The following specification particularly describes the nature of the invention and the manner in which it is to be performed.

ENZYMES AND METHODS FOR RESOLVING AMINO VINYL CYCLOPROPANE CARBOXYLIC ACID DERIVATIVES

INTRODUCTION
Aspects of the present invention relate to the preparation and isolation of amino vinyl cyclopropane carboxylic acid derivatives and salts thereof  methods of resolving enantiomers  and methods of identifying compositions and/or enzymes that are capable of resolving racemic or partially enantiomerically enriched mixtures. In aspects  the salts of amino vinyl cyclopropane carboxylic acid derivatives are utilized in a hydrolase-catalysed bioresolution process  without the need for additional buffering capacity  to produce enantiomerically enriched 1-amino-2-vinylcyclopropane carboxylic acid derivatives.
Chemical synthesis of many compounds fails to selectively produce a desired enantiomer  thus resulting in racemic or enantiomeric mixtures that must be separated or resolved before further processing. Amino vinyl cyclopropane carboxylic acid derivatives have been taught to be key intermediates for the preparation of inhibitors of the Hepatitis C virus NS3 protease. See P. L. Beaulieu et al.  “Synthesis of (1R 2S)-1-Amino-2-vinylcyclopropanecarboxylic Acid Vinyl-ACCA) Derivatives: Key Intermediates for the Preparation of Inhibitors of the Hepatitis C Virus NS3 Protease ” Journal of Organic Chemistry  Vol. 70(15)  pp. 5869-5879  2005.
The Beaulieu et al. article teaches an approach to manufacture such derivatives  involving condensation of benzaldehyde with ethyl glycinate hydrochloride  followed by reaction with trans-1 4-dibromobut-2-ene  to form a racemic amino vinyl cyclopropane carboxylic acid ethyl ester (1). The amine functionality on this compound is then protected by addition of a BOC group (i.e.  a -C(O)OC(CH3)3 group) on the nitrogen atom. The protected compound (2) is subjected to enzymatic resolution and optionally is converted to the tosylate salt. Beaulieu et al. teach that  when handling solutions of amino ester (1)  solvent must be removed under reduced pressure at room temperature.

A direct resolution method  using unprotected compounds  will provide a simpler and more efficient route to such derivatives.

SUMMARY
An alternative approach has been found for producing enantiomerically enriched amino vinyl cyclopropane carboxylic acid derivatives. Specifically  an approach involves a hydrolase catalysed bioresolution of amino ester (3).

To obtain large quantities of an amino ester (3)  one might consider distillation to separate the compound from solvent and reaction mixture  prior to enzymatic resolution. However  the room temperature vacuum separation of an amino ester (3) from solvents is not suitable for efficient large scale production. Moreover  it now has been discovered that 1-amino-2-vinylcyclopropane carboxylic acid esters  e.g.  (3) where R is methyl or ethyl  have relatively low thermal stability  showing an exotherm at 50°C under accelerated rate calorimetry (ARC). These factors limit processing options that would normally be desirable for use in large scale production.
The present applicants have discovered a number of novel salts that enable efficient large-scale production. Surprisingly  these salts have been found to have certain advantages  which allow a viable manufacturing process for enantio-enriched amino ester (3). These advantages include one or more of the following: enhanced thermal stability to temperatures beyond the melting point of the salt (typically >100°C); avoidance of expensive BOC anhydride reactants; improved form of compound (solid  as opposed to the oil form of the free amine or the BOC protected amine) for better ease of handling and storage; avoidance of a time consuming low temperature vacuum separation step; avoidance of a distillation step that raises the temperature of the low thermal stability compound; and suitability for direct use of the salts in subsequent bioresolution steps  without the need for additional buffer.
In an aspect  the invention comprises a compound of formula (4) 

where R is an alkyl group  n is an integer of 1-3  and HX is an acid such as phosphoric acid  sulfuric acid  β β-dimethylglutaric acid  citric acid  boric acid  acetic acid  maleic acid  malic acid  succinic acid  3-(N-morpholino)propane sulfonic acid  2-(N-morpholino)ethane sulfonic acid  4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid  etc..
In an aspect  the invention comprises methods of making the above compound (4) by reacting a compound of formula (3) with an acid HX  as defined above.

