-Knorr reaction

Example 11

Pyrrolyl cvclopentvhdene isopropyl ester (CIE)

4-fluoroalpha-[2-methyl-1 -oxopropyl]-gamma-oxo-N, beta- diphenylbenzeπebutanamide (4 64 grams, 11 1 mmol, 1 03 eq ) was weighed into a one-neck 50 mL rbf Amino cyclopentylidene isopropyl ester (3 08 grams, 10 8 mmol) in MTBE (11 ml_) was added followed by a tetrahydrofuran flush (4 2 mL) Tπethylamine (1 09 grams, 10 8 mmol, 1 eq ) was added and the slurry was heated to 5O ⁰ C Pivalic acid (1 10 grams, 10 8 mmol, 1 eq ) was added and the mixture was heated at reflux (67-68 ⁰ C) for 88 hours On cooling, the volatiles were removed in vacuo and the residue was taken up in isopropyl alcohol (17 5 mL) and heated to 80 ⁰ C Further isopropyl alcohol (10 mL) was required to give a clear solution The solution was seeded with authentic product and crystallisation occurred The slurry was cooled to O ⁰ C and held for 30 minutes The product was collected on a grade 2 sinter funnel and washed with isopropyl alcohol (3 times 10 mL) The product was dned in a vacuum oven at 40-50 ⁰ C for 18 hours to give a pale yellow solid (4 31 grams, 60 0 % yield) Purity by high pressure liquid chromatography was greater than 99% pure

Example 12

4-fluoro-alpha-[2-methyl-1-oxopropyll-qamma-oxo-N, beta-diphenylbenzene butanamide

A reaction vessel is iπerted using at least 4 cycles of vacuum, releasing the vacuum each time with nitrogen 250 liters of tetrahydrofuran is charged to the reaction vessel via spray nozzles Spray ball nozzles ensure that all areas of the reaction vessel are penetrated in particular the top inner surface of the vessel and the agitator device also present inside the reaction vessel The tetrahydofuran washings are drained off and collected for waste recycling

When the reaction vessel is dry 480kgs 2-benzylιdιne isobutyrylacetamide (BIBEA), 60kgs ethyl hydroxyethylmethyl thiazolium bromide (MTB or ethyl hydroyethyl MTB), 200 liters, 216kgs of 4-fluorobenzaldehyde and 120kgs of triethylamine are charged to the reaction vessel and heated with agitation to between 60 and 70 ⁰ C The reaction mixture is aged for 16 to 24 hours maintaining the temperature at 65 +/- 5 ⁰ C The contents re then cooled to 60 +/- 5 ⁰ C for 54 to 66 minutes 600 liters of isopropanol is charged to the reaction mixture and the mixture is heated to about 100 ⁰ C to achieve a solution

600 liters of deionised water is charged to the reaction vessel over 30 minutes while maintaining the temperature at 60 +/- 5 ⁰ C The batch is aged for 54 to 66 minutes and the contents cooled to between 25 +/- 5 ⁰ C over a 2 to 4 hour period at a rate of 15/2O ⁰ C per hour The batch is aged at this temperature for at least 1 hour and the contents cooled further to 0 +/- 5 ⁰ C and aged for at least 1 hour

The batch is isolated on a filter and washed with isopropanol The product is dried under vacuum at 50 +/- 5 ⁰ C to a water content of less than 0 5% The contents are then cool to approximately less than 30 ⁰ C before discharging

Example 13

PXRD of 4-fluoro-alpha-r2-methyl-1-oxopropyπ-qamma-oxo-N. beta-diphenylbenzeπe butanamide

The powder X-ray diffraction pattern was determined using a Bruker-AXS Ltd D4 powder X-ray diffractometer fitted with an automatic sample changer, a theta-theta goniometer, automatic beam divergence slit, and a PSD Vantec-1 detector The sample was prepared for analysis by mounting on a low background silicon wafer specimen mount The specimen was rotated whilst being irradiated with copper K-alpha, X-rays (wavelength = 1 5406 Angstroms) with the X-ray tube operated at 40kV/30mA The analyses were performed with the goniometer running in continuous mode set for a 0 2 second count per 0 018° step over a two theta range of 2° to 55° Peaks were selected using Bruker-AXS Ltd Evaluation software with a threshold of 1 and a peak width of 0 3° 2-theta The data were collected at 21°C

As will be appreciated by the skilled person, the relative intensities of the various peaks within Table 1 given below may vary due to a number of factors such as for example oπentation effects of crystals in the X-ray beam or the purity of the mateπal being analysed or the degree of crystallinity of the sample The peak positions may also shift for variations in sample height but the peak positions will remain substantially as defined in given Table

Example 14

DSC of 4-fluoro-alpha-f2-methyl-1-oxopropyl]-qamma-oxo-N. beta-dipheπylbenzene butanamide

3 117mg of 4-fluoro-alpha-[2-methyl-1-oxopropyl]-gamma-oxo-N, beta- diphenylbenzene butanamide was heated from 10 to 250°C at 20°C per minute using a Perkin Elmer Diamond DSC with autosampler and a 4 hole side wall vented aluminium pan and lid with nitrogen flow gas

Example 15

FT-IR of 4-fluoro-alpha-[2-methyl-1-oxopropyll-qamma-oxo-N, beta-diphenylbenzene butanamide