Another aspect of the invention comprises methods of making the above salt compound (4) by dialkylation of the appropriate (E)-N-phenylmethyleneglycine alkyl ester with trans-1 4-dihalobut-2-ene in a solvent  hydrolysis of the intermediate imino ester of formula (5) with an acid HX  and isolation of the resulting salt  such as by filtration.

A further aspect of the invention comprises use of the above salt compound (4) in a hydrolase catalysed bioresolution process  without the need for additional buffer.
A further aspect of the present invention includes methods of identifying an enzyme capable of resolving a racemic or partially enantiomerically enriched mixture. Embodiments of a method include: providing a racemic or partially enantiomerically enriched mixture; exposing cell constituents to the racemic or partially enantiomerically enriched mixture; examining the racemic or partially enantiomerically enriched mixture for a change in the enantiomeric ratio; isolating an enzyme having resolving activity for the racemic or partially enantiomerically enriched mixture; and identifying said enzyme.
Aspects of the present invention include methods of resolving a racemic or partially enantiomerically enriched mixture of an ester of 1-amino-2-vinylcyclopropane carboxylic acid. Embodiments include: providing a racemic or partially enantiomerically enriched mixture of an ester of 1-amino-2-vinylcyclopropane carboxylic acid; and exposing said racemic or partially enantiomerically enriched mixture of an ester of 1-amino-2-vinylcyclopropane carboxylic acid to cell constituents.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a graphic representation of rates of hydrolysis of 50 g/L 1-amino-2-vinylcyclopropane carboxylic acid methyl ester using different enzymes  as measured using achiral high performance liquid chromatography (HPLC).
Fig. 2 is a sequence listing of a protein derived from Leuwenhoekiella blandensis and useful for producing enantiomerically enriched amino vinyl cyclopropane carboxylic acid derivatives.
Fig. 3 is a sequence listing of a protein derived from Crocibacter atlanticus and useful for producing enantiomerically enriched amino vinyl cyclopropane carboxylic acid derivatives.

DETAILED DESCRIPTION
According to a first embodiment  this invention comprises a compound of formula (4) 

where R is an alkyl group. In embodiments  R has 1 to about 20 carbon atoms  or 1 to about 6 carbon atoms  or R is methyl or ethyl. In certain embodiments when R has more than 2 carbon atoms  R is an n-alkyl group. HX is an acid such as phosphoric acid  sulfuric acid  β β-dimethylglutaric acid  citric acid  boric acid  acetic acid  maleic acid  malic acid  succinic acid  3-(N-morpholino)propanesulfonic acid  2-(N-morpholino)ethanesulfonic acid  4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid  etc.  and n is an integer of about 1-3.
Salts can be made by reaction of an acid of formula HX to a solution containing a compound of formula (3).

The concentration of the free amino ester (3) can be at least about 20 g/L  or at least about 50 g/L  or at least about 75 g/L  and generally less than about 200 g/L  or less than about 100 g/L. The amount of acid added is about 0.3-2 mole equivalents  or about 0.5-1.5 mole equivalents  or about 1 mole equivalent  per mole of amino ester.
The salt formation is undertaken in an organic solvent. A suitable solvent is one in which the free base (i.e.  free amino ester) has good solubility  but in which the salt has low solubility. Useful solvents include  but are not limited to  ethers such as methyl t-butyl ether (MTBE)  esters such as ethyl acetate and isopropyl acetate  halogenated hydrocarbons such as dichloromethane  and hydrocarbons such as toluene. Salt formation can be carried out using a combination of a solvent and a water soluble co-solvent (e.g.  methanol  ethanol  acetone  and the like). The amount of the co-solvent is generally about 5-20%  based on the total volume of solvent.
Thus  according to embodiments  compound (4) may be made as illustrated in Scheme 1 below:

Scheme 1
According to an approach  beginning with (E)-N-phenylmethylene glycine alkyl ester and trans-1 4-dihalobut-2-ene  the compound (4) may be made by dialkylation of the appropriate (E)-N-phenylmethyleneglycine alkyl ester with a trans-1 4-dihalobut-2-ene in solvent  hydrolysis of the intermediate imino ester using an acid  provided that if an acid other than a desired HX is used  the hydrolysis step is followed by adjusting the pH to about 8-9  solvent extraction  addition of a lower alcohol and acid HX; and isolation of the salt  such as by centrifugation  filtration  decantation  etc. This approach is illustrated in Scheme 2 below  which shows certain specific reagents.

Scheme 2
The dialkylation step is facilitated by bases. Useful bases include  but are not limited to  potassium hydroxide  sodium t-butoxide  potassium t-butoxide  lithium t-butoxide  lithium hexamethylsilazane  sodium hexamethylsilazane and potassium hexamethylsilazane  and the like. The trans-1 4-dihalobut-2-ene can be trans-1 4-dibromobut-2-ene.
The dialkylation step typically occurs in a suitable solvent. Non-limiting examples of such solvents are toluene  MTBE  hexane  and tetrahydrofuran (THF). An example of a useful solvent is a mixture of toluene and MTBE  containing 50-70% by volume MTBE.
Useful amounts of lithium t-butoxide or other base  per mole of trans-1 4-dibromobut-2-ene  are about 2.1-2.6 mole equivalents  Useful amounts of the (E)-N-phenylmethyleneglycine alkyl ester  per mole of trans-1 4-dibromobut-2-ene  are about 1.05-1.5 mole equivalents.
The solution of the imino ester (5) resulting from the dialkylation step is then hydrolyzed. According to one approach  the hydrolysis step comprises using an appropriate acid such as  but not limited to  hydrochloric acid  sulfuric acid  nitric acid  or phosphoric acid  including aqueous HCl in concentrations of 0.1M to 12M  or 2M to 6M.
After hydrolysis  the organic phase is discarded and base is added to raise the pH to about 8-9. Suitable bases include  but are not limited to  sodium hydroxide  potassium hydroxide  sodium carbonate  and sodium bicarbonate. The amino ester may then be extracted into a suitable organic solvent  such as MTBE. In this approach (using an acid other than a desired HX)  one then adds an acid HX to form the salt according to the procedure set forth above.
According to an alternate approach illustrated in Scheme 3  the hydrolysis of 5 can be undertaken directly with HX to form the corresponding salt. The amount of acid HX added  per mole of amino ester  in this approach is in the range of about 0.3-2 mole equivalents  or about 0.5-1.5 mole equivalents  or about 1 mole equivalent.

Scheme 3
The salt is then isolated  such as by centrifugation  filtration  decantation  etc.  as a solid that is thermally stable and can be easily handled for future reactions. The compound (4) can be used in enzymatic resolution of the racemic species to preferentially obtain a desired single enantiomer form  as represented in Scheme 4.

Scheme 4
These salts can be used directly in a hydrolase catalysed enzymatic resolution by dissolution of the salt in water  adjustment of pH to the range of 6-9 by addition of base  and addition of a hydrolase enzyme. No additional buffer is needed. Examples of organisms from which suitable enzymes can be obtained include Formosa sp.  Psychroserpens sp.  Shewanella sp.  Winogradskyella sp.  Leeuwenhoekiella blandensis  Croceibacter atlanticus and Leeuwenhoekiella aequorea and Aquamarina  sp.
In a further aspect the present invention relates to methods of identifying compositions and/or enzymes capable of resolving racemic or partially enantiomerically enriched mixtures.
In embodiments  methods of identifying compositions and/or enzymes capable of resolving a racemic or partially enantiomerically enriched mixture of an ester of 1-amino-2-vinylcyclopropane carboxylic acid  as schematically represented in scheme 5  are provided.