The IR spectrum was acquired using a ThermoNicolet Nexus FTIR spectrometer equipped with a 'DurasampllR' single reflection ATR accessory (diamond surface on zinc selenide substrate) and d-TGS KBr detector The spectrum was collected at 2cm ¹ resolution

and a co-addition of 256 scans Happ-Genzel apodization was used Because the FT-IR spectrum was recorded using single reflection ATR, no sample preparation was required Using ATR FT-IR will cause the relative intensities of infrared bands to differ from those seen in a transmission FT-IR spectrum using KBr disc or nujol mull sample preparations Due to the nature of ATR FT-IR, the bands at lower wavenumber are more intense than those at higher wavenumber Experimental error, unless otherwise noted, was ± 2 cm ' Peaks were picked using ThermoNicolet Omnic 6 Oa software Intensity assignments are relative to the major band in the spectrum, so are not based on absolute values measured from the baseline

Example 16 FT-Raman IR of 4-fluoro-alpha-f2-methyl-1-oxopropyn-qamma-oxo-N, beta- diphenylbenzene butanamide

The Raman spectrum was collected using a Bruker Vertex70 with Ramll module FT-Raman spectrometer equipped with a 1064nm NdYAG laser and LN-Germanium detector

All spectra were recorded using 2cm ^" resolution and Blackman-Harπs 4-term apodization

The spectrum was collected using laser power of 30OmW and 4096 co-added scans The sample was placed in a glass vial and exposed to the laser radiation The data is presented as intensity as a function of Raman shift (cm ¹ ) and is corrected for instrument response and frequency dependent scattering using a white light spectrum from a reference lamp The Bruker Raman Correct function was used to do the correction (Bruker software - OPUS 6 0) Experimental error, unless otherwise noted, was ± 2 cm ¹ Peaks were picked using ThermoNicolet Omnic 6 Oa software Intensity assignments are relative to the major band in the spectrum, so are not based on absolute values measured from the baseline

Example 17 (2R-trans)-5-(4-fluorophenyl)-2-(1-methγlethylVN.4-dιpheny l-1-r2-(tetrahvdro-4-hvdroxy-6- oxo-2H-pyran-2-vπethvπ-1H-pyrrole-3-carboxamιde

50 grams tert-butyl isopropylidene (TBIN), prepared as described in Tetrahedron Letters, 2279 (1992), 13 25 grams wet sponge nickel catalyst, 28% ammonia solution (137 5 ml) and 375 ml isopropyl alcohol (IPA) are added to a pressure vessel The mixture is reduced with 50 psi of hydrogen, then filtered and concentrated in vacuo The resulting oil is dissolved in 250 ml warm toluene, water washed and again concentrated in vacuo to give an ammo ester The amino ester, 85 grams 4-fluoro-alpha-[2-methyl-1-oxopropyl]-gamma-oxo- N, beta-diphenylbenzene butanamide (US Pat No 5,155,251 and Bauman K L , Butler D E , Deeπng C F et al Tetrahedron Letters 1992,33 2283-2284 both references incorporated by reference in their entirety), 12 5 grams pivalic acid, 137 5 ml tetrahydrofuran and 137 5 ml hexanes are charged to an argon inerted pressure vessel which is sealed and heated to 75°C for 96 hours After cooling, the solution is diluted with 400 ml methyl tert-butyl ether (MTBE) and washed firstly with dilute aqueous sodium hydroxide followed by dilute aqueous hydrochloric acid The mixture is then concentrated in vacuo to give an acetonide ester

The acetonide ester is dissolved in 275 ml warm methanol and aqueous hydrochloπc acid (5 grams of 37% hydrochloric acid in 75 ml of water) is added The mixture is stirred at 30 °C to produce a diol ester 100 ml methyl tert-butyl ether and aqueous sodium hydroxide (150 ml of water and 25 grams of 50% aqueous sodium hydroxide) are then added and the mixture stirred at 3O ⁰ C to produce the sodium salt 600 ml water is added and the mixture washed twice with 437 5 ml methyl tert-butyl ether

In this case, the mixture is distilled under atmospheric pressure to a batch temperature of 99°C Distillation is continued until the methanol content of the mixture is reduced to 04 w/v The batch is stirred at 75-85% for 18 hours, then cooled, acidified and extracted into 875 ml toluene The mixture is heated at reflux for 4 hours and water is removed azeotropically After cooling, the mixture is filtered, washed with toluene and dried directly The titled compound is isolated as a white solid (Yield 37 9 grams)

Example 18

PXRD of (2R-transV5-(4-fluorophenyl)-2-(1-methylethyl)-N.4-diphenyl- 1-r2- (tetrahvdro-4-hvdroxy-6-oxo-2H-pyran-2-yl)ethvn-1H-pyrrole-3 -carboxamιde

The powder X-ray diffraction pattern was determined using a Bruker-AXS Ltd D4 powder X-ray diffractometer fitted with an automatic sample changer, a theta-theta goniometer, automatic beam divergence slit, and a PSD Vantec-1 detector The sample was prepared for analysis by mounting on a low background silicon wafer specimen mount The specimen was rotated whilst being irradiated with copper K-alphai X-rays (wavelength = 1 5406 Angstroms) with the X-ray tube operated at 40kV/30mA The analyses were performed with the goniometer running in continuous mode set for a 0 2 second count per 0 018° step over a two theta range of 2° to 55° Peaks were selected using Bruker-AXS Ltd Evaluation software with a threshold of 1 and a peak width of 0 3° 2-theta The data were collected at 21°C

As will be appreciated by the skilled person, the relative intensities of the various peaks within Table 1 given below may vary due to a number of factors such as for example orientation effects of crystals in the X-ray beam or the puπty of the mateπal being analysed or the degree of crystallinity of the sample The peak positions may also shift for variations in sample height but the peak positions will remain substantially as defined in given Table