Scheme 5
In particular embodiments  racemic or partially enantiomerically enriched mixtures can be exposed to cell constituents from one or more organisms. In further embodiments  racemic or partially enantiomerically enriched mixtures can be examined to determine if there are changes in the enantiomeric ratio or resolution of the mixtures.
In alternative embodiments  cell constituents shown to have resolving activity can be fractionated or separated and can be further tested for resolving activity  so as to isolate or identify one or more enzymes having the resolving activity. In additional embodiments  one or more enzymes having resolving activity can be  by way of non-limiting examples  in addition to an enzyme  a peptide or an RNA. In certain embodiments of the invention  a gene encoding one or more enzymes having resolving activity may be identified and cloned using techniques standard in the art. See  e.g.  J. Sambrook et al. (eds)  Molecular Cloning: A Laboratory Manual  Cold Spring Harbor Laboratory Press  1.84-1.88  2001.
Resolution  as used herein  relates to a change in the level of one of a pair of enantiomers relative to the other (the enantiomeric ratio). Thus  resolution might result from the modification of one enantiomer  thus making it no longer part of an enantiomeric pair  or the conversion of one enantiomer into the other enantiomer. One non-limiting example of resolution includes the cleaving of an ester to form 1-amino-2-vinylcyclopropane carboxylic acid.
Enantiomerically enriched  as used herein  refers to mixtures comprising a pair of enantiomers wherein the enantiomeric ratio is other than 1:1.
In embodiments of the invention  a racemic or enantiomerically enriched mixture may include any composition or solution containing the two species of an enantiomeric pair. In further embodiments  one or more groups of the enantiomeric pair may be protected by  for example  a BOC group. In additional embodiments  the molecules of the enantiomeric pair can be  or can exist as  part of a salt  such as  by way of non-limiting examples  a phosphate  sodium  nitrate  or calcium salt. In further embodiments  the molecules of the enantiomeric pair can be in the free amine form. In particular embodiments  the mixture or solution comprising the enantiomeric pair may comprise any solvent or solution. Examples of solvents or solutions include  but are not limited to  water  saline  buffered saline  phosphate buffered saline  and/or solutions comprising a polysorbate surfactant (e.g.  a TWEEN® product).
In particular embodiments of the invention  exposing a racemic or partially enantiomerically enriched mixture to cell constituents may include any method or technique for bringing the mixture and the cell constituents in contact with each other  such that the cell constituents may at least partially resolve the mixture. Examples of methods of exposure include  but are not limited to  fluid contact and physical contact.
Exposure to cell constituents may take place for any period of time required to recognize or determine a statistically significant change in the enantiomeric ratio. Examples of time periods of exposure include  but are not limited to  from about 0.1 hours to about 72 hours  about 1 hour to about 48 hours  about 8 hours to about 30 hours  about 30 hours  and about 12 hours.
Exposure may also take place at any temperatures. In embodiments  exposure occurs approximately at temperatures that are the normal living environment of the organisms from which the cell constituents are obtained. Examples of temperatures at which exposure may occur include  but are not limited to  about 1°C to about 99°C  about 10°C to about 50°C  and about 30°C.
Exposure to cell constituents may take place at any pH values. Examples of pH values at which exposure may occur include  but are not limited to  about pH 1 to about pH 12  about pH 3 to about pH 11  about pH 6 to about pH 9  about pH 9  and about pH 7.
In certain embodiments of the invention  cell constituents can include  but are not limited to  cell extracts  cell pastes  cell lysates  cell free extracts  lyophilized cell free extracts  lyophilized cell extracts  lyophilized cell pastes  lyophilized cell lysates  sonicated cells  isolated proteins  and/or combinations  and/or fractions  and/or fragments thereof.
The cell constituents can be from any organism or a combination of organisms. Examples of organisms from which cell constituents might be obtained include  but are not limited to  animals  plants  bacteria  archea  fungi  marine organisms  marine algae  Formosa sp.  Psychroserpens sp.  Shewanella sp.  Winogradskyella sp.  Leeuwenhoekiella blandensis  Croceibacter atlanticus and Leeuwenhoekiella aequorea  Aquamarina  sp.  AQP317  and AQP383.
AQP317 was deposited with the National Collection of Industrial  Food and Marine Bacteria  Aberdeen  Scotland (“NCIMB”)  under the Budapest treaty  as NCIMB 41475 on March 9  2007  and AQP383 was deposited with NCIMB as NCIMB 41476 on March 9  2007.
Leewenhoekiella blandensis was deposited with NCIMB  under the Budapest Treaty  in 2010. Croceibacter atlanticus was deposited with NCIMB  under the Budapest Treaty  in 2010. Leewenhoekiella aquorea was deposited with the NCIMB  under the Budapest Treaty  in 2010.
In alternative embodiments of the invention  cell constituents may be further fractionated or separated. Methods of fractionation and separation include  but are not limited to  various forms of chromatography  size exclusion  gel electrophoresis  iso-electric and precipitate separations. Cell constituents can be fractionated by ammonium sulfate precipitation.
In additional embodiments  enzymes having resolving activity can preferentially precipitate at ammonium sulfate concentrations of approximately 30% to 40%  or higher  and/or can preferentially precipitate at ammonium sulfate concentrations of approximately 50% to 60%  or higher.
The following examples will further illustrate certain specific aspects and embodiments. These examples are provided only for purposes of illustration  and should not be construed as limiting the scope of the invention in any manner.