Such further PXRD patterns generated by use of alternative wavelengths are considered to be alternative representations of the PXRD patterns of the crystalline matenals of the present invention and as such are within the scope of the present invention

Example 19

DSC of (2R-trans)-5-(4-fluorophenyl)-2-(1-methylethyl)-N.4-dιpheny l-1 -β-ftetrahvdro- 4-hvdroxγ-6-oxo-2H-pyran-2-yl)ethyll-1 H-pyrrole-3-carboxamιde

2 893mg of the sample was heated from 10 to 300°C at 20°C per minute using a Perkin Elmer Diamond Differential Scanning Calorimetry (DSC) with autosampler and a 4 hole side wall vented aluminium pan and lid with nitrogen flow gas

Example 20

FT-IR of (2R-trans V5-(4-fluorophenyl)-2-(1-methylethyl)-N.4-dιphenyl-1-r2- (tetrahvdro-4-hvdroxy-6-oxo-2H-pyran-2-yl)ethyll-1 H-Pvιτole-3-carboxamιde

The IR spectrum was acquired using a ThermoNicolet Nexus FTIR spectrometer equipped with a 'DurasampllR' single reflection ATR accessory (diamond surface on zinc selenide substrate) and d-TGS KBr detector The spectrum was collected at 2cm ^'1 resolution and a co-addition of 256 scans Happ-Genzel apodization was used Because the FT-IR spectrum was recorded using single reflection ATR, no sample preparation was required Using ATR FT-IR will cause the relative intensities of infrared bands to differ from those seen in a transmission FT-IR spectrum using KBr disc or nujol mull sample preparations Due to the nature of ATR FT-IR, the bands at lower wavenumber are more intense than those at higher wavenumber Experimental error, unless otherwise noted, was ± 2 cm ¹ Peaks were picked using ThermoNicolet Omnic 6 Oa software Intensity assignments are relative to the major band in the spectrum, so are not based on absolute values measured from the baseline

Example 21

FT-Raman of (2R-traπsV5-(4-fluorophenyl)-2-(1-methylethyl)-N.4-diphenyl -1-f2- (tetrahvdro-4-hvdroxy-6-oxo-2H-pyran-2-yl)ethyll-1 H-pyrrole-3-carboxamide

The Raman spectrum was collected using a Bruker Vertex70 with Ramll module FT-Raman spectrometer equipped with a 1064nm NdYAG laser and LN-Germanium detector

The spectrum was recorded using 2cm resolution and Blackman-Harπs 4-term apodization The spectrum was collected using laser power of 30OmW and 4096 co-added scans The sample was placed in a glass vial and exposed to the laser radiation The data is presented as intensity as a function of Raman shift and is corrected for instrument response and frequency dependent scattering using a white light spectrum from a reference lamp The Bruker Raman Correct function was used to do the correction (Bruker software - OPUS 6 0) Experimental error, unless otherwise noted, was ± 2 cm ^'1 Peaks were picked using ThermoNicolet Omnic 6 Oa software Intensity assignments are relative to the major band in the spectrum, so are not based on absolute values measured from the baseline

Example 22

Phthalimide acetal

Slurry 50.0 gm of Potassium Phthalimide (1 eq.) in 400 mis (8 vol.) of N,N dimethyformamide at room temperature, a slurry. 3-Bromopropionaldehyde dimethyl acetal 54.4 grams (1.1 eq.) was added dropwise at room temperature, a slurry. The reaction was held for approximately 15 hours and called complete. 2-Methyltetrahydrofuran 250 mis, and water 250 mis, were added and stirred, allowed to settle and separated. The aqueous layer was rewashed twice with 100 mis 2-MTHF, the organic layers combined and washed with 70% saturated brine to remove water. The organic layer was then dried over sodium sulfate, distilled at atmospheric pressure to a slurry. The white slurry granulated at reduced temp 0-5 °C for 1 hr., filtered on a paper covered Buckner funnel and washed with 2-MTHF. The white solids were vac oven dried at less than 40 °C, resulting in a yield of 46.5% of the titled product.

Example 23 3-phthalimido-propionaldehvde

15.0 grams of Phthalimide Acetal (1 eq.) were added to 700 mis (approximately 47 vol.) glacial acetic acid and 70 mis (approximately 5 vol.) water. This reaction was held for 48 hours at room temperature up to 30 °C, and called complete. Saturated sodium bicarbonate was added to a pH of 7, and extracted with 500 mis 2-MTHF, reextracted with 500 mis 2- MTHF. The organic layer was then dried over sodium sulfate, vacuum distilled to a slurry. The white slurry granulated at reduced temperature 0-5 ⁰ C for 1 hour, filtered on a paper covered Buckner funnel and washed with 2-MTHF. The white solids were vac oven dried at room temperature, resulting in a yield of 47% of the titled product.

Example 24

SEQ ID NO: 1 - Nucleotide sequence of DERA03 atgactgatctgaaagcaagcagcctgcgtgcactgaaattgatggacctgaccaccctg aatgacgacgacaccgacgagaa agtgatcgccctgtgtcatcaggccaaaactccggtcggcaataccgccgctatctgtat ctatcctcgctttatcccgattgctcgca aaactctgaaagagcagggcaccccggaaatccgtatcgctacggtaaccaacttcccac acggtaacgacgacatcgacatc gcgctggcagaaacccgtgcggcaatcgcctacggtgctgatgaagttgacgttgtgttc ccgtaccgcgcgctgatggcgggtaa cgagcaggttggttttgacctggtgaaagcctgtaaagaggcttgcgcggcagcgaatgt actgctgaaagtgatcatcgaaacc ggcgaactgaaagacgaagcgctgatccgtaaagcgtctgaaatctccatcaaagcgggt gcggacttcatcaaaacctctacc ggtaaagtggctgtgaacgcgacgccggaaagcgcgcgcatcatgatggaagtgatccgt gatatgggcgtagaaaaaaccgt tggtttcaaaccggcgggcggcgtgcgtactgcggaagatgcgcagaaatatctcgccat tgcagatgaactgttcggtgctgact gggcagatgcgcgtcactaccgctttggcgcttccagcctgctggcaagcctgctgaaag cgctgggtcacggcgacggtaaga gcgccagcagctactaa

Example 25

SEQ ID NO: 2 - Nucleotide Sequence of DERA04 atgggtaatatcgcgaaaatgattgatcacaccctcttaaaacccgaagcaaccgaacaa caaattgtacaattatgcacggaag cgaaacaatatggctttgcagcagtatgcgtaaatccgacatgggttaaaaccgccgcac gtgaattaagcgggacagacgttcg tgtgtgtactgtaattggatttcccttgggcgctacgactccagaaactaaagcattcga aactactaacgcgattgaaaatggagca cgggaagtagatatggtaattaatattggtgcattgaaatctggacaagatgaactggtg gaacgtgatattcgtgccgttgttgaag ctgcagcaggccgcgcgcttgtgaaagtaattgtagaaacagcccttcttactgatgaag aaaaagttcgcgcttgtcaattagcag taaaagcgggtgccgattatgtgaagacgtcgacaggatttagcggtggtggtgcaacgg tggaagatgtggctttaatgcggaa aacggttggtgatcgtgcaggggtcaaagcaagcggcggagtacgtgactggaaaacagc agaagcaatgattaacgcagga gcaacgcgcattggcacaagttctggagtagcaatcgtaacaggtggaaccggccgggca gactattaa

Example 26

SEQ ID NO: 3 - Nucleotide Sequence of DERA06 atgggactcgcctcctacatcgaccacacgctgcttaaggccaccgccacgctcgccgac atccgcacgctgtgtgaggaagcc cgcgagcactcgttctacgcggtgtgcatcaacccggtctttattccccacgcccgcgcc tggctcgaaggcagcgacgtgaaggt (^ccafxgtdgc^gcttt∞∞tcggcgccatcagctccgagcagaaagctctggaag cccgcctgagcgccgaaacgggcg ccgacgaaatcgatatggtcatccacatcggctcggcgcttgccggcgactgggacgcgg tggaagccgacgtgcgggcagtg cgccgcgcggtgcccgagcaggtgctcaaggtgattatcgaaacctgctacctgaccgac gagcaaaagcgcttggcgactga ggtcgccgtacagggcggcgccgacttcgtgaagacgagcacaggcttcggcaccggcgg cgccaccgtggacgacgtgcgc ctgatggcggaagtgatcgggggccgcgccggactcaaggcggcgggcggcgtccgcact cctgccgacgcgcaagccatg atcgaggcgggcgcgacccggctgggcacctcgggcggcgtgggtctggtgtcgggcggc gaaaacggagccggctactga

Example 27

SEQ ID NO: 4 - Nucleotide Sequence of DERA08 atgggaattgctaaaatgatcgatcacactgctttaaaaccagacacaacgaaagaacaa attttaacactaacaaaagaagca agagaatacggttttgcttccgtatgcgtaaatccaacttgggtaaaactatccgctgaa caacttgctggagcagaatctgtagtat gtactgttatcggtttcccactaggagcgaatacccctgaagtaaaagcatttgaagtaa aagatgctatccaaaacggtgcaaaa gaagtggatatggttattaatatcggcgcactaaaagacaaagacgacgaactagtagaa cgtgatattcgcgctgtagtcgatgc tgccaaaggaaaagcattagtaaaagtaattatcgaaacttgcctattaacagacgaaga aaaagttcgcgcatgtgaaatcgct gtaaaagcgggaacagacttcgttaaaacatccactggattctccacaggtggcgcaact gccgaagatatcgccttaatgcgta aaactgtaggaccaaacatcggcgtaaaagcatctggtggggttcgtacgaaagaagacg tagaaaaaatgatcgaagcagg cgcaactcgtattggcgcaagtgcaggtgtcgcaattgtttccggcgaaaaaccagccaa accagataattactaa

Example 28

SEQ ID NO: 5 - Nucleotide Sequence of DERA11 atgacatcaaatcaacttgctcaatatatcgatcacaccgcacttaccgcagaaaaaaat gaacaagatatttcgacactctgtaat gaagcgattgaacacggattttattctgtatgtatcaattctgcttatattccactcgct aaagaaaaacttgctggctcaaatgtaaaa atttgcaccgtagttggattccctttgggggcgaatttaacctcagtcaaagcatttgaa acgcaagaatctattaaagcgggtgcaa atgaaattgatatggtgattaatgtaggttggataaaatcgcaaaaatgggatgaagtaa aacaagatattcaagcggtatttaatg

cttgtaatggcacgccattaaaagtgattttagaaacttgtttgctcactaaagatg aaatagtgaaagcctgcgaaatttgtaaaga aatcggtgtagcttttgttaaaacatcaacaggctttaataaaggtggtgcgaccgtaga agatgttgcattgatgaaaaacacggtc ggcaatattggtgttaaagcatcaggtggtgtgcgtgatactgaaactgcacttgcaatg attaaggcgggtgcgactcgcattggtg caagcgctggcattgcgattattagcggtactcaagacactcaaagcacttactaa