EXAMPLE 1: Synthesis of 1-amino-2-vinylcyclopropanecarboxylic acid methyl ester.
To a stirred solution of trans-1 4-dibromo-2-butene (340.6 g  1.33 mol) in MTBE (1.5 L) is added lithium tert-butoxide (318 g  3.325 mol). The resulting suspension is cooled below 15°C and a solution of (E)-N-phenylmethyleneglycine methyl ester (310 g  1.75 mol) in toluene (875 mL) is slowly added over 60 minutes  ensuring that the reaction temperature remains at 15-20°C. After stirring for an additional 2 hours at 20-25°C  the reaction is quenched by adding NaCl solution (20% by weight  2L). The organic phase is mixed with 1M HCl solution (1.75 L  1.75 mol) and vigorously stirred at 20°C for 2 hours. Aliquots are taken from the mass to ensure that all of the intermediate imine has been hydrolysed. Phases are then separated and the aqueous phase is washed with 500 ml MTBE. The aqueous phase is then cooled to 13°C and pH is adjusted to about 9 with NaOH solution (6M  200 mL). The mixture is then extracted with MTBE (5 L)  yielding a solution containing 186 g of 1-amino-2-vinylcyclopropanecarboxylic acid methyl ester.

EXAMPLE 2: Synthesis of 1-amino-2-vinylcyclopropanecarboxylic acid methyl ester phosphate salt.
Into a round bottom flask equipped with an overhead stirrer is placed a solution of 1-amino-2-vinylcyclopropane carboxylic acid methyl ester (7 g) in MTBE (110 mL). Methanol (10 mL) is then added and the mixture is stirred at room temperature. Orthophosphoric acid (3.5 mL) is added drop-wise and a precipitate forms. After the addition is complete  stirring is continued for a further 60 minutes. The phosphate salt is then recovered by filtration and dried in vacuo. The phosphate salt is obtained as a beige powder in a yield of 11.72 g (~98%).
1H NMR (D4-MeOH): δ 5.82-5.70 (m  1H)  5.42 (dd  J=17 & 1  1H)  5.22 (dd  J=10 & 1  1H)  3.86 (s  3H)  2.49 (q  J=9  1H)  1.85-1.80 (m  1H)  1.80-1.73 (m  1H).

EXAMPLE 3: Synthesis of 1-amino-2-vinylcyclopropane carboxylic acid methyl ester phosphate salt.
In a glass-lined reactor  a solution of approximately 1-amino-2-vinyl cyclopropane carboxylic acid methyl ester (70 Kg) in MTBE (566 Kg) is prepared via the synthesis procedure described in Example 1. After drying the solution with magnesium sulphate and filtering  methanol (40 Kg) is added. At a temperature below 15°C  80% phosphoric acid (60 Kg) is added gradually  over 40 minutes. During this time a precipitate is formed. Once the addition is complete  the slurry is stirred below 10°C for at least an additional hour. The solid is collected by filtration  washed with MTBE  and discharged from the filter to storage containers as an off-white damp filter cake with typically 23% MTBE content. About 138.5 kg of damp filter cake is isolated  equivalent to about 106.5 kg (~90% yield) of 1-amino-2-vinylcyclopropane carboxylic acid methyl ester phosphate  on a solvent-free basis.

EXAMPLE 4: Synthesis of 1-amino-2-vinylcyclopropane carboxylic acid ethyl ester phosphate salt.
Into a round bottom flask is placed a solution of 1-amino-2-vinyl cyclopropane carboxylic acid ethyl ester (8.5 g) in MTBE (100 mL). Methanol (12 mL) is then added and the mixture is stirred at room temperature. Orthophosphoric acid (6 mL) is added drop-wise and a precipitate starts to form. After the addition is complete  stirring is continued for a further 2 hours. The phosphate salt is recovered by filtration  washed with MTBE  and dried in vacuo. About 9.4 g (70% yield) of an off-white solid is obtained.
1H NMR (D4-MeOH): δ 5.81-5.67 (m  1H)  5.38 (dd  J=16 & 1  1H)  5.19 (dd  J= 11 & 1  1H)  4.28 (q  J= 7  2H)  2.43 (q  J=9  1H)  1.80-1.68 (m  2H)  1.31 (t  J= 7  3H).