Example 29

SEQ ID NO: 6 - Nucleotide Sequence of DERA12 atgatagagtacaggattgaggaggcagtagcgaagtacagagagttctacgaattcaag cccgtcagagaaagcgcaggtatt gaagatgtgaaaagtgctatagagcacacgaatctgaaaccgtttgccacaccagacgat ataaaaaaactctgtcttgaagca agggaaaatcgtttccatggagtctgtgtgaatccgtgttatgtgaaactggctcgtgaa gaactcgaaggaaccgatgtgaaagtc gtcaccgttgttggttttccactgggagcgaacgaaactcggacgaaagcccatgaggcg attttcgctgttgagagtggagccgat gagatcgatatggtcatcaacgttggcatgctcaaggcaaaggagtgggagtacgtttac gaggatataagaagtgttgtcgaatc ggtgaaaggaaaagttgtgaaggtgatcatcgaaacgtgctatctggatacggaagagaa gatagcggcgtgtgtcatttccaaa cttgctggagctcatttcgtgaagacttccacgggatttggaacaggaggggcgaccgca gaagacgttcatctcatgaaatggat cgtgggagatgagatgggtgtaaaagcttccggagggatcagaaccttcgaggacgctgt taaaatgatcatgtacggtgctgata gaataggaacgagttcgggagttaagatcgttcaggggggagaagagagatatggaggtt ga

Example 30

SEQ ID NO: 7 - Nucleotide Sequence of DERA15 atgccgtcggccagggatatactgcagcagggtctagacaggctagggagccctgaggac ctcgcctcgaggatagactctacg ctactaagccctagggctacggaggaggacgttaggaatcttgtgagagaggcgtcggac tacgggtttagatgcgcggttctga ctccagtgtacacagtaaagatttctgggctggctgagaagcttggtgtgaagctatgta gcgttataggctttcccctgggccaggc cccgctcgaggtaaagctagttgaggcacaaactgttttagaggctggggctactgagct tgatgttgtcccccatctctcactaggc cccgaagctgtttacagggaggtctcagggatagtgaagttggcgaaaagctatggagcc gttgtgaaagtaatattagaagcgc cactctgggatgacaaaacgctctccctcctggtggactcgtcgaggagggcgggggcgg atatagtgaagacaagcaccggg gtctatacaaagggtggtgatccagtaacggtcttcaggctggccagtcttgccaagccc cttggtatgggtgtaaaggcaagcgg cggtataaggagtggcatcgacgccgtcctcgccgtaggagctggcgcggatatcatagg gacaagcagtgctgtaaaggttttg gagagcttcaaatccctagtctaa

Example 31

SEQ ID NO: 8 - Nucleotide Sequence of DERA 101 atggctgcaaacaaatatgaaatggccttcgcacagttcgatccagctgaaagcgaagaa cgcatcctg ctgaaaactgaccagatcattcgtgaccactattcccgtttcgacactccagaaactaaa aagttcctg catggcgttatcgatctgacgtctctgaacgccaccgactctgaggaatctatcactaaa ttcaccgaa tctgtaaacgatttcgaagataccgacccgactatccctagcgttgcggcgatctgcgtt tatccgaac tttgtcagcaccgtgcgtgaaaccctgactgccgagaatgtgaaagttgcaagcgtcagc ggttgcttc ccggcctcccagagcttcatcgaagtgaaactggcagaaaccgcactggcggttagcgac ggtgcggat gaaattgacattgttctgaacatgggtaaattcctgtccggtgattacgaggccgcagcc actgagatc gaggaacagatcgctgcggcgaagggtgcgaccgtaaaagttatcctggagactggtgct ctgaagacg ccggaaaacattcgccgcgcaaccatcctgtctctgttttgtggcgcccatttcgttaaa acctctact

ggcaaaggctacccgggcgcctctctggaagcagcttacactatgtgtaaagtcctg aaacagtactac ggcctgttcggtgaagttcgtggcatcaagctgagcggcggtatccgtaccaccgaagac gcggttaag tactactgcctgatcgaaacgctgctgggcaaagaatggctgaccccggcgtacttccgc atcggcgcc tcctctctggttgatgctctgcgccaggatattatggtttaa

Example 32

SEQ ID NO: 9 - Nucleotide Sequence of DERA 102

Atggaactgaaccgcatgattgaccacactattctgaaaccggaagccaccgaggcg gctgtgcagaaa attatcgatgaagctaaagaatacaacttcttcagcgtctgtatcaacccgtgttgggtt gcttttgcc tccgagcagctggctgatactgatgttgccgtctgtaccgtaatcggtttcccgctgggc gcgaacacg ccggaggttaaagcgtacgaagcagctgacgccattaaaaacggtgctaatgaggtggat atggtgatc aatattggtgctctgaaatcccaacagtacgactacgtgcgccaagacatccagggtgtg gttgacgcc gcaaaaggtaaagcactggttaaagttatcatcgaaactgccctgctgaccgatgaagag aaagttaag gcttgcgaactggcgaaagaagcaggcgctgatttcgtgaaaaccagcaccggtttttcc actggcggt gcaaaagttgctgacattcgtctgatgcgcgaaaccgtgggtccggatatgggcgttaaa gcatccggt ggcgtacacaacgcagaagaagcactggccatgatcgaagcgggcgcaactcgtatcggc gcttccacc ggtgtagccatcgtaagcggtgctactggtgagggtaccaaatggtaa

Example 33

SEQ ID NO: 10 - Nucleotide Sequence of DERA 103 atgactattgaatccgctatcgcgctggcacctgcagaacgtgctgttaacctgattggt agcgacctg accgaaaaatctctgaaactgcacctggaaggcctgtctggtgtcgacgcggttggtctg gaacagcgt gctgccggtctgtccacccgctctatcaaaaccacctccaaagcttgggccctggacacc atcatcaaa ctgatcgatctgactactctggagggcgcagatactccgggcaaggttcgttctctggct gcgaaagca atgctgccggacgcctctgatgtgtccgctccgcaggtggcagctgtgtgcgtttacggt gatatggtg ccatacgcggcggaagcactgggctcctcttggtctaatggttctgacaacggcattaac gttgctgcg gtggcaactgcgttcccatccggtcgcagctccctgccaatcaaaatcgctgacaccaag gaagccgtt gcccacggtgctgacgaaatcgacatggtaatcgatcgtggtgcgttcctgagcggcaaa tacggtgtt gtgttcgaccagatcgtagctgtgaaagaagcttgccgccgcgaaaacggcacttacgcg cacctgaaa gttatcctggaaaccggcgaactgaacacctatgacaacgtccgccgtgcctcctggctg gcgatcctg gcgggtggtgactttgtgaaaacctctaccggcaaggttagcccggccgcaaccctgccg gttacgctg ctgatgctggaagtcgttcgcgattggcatgtgctgactggcgagaaaatcggtgtgaaa ccagccggt ggtatccgctcctccaaagacgcgattaaatacctggtcaccgtggcggaaaccgtaggt gaagagtgg ctgcaaccgcacctgtttcgctttggcgcctcctccctgctgaacgacgttctgatgcag cgtcagaag ctgtctaccggccactactccggcccagattacgtgaccatcgactaa

Example 34

SEQ ID NO: 11 - Nucleotide Sequence of DERA 104 atgtcttctactccaactattctggatccggcgtttgaggacgttacccgttctgaagca tctctgcgc cgtttcctgcacggcctgccgggtgtcgatcaggtgggcgcagaggcccgtgccgctggt ctggcaacc cgttccattaaaacgtccgcaaaagaatttgcactggacctggcgattcgtatggttgac ctgaccacg

ctggagggccaggatacgccgggtaaggttcgtgccctgagcgcgaaagcaatgcgt ccggatccgtct gatccaacctgtcctgctactgctgctgtatgtgtttacccggacatggttggcatcgcg aaacaggcg ctgggtactagcggcgtacacgtagctgctgtggctactgctttcccgtctggccgtgcc gctctggac atcaaactggcggacgttcgtgatgcggtggacgcaggcgctgacgaaatcgatatggtt atcgaccgc ggtgcttttctggctggtcgttaccaacacgtatacgacgaaattgttgcggtgcgcgaa gcctgccgc cgtgaaaacggtgaaggcgctcacctgaaggtaatcttcgagactggtgagctgcagacc tacgacaac gttcgccgtgcgagctggctggcgatgatggctggtgcacacttcgttaaaacgtccacc ggcaaagtc cagccggcagctaccctgccggttaccctggttatgctgcaggccgtacgtgactttcgt ggcgcaacg ggccgtatggttggcgttaaacctgctggcggtatccgtaccgccaaggacgcaatcaaa tacctggtt atggtaaacgaggtagcgggcgaagattggctggacccggactggtttcgttttggtgca tctactctg ctgaacgacctgctgatgcagcgtacgaagatgaaaaccggccgttacagcggcccagac tactttacc ctggactaa

Example 35

SEQ ID NO: 12 - Nucleotide Sequence of DERA 105 atggaactgatcactcagccgtcttgttgggtattttccgtctttttccgccgtcagtac ggctggctg gtttttgtggaaggtgcttggtacgatggtcgccgtcaaactttccacctggatggtaac ggccgcaaa ggcttcctgcgcatgactatgaatatcgcaaaaatgatcgatcacaccctgctgaaaccg gaagcgact gagcagcagatcgtacaactgtgcaccgaagctaaacagtatggttttgcttccgtttgt gtgaaccct acgtgggtgaaaaccgccgcacgcgaactgtctggtaccgacgttcgtgtttgtaccgta attggcttc ccgctgggcgcgactaccccagaaaccaaagcgttcgaaactaccaacgcgatcgaaaac ggcgctcgt gaagtcgacatggtaatcaacattggcgctctgaaatctggtcaggacgaactggtagag cgtgacatc cgcgccgtcgtagaagctgcggcaggccgtgcactggtaaaagtaatcgttgaaaccgct ctgctgact gatgaagagaaagttcgtgcgtgtcagctggcggttaaagctggtgcagattacgtgaaa acgagcact ggtttctccggtggtggcgctactgtcgaagacgtggcgctgatgcgtaaaaccgtaggc gatcgcgca ggcgttaaagcgagcggcggtgttcgtgattggaagactgccgaagctatgattaacgca ggcgcgact cgtatcggcacttctagcggcgtggcaattgttactggcggcaccggtcgcgctgacact aaatggtaa

Example 36

SEQ ID NO: 13 - Nucleotide Sequence of DERA 106 atgactatcgctaaaatgattgatcacacggcgctgaagccagataccaccaaagaacaa atcctgacg ctgaccaaagaagcacgtgaatatggctttgctagcgtctgtgtgaatccgacttgggtg aaactgtct gcggaacagctgagcggcgctgaatctgtggtgtgcaccgtcatcggttttccgctgggc gcgaatact ccggaagtgaaggcattcgaagtaaaaaacgctatcgaaaacggcgcgaaggaagtagat atggttatc aacattggtgctctgaaggataaggacgacgaactggtggaacgtgatatccgtgccgtc gtggatgct gctaaaggtaaagcgctggtgaaagtcattatcgaaacctgcctgctgaccgatgaagag aaggtccgt gcttgcgaaatcgccgtgaaagctggcactgatttcgttaaaacttctactggcttttct actggtggc gcgactgcagaagacatcgcactgatgcgtaagactgtcggtccgaacatcggtgtaaaa gcgtccggt ggtgttcgtactaaagaagacgttgagaagatgatcgaagcgggtgccacccgtatcggc gcttctgca ggtgtggcaatcgtatccggtgaaaaaccggcgaaacctgacaacaccaagtggtaa