EXAMPLE 5: Synthesis of 1-amino-2-vinylcyclopropane carboxylic acid methyl ester phosphate salt from N-phenyl methylene glycine methyl ester.
To lithium t-butoxide (31.8 g) slurried in MTBE (50 mL) is added trans-1 4-dibromo-2-butene (34 g) dissolved in MTBE (100 mL). To the stirred reaction mixture is then added a solution of N-phenyl methyleneglycine methyl ester (31 g) in toluene (90 g). The temperature is maintained below 15°C during the addition and subsequently the reaction is stirred at ambient temperature for 2 hours. The reaction is quenched by adding NaCl solution (20% by weight  200 mL). The aqueous phase is discarded and to the organic phase is added approx 15% by volume of methanol. The solution is cooled below 5°C and a molar equivalent of 85% phosphoric acid is added slowly to precipitate the phosphate salt. The mixture is stirred for 60 minutes  and precipitate is recovered by filtration and washed with MTBE. An amount of 32.3 g of 1-amino-2-vinylcyclopropane carboxylic acid methyl ester phosphate salt (85% yield  based on trans-1 4-dibromo-2-butene) with purity about 97% by HPLC is obtained.

EXAMPLE 6: Esterase catalysed bioresolution of 1-amino-2-vinylcyclopropane carboxylic acid ethyl ester phosphate salt.
Into a 20 mL stem block tube is placed 1-amino-2-vinylcyclopropane carboxylic acid ethyl ester phosphate salt (0.5 g  2 mmol) dissolved in deionised water (4 mL). The pH of the solution is adjusted to 8 by drop-wise addition of 1M NaOH solution (2 mL). The mixture is continuously stirred at 25°C and lyophilised AQP317 (Formosa algae) (200 mg) is added. Stirring is continued at 25°C for 41 hours  after which HPLC analysis determines that conversion has reached about 50% and gas chromatography (GC) analysis indicates that the enantiomeric excess of the residual ester has reached 99%.

EXAMPLE 7: Primary screen for esterase activity.
Substrate  1-amino-2-vinyl-cyclopropane carboxylic acid methyl ester  (200 μL  10 g/L in phosphate buffered saline) is applied to screening plates containing 1 mg per well of lyophilized cell paste. After overnight incubation at 30°C  the reactions are sampled into HPLC mobile phase and analyzed for amino acid formation. Further analysis of suspected hits is performed by analysis of residual ester as a trifluoroacetate by chiral GC on a Chirasil Dex CB column  helium carrier gas at 830 KPa (20 p.s.i.)  oven temperature isothermal at 100°C. From a screen of 230 marine microorganisms  twelve confirmed hits are identified  wherein the residual ester enantiomeric excess is greater than 90%.

EXAMPLE 8: AquapharmTM organisms screen.
Approximately 4 mg of a subset consisting of 8 of the 12 confirmed hits of lyophilized organism preparations (obtained from Aquapharm Biodiscovery Ltd.) is weighed into glass scintillation vials along with 1 mL of 10 g/L 1-amino-2-vinyl-cyclopropane carboxylic acid methyl ester  in phosphate buffered saline + 0.1% by volume Tween 80 (pH 7)  or in phosphate buffer pH 9. These mixtures are incubated for 30 hours at 30°C  and 300 rpm. Post-reaction residual ester is analyzed by GC as a trifluoroacetic acid derivative. The results are shown in Table 1. All samples are more active at pH 7 than pH 9.
AQP250 is found to not be viable when recovery is attempted from primary culture plates. An alternative organism  identified as a Psyhcroserpens sp  and designated AQP 383  having similar morphology and isolated from the same original source  is used as an alternative. When assayed  this is also demonstrated to have the desired activity.
Table 1
Organism Residual Ester Enantiomeric Excess
pH 7 pH 9
3 Hours 23 Hours 46 Hours 96 Hours 3 Hours 23 Hours
AQP029 15% ND ND 2% Shewanella baltica
AQP246 0% 11% ND ND -3% 1% Shewanella baltica
AQP237 0% 9% ND ND -2% 0%
AQP250 2% 32% ND ND -1% 2%
AQP272 0% 7% 14.6% 55.8% -1% 5% Winogradskyella thalassocola
AQP317 2% 25% 60.4% 93.5% 1% 22% Formosa algae
AQP331 2% 17% 39.1% 92.0% -10% 10% Winogradskyella thalassocola
AQP332 1% ND ND ND -2% ND Aquamarina sp.