Example 37

SEQIDNO 14 -Nucleotide Sequence of DERA 107 atgtctcgctctattgcacaaatgatcgatcacaccctgctgaaacctaataccaccgaa gaccagatc gtgaaactgtgcgaagaggctaaagaatactctttcgcctccgtatgcgtcaacccaacg tgggtcgcg ctggcagcgcagctgctgaaagacgctcctgatgtgaaagtgtgcactgttatcggcttc ccactgggt gcaaccacgcctgaagtaaaagcgtttgaaaccactaacgcaatcgagaacggcgcaacg gaggttgat atggttatcaacatcggtgccctgaaggacaaacagtacgaactggttggtcgtgatatc caggctgtt gtgaaggcagcagaaggcaaagccctgaccaaagtgattatcgaaacctccctgctgacc gaagaagaa aagaaggcggcttgtgaactggcggtaaaagcaggtgctgatttcgtcaaaacgtctacc ggtttctct ggtggcggtgcaaccgcagaagacattgccctgatgcgtaaggttgttggtcctaacctg ggcgttaag gccagcggcggtgtgcgtgacctgtctgacgcgaaggcgatgattgacgcgggcgcgact cgtatcggc gcttccgcaggtgttgcgatcgttaatggtgaacgctctgaaggttccacgaaatggacc gcagctggt gcggcgacgacgtgcgcttgtacgggcggctaa

Example 38

SEQIDNO 15 -Nucleotide Sequence of DERA 108 atgaaactgaacaaatacatcgatcacaccatcctgaaaccggaaacgactcaggaacag gtggagaaa atcctggctgaagcgaaagaatacgatttcgcgtccgtctgcgttaacccgacgtgggta gctctggca gctgaaagcctgaaagatagcgacgtcaaagtctgcactgtcatcggcttcccgctgggc gctaacact ccggcagtgaaggcgttcgaaactaaagacgctattagcaacggcgcggatgaaatcgac atggtgatt aacatcggcgcactgaaaacgggtaactacgatctggttctggaagatattaaggctgtc gttgcagca agcggcgataaactggtaaaggtaatcatcgaagcgtgcctgctgaccgacgatgaaaag gttaaagcg tgccagctgtctcaggaagcgggcgctgactacgtcaagacgagcactggcttctctacc ggcggtgcg acggtcgcagatgttgctctgatgcgtaaaactgttggcccggacatgggcgtaaaagcg tctggcggt gcgcgctcttacgaagacgctatcgcgttcattgaagctggcgcaagccgtattggcgcc agctctggc gtggcgatcatgaatggtgcgcaggctgatggcgacaccaagtggtaa

Example 39

SEQ ID NO 16 - Amino Acid Sequence of DERA03

Mtdlkasslralklmdlttlndddtdekvialchqaktpvgntaaiciyprflpiar ktlkeqgtpeir latvtnfphgnddidialaetraaiaygadevdvvfpyralmagneqvgfdlvkackeac aaanvllkv iietgelkdealirkaseisikagadflktstgkvavnatpesaπmmevirdmgvektv gfkpaggvr taedaqkylaiadelfgadwadarhyrfgassllasllkalghgdgksassy .

Example 40

SEQ ID NO 17 - Ammo Acid Sequence of DERA04

Mgniakmidhtllkpeateqqivqlcteakqygfaavcvπptwvktaarelsgtdv rvctvigfplgattpetkafettnaieπgarev dmvinigalksgqdelverdiraweaaagralvkvivetalltdeekvracqlavkagad yvktstgfsgggatvedvalmrktvgd ragvkasggvrdwktaeaminagatπgtssgvaivtggtgrady

Example 41

SEQ ID NO 18 - Ammo Acid Sequence of DERA06

Mglasyidhtllkatatladirtlceearehsfyavcinpvfipharawlegsdvkv atvcgfplgaisseqkalearlsaetgadeidm vihigsalagdwdaveadvravrravpeqvlkviietcyltdeqkrlatevavqggadfv ktstgfgtggatvddvrlmaeviggragl kaaggvrtpadaqamieagatrlgtsggvglvsggengagy

Example 42

SEQ ID NO 19 - Amino Aαd Sequence of DERA08

Mgiakmidhtalkpdttkeqiltltkeareygfasvcvnptvwklsaeqlagaesvv ctvigfplgantpevkafevkdaiqngakev dmvinigalkdkddelverdiravvdaakgkalvkvnetclltdeekvraceiavkagtd fvktstgfstggataedialmrktvgpnig vkasggvrtkedvekmieagatπgasagvaivsgekpakpdny

Example 43

SEQ ID NO 20 - Amino Acid Sequence of DERA11

Mtsnqlaqyidhtaltaekneqdistlcneaiehgfysvcinsayiplakeklagsn vkictwgfplganltsvkafetqesikagane idmvinvgwiksqkwdevkqdiqavfnacngtplkviletclltkdeivkaceickeigv afvktstgfnkggatvedvalmkntvgni gvkasggvrdtetalamikagatπgasagiansgtqdtqsty