EXAMPLE 9: Activity at 50 g/L substrate concentration.
Reactions containing 200 mg of lyophilized cell paste and 250 mg of methyl-1-amino-2-vinyl-cyclopropane carboxylate-phosphate salt are prepared in a total of 5 mL of phosphate buffered saline and monitored for conversion over time using achiral HPLC. Two organisms  AQP317 and AQP383  demonstrate significantly greater rate of activity compared to the other confirmed hits  as judged by achiral HPLC  and completely resolve the substrate at these concentrations. The results are plotted in Fig. 1.

EXAMPLE 10: Alcalase-catalyzed bioresolution of 1-amino-2-vinylcyclopropane carboxylic acid ethyl ester phosphate salt.
1-Amino-2-vinylcyclopropane carboxylic acid ethyl ester phosphate salt (2 g  7.9 mmol) in deionized water (45 mL) is placed into a jacketed vessel and pH is adjusted to 8 by adding 2M sodium hydroxide solution (5 mL). The mixture is continuously stirred at 35°C and Alcalase® enzyme solution sold by Novozymes (6 mL) is added. The mixture is continuously stirred and pH is maintained at 8.15. Aliquots (100 μL) are periodically taken and GC analysis indicates e.e. = 5.7% (t =24 hours)  e.e. = 14.3% (t =48 hours)  and e.e. =21.4% (t =72 hours). No significant conversion has occurred after 72 hours and the reaction is halted. The reaction is represented in Scheme 5.

EXAMPLE 11: Preparation of cell-free extract of Formosa algae AQP317.
A cell free extract of AQP317  Formosa algae  is prepared by re-suspending 500 mg of lyophilized cell paste  ex of 500 mL culture  in 50 mL of PBS. The cell suspension is sonicated  15 μm amplitude  for 30 minutes with 10 seconds on and 15 seconds off  at 4°C. Debris is removed by centrifugation at 10 000 G for 10 minutes at 4°C.
The cell-free extract is subjected to a series of ammonium sulfate precipitations  precipitate is recovered by centrifugation at 10 000 G for 20 minutes at 4°C. See R. M. C. Dawson et al.  eds.  Data for Biochemical Research  Third Edition  pp 537-539  1986. Each pellet is re-suspended in 5 mL of phosphate buffered saline (Sigma P4417). Protein content is assayed using Coomassie Plus reagent from Pierce. A similar experiment is performed using Psychroserpens AQP383  in which activity is demonstrated to precipitate at 30-40%.

EXAMPLE 12: Biotransformation assay.
Activity is assayed in 1 mL scintillation vials containing 10 mM 1-amino 2-vinylcyclopropane carboxylic acid ethyl ester phosphate salt in phosphate buffered saline. Reactions are sampled after 2.5 hours and diluted 1:5 into a HPLC mobile phase prior to analysis. Percentage conversion is then used to calculate the number of enzyme activity units present  with results as shown in Table 2.

EXAMPLE 13: Isolation of resolving activity.
Cell free extracts of AQP317 and AQP383 are fractionated using standard techniques. Various fractions are exposed to a racemic or partially enantiomerically enriched mixture of an ester of 1-amino-2-vinylcyclopropane carboxylic acid and incubated at 30°C for 30 hours. The different fractions are then tested for alteration of the enantiomeric ratio  as previously described. Fractions having resolving activity are further fractionated and/or separated via gel electrophoresis. Further fractions and gel isolates are tested for resolving activity.
A single compound having resolving activity is isolated and identified as an esterase. The isolated esterase is subjected to carboxyl and/or amino peptide sequencing or protein mass spectrometry. The sequence information is then used to generate putative primer pairs for the isolation of the gene encoding the esterase or to enable synthesis of the whole gene. DNA extracts of AQP317 and AQP383  Leuwenhoekiella blandensis  Croceibacter atlanticus  and Leuwenhoekiella aquorea are created and PCR is performed using the putative primer pairs. The PCR products are analyzed and the sequence encoding the esterase is isolated and cloned into a vector before sequencing.

EXAMPLE 14: Demonstration of activity of the cloned polypeptide
A nucleotide sequence encoding the polypeptide is cloned into a Pseudomonas expression system  using standard techniques known to those skilled in the art.
Cultures of the recombinant esterase are grown at 25°C  induced after 24 hours growth and 1 mL samples are taken at intervals post induction and micro centrifuged. Pellets are resuspended in 1 mL of 20 g/L 1-amino-2-vinylcyclopropane carboxylic acid methyl ester phosphate salt pH 7 and incubated at 25°C. After 144 hours reaction time  the residual ester is extracted with MTBE  derivitised with trifluoroacetic anhydride  and analysed by gas chromatography. The residual ester has an enantiomeric excess of 87%.

Table 2
Fraction Whole Cells Whole Sonicate Sonicate S/N 0-20 20-30 30-40 40-50 50-60 60-70 70+ S/N
Volume (mL) 50 50 50 5 5 5 5 5 5 50
Protein (mg/mL) 10 0.71 0.71 0.17 0.13 0.11 0.15 3.51 1.44 0.09
Total Protein (mg) 35.5 0.8 0.6 0.5 0.7 17.6 7.2 4.6
Protein Yield (%) 2 2 2 2 49 20 13
Cumulative Protein Yield (%) 2 4 6 8 57 77 90
Conversion* 17 15 21 1 1 1 1 10 6 5
Volume Assayed (mL) 0.5 0.5 0.5 0.05 0.05 0.05 0.05 0.05 0.05 0.5
Volumetric Activity** 0.0229 0.0194 0.0277 0.0165 0.0123 0.0089 0.0101 0.1288 0.0809 0.0070
Total Units 1.15 0.97 1.39 0.08 0.06 0.04 0.05 0.64 0.40 0.35
Activity Yield (%) 6 4 3 4 46 29 25
Cumulative Activity Yield (%) 6 10 14 17 64 93 118
* Percent at 2.5 hours.
** Units are μmol/minute/mL.

We claim:
1. A method of making a compound of formula (4)  comprising reacting a compound of formula (3) with an acid HX  wherein R is an alkyl group  n is an integer of 1 -3  and HX is phosphoric acid  sulfuric acid  β β-dimethylglutaric acid  citric acid  boric acid  acetic acid  maleic acid  malic acid  succinic acid  3-(/V-morpholino)propanesulfonic acid  2-(/V-morpholino)ethanesulfonic acid  or 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid.

2. The method of claim 1  wherein reacting is conducted in an organic solvent.
3. The method of claim 1  wherein reacting is conducted in a solvent comprising MTBE  ethyl acetate  dichloromethane  isopropyl acetate  or toluene.
4. The method of claim 1  wherein reacting is conducted in a solvent comprising MTBE  ethyl acetate  dichloromethane  isopropyl acetate  or toluene  in combination with a water soluble co-solvent.
5. A method of making a compound of formula (4)  comprising hydrolysing an imino ester intermediate of formula (5) with an acid HX  wherein R is an alkyl group  n is an integer of 1 -3  and HX is phosphoric acid  sulfuric acid  β β-dimethylglutahc acid  citric acid  boric acid  acetic acid  maleic acid  malic acid  succinic acid  3-(Λ/-morpholino)propanesulfonic acid  2-(/V-morpholino)ethanesulfonic acid  or 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid.

6. The method of claim 5  wherein the imino ester intermediate of formula (5) is obtained by dialkylation of (E)-Λ/-phenylmethyleneglycine alkyl ester (6) with trans-λ  4-dihalobut-2-ene of formula (7).

7. The method of claim 5  wherein the imino ester intermediate of formula (5) is hydrolysed without isolation  following the dialkylation.
8. The method of claim 5  wherein HX is phosphoric acid.
9. A method of resolving a racemic or partially enantiomerically enriched mixture of an ester of 1-amino-2-vinylcyclopropane carboxylic acid  comprising:
a) providing a racemic or partially enantiomerically enriched mixture of an ester of 1 -amino-2-vinylcyclopropane carboxylic acid; and
b) exposing the racemic or partially enantiomerically enriched mixture of an ester of 1 -amino-2-vinylcyclopropane carboxylic acid to cell constituents.
10. The method of claim 9   wherein cell constituents are obtained from animals  plants  bacteria  archea  fungi  marine organisms  marine algae  Formosa sp.  Psychroserpens sp.  Shewanella sp.  Winogradskyella sp.  Leeuwenhoekiella blandensis  Croceibacter atlanticus  Leeuwenhoekiella aequorea  Aquamarina sp.  AQP317  and AQP383.