Example 44

SEQ ID NO 21 - Amino Aαd Sequence of DERA12

Mieyrieeavakyrefyefkpvresagiedvksaiehtnlkpfatpddikklclear enrfhgvcvnpcyvklareelegtdvkvvtvv gfplganetrtkaheaifavesgadeidmviπvgmlkakeweyvyedirswesvkgkvv kviietcyldteekiaacvisklagah fvktstgfgtggataedvhlmkwivgdemgvkasggirtfedavkmimygadπgtssgv kivqggeerygg

Example 45

SEQ ID NO 22 - Amino Acid Sequence of DERA15

Mpsardilqqgldrlgspedlasridstllsprateedvrnlvreasdygfrcavlt pvytvkisglaeklgvklcsvigfplgqaplevklv eaqtvleagateldvvphlslgpeavyrevsgivklaksygavvkvileaplwddktlsl lvdssrragadivktstgvytkggdpvtvf rlaslakplgmgvkasggirsgidavlavgagadiigtssavkvlesfkslv

Example 46

SEQ ID NO 23 - Amino Acid Sequence of DERA101 maankyemafaqfdpaeseenllktdqiirdhysrfdtpetkkflhgvidltslnatdse esitkfte svndfedtdptipsvaaicvypnfvstvretltaenvkvasvsgcfpasqsflevklaet alavsdgad eidivlnmgkflsgdyeaaateieeqiaaakgatvkviletgalktpemrratilslfcg ahfvktst gkgypgasleaaytmckvlkqyyglfgevrgiklsggirttedavkyyclietllgkewl tpayfriga sslvdalrqdirav

Example 47

SEQ ID NO 24 - Amino Acid Sequence of DERA102 melnrmidhtilkpeateaavqkndeakeynffsvcinpcwvafaseqladtdvavctvi gfplgant pevkayeaadaiknganevdmvinigalksqqydyvrqdiqgvvdaakgkalvkviieta lltdeekvk acelakeagadfvktstgfstggakvadirlmretvgpdmgvkasggvhnaeealamiea gatrigast gvaivsgatgegtkw.

Example 48

SEQ ID NO: 25 - Amino Acid Sequence of DERA103 mtiesaxalapaeravnligsdltekslklhleglsgvdavgleqraaglstrsxkttsk awaldtiik lidlttlegadtpgkvrslaakamlpdasdvsapqvaavcvygdmvpyaaealgsswsng sdnginvaa vatafpsgrsslpikiadtkeavahgadeidmvidrgaflsgkygvvfdqivavkeacrr engtyahlk viletgelntydnvrraswlaxlaggdfvktstgkvspaatlpvtllmlevvrdwhvltg ekigvkpag girsskdaxkylvtvaetvgeewlqphlfrfgassllndvlmqrqklstghysgpdyvtx d.

Example 49

SEQ ID NO: 26 - Amino Acid Sequence of DERA104 msstptildpafedvtrseaslrrflhglpgvdqvgaearaaglatrsiktsakefaldl airmvdltt legqdtpgkvralsakamrpdpsdptcpataavcvypdmvgiakqalgtsgvhvaavata fpsgraald xkladvrdavdagadexdmvxdrgaflagryqhvydexvavreacrrengegahlkvxfe tgelqtydn vrraswlammagahfvktstgkvqpaatlpvtlvmlqavrdfrgatgrmvgvkpaggirt akdaikylv mvnevagedwldpdwfrfgastllndllmqrtkmktgrysgpdyftld.

Example 50

SEQ ID NO. 27 - Amino Acid Sequence of DERA105 melitqpscwvfsvffrrqygwlvfvegawydgrrqtfhldgngrkgflrmtmniakmid htllkpeat eqqxvqlcteakqygfasvcvnptwvktaarelsgtdvrvctvigfplgattpetkafet tnaxengar evdmvxnigalksgqdelverdxravveaaagralvkvxvetalltdeekvracqlavka gadyvktst gfsgggatvedvalmrktvgdragvkasggvrdwktaeaminagatrigtssgvaivtgg tgradtkw.

Example 51

SEQ ID NO' 28 - Amino Acid Sequence of DERA106 mtiakmidhtalkpdttkeqiltltkeareygfasvcvnptwvklsaeqlsgaesvvctv igfplgant pevkafevknaiengakevdmvinigalkdkddelverdiravvdaakgkalvkviietc lltdeekvr aceiavkagtdfvktstgfstggataedialmrktvgpnigvkasggvrtkedvekmiea gatrigasa gvaivsgekpakpdntkw.

Example 52

SEQ ID NO 29 - Ammo Acid Sequence of DERA107 msrsiaqmidhtllkpnttedqivklceeakeysfasvcvnptwvalaaqllkdapdvkv ctvigfplg attpevkafettnaiengatevdmvinigalkdkqyelvgrdiqavvkaaegkaltkvii etsllteee

kkaacelavkagadfvktstgfsgggataedialmrkvvgpnlgvkasggvrdlsda kamidagatrig asagvaivngersegstkwtaagaattcactgg.

Example 53

SEQ ID NO 30 - Ammo Acid Sequence of DERA108 mklnkyidhtilkpettqeqvekilaeakeydfasvcvnptwvalaaeslkdsdvkvctv igfplgant pavkafetkdaisngadeidmvinigalktgnydlvledikavvaasgdklvkviieacl ltddekvka cqlsqeagadyvktstgfstggatvadvalmrktvgpdmgvkasggarsyedaiafieag asngassg vaimngaqadgdtkw.

All publications, including but not limited to, issued patents, patent applications, and journal articles, cited in this application are each herein incorporated by reference in their entirety

Although the invention has been described above with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention Accordingly, the invention is limited only by the following claims