BIOCATALYSTS AND METHODS FOR HYDROXYLATION OF CHEMICAL COMPOUNDS

Abstract

The present disclosure provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

Claims

1. An engineered polypeptide having proline hydroxylase activity, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 and one or more residue differences as compared to the sequence of SEQ ID NO:2 at residue positions selected from: X2; X3; X4; X5; X9; X13; X25; X26; X29; X30; X36; X42; X52; X57; X58; X59; X66; X86; X92; X95; X103; X112; X113; X115; X116; X121; X131; X150; X151; X225; X230; X270; and X271.

2. The engineered polypeptide of claim 1 in which the residue differences at residue positions X2; X3; X4; X5; X9; X13; X25; X26; X29; X30; X36; X42; X52, X57; X58; X59; X66; X92; X95; X103; X112; X115; X116; X121; X131; X150; X151; X225; X230; and X271 are selected from X2K; X2T; X3S; X4Q; X4L; X4E; X4S; X51; X5L; X5M; X91; X13T; X25R; X26T; X29A; X30V; X30P; X36T; X42E; X52P; X57T; X57A; X58A; X59G; X66Q; X86S; X92V; X95M; X103L; X103Q; X112T; X112V; X113E, X115E; X115H; X115D; X115G; X1155; X115A; X116L; X121F; X131Y; X131F; X1505; X1515; X225L; X225Y; X225W; X230V; X270E; X271K; and X271R.

3. The engineered polypeptide of claim 1, in which the amino acid sequence comprises at least a combination of features selected from: (a) X103L and X166Q; (b) X52P and X255Y; (c) X4E/L/S and X115A; (d) X25R and X58A; (e) X29A and X166T/Q/L; (f) X115H/D/G and X121F; (g) X3S, X103L, and X166Q; (h) X103L, X131Y/F, and X166T/Q/L; (i) X26T, X103L and X166T/Q/L; (j) X25R, X66Q, X92V and X115E; (k) X25R, X66Q, X92V, X103L, X115E, and X166Q; and (l) X3S, X25R, X66Q, X92V, X103L, X115E, and X166Q.

4. The engineered polypeptide of claim 1, which further comprises one or more residue differences as compared to the sequence of SEQ ID NO: 2 at residue positions selected from: X17, X24, X26, X62, X88, X98, X114, X140, X151, X186, X188, and X205.

5. The engineered polypeptide of claim 4 in which the residue differences at residue positions X17, X24, X26, X62, X88, X98, X114, X140, X151, X186, X188, and X205 are selected from X17V, X24R, X24S, X26R, X26W, X62Q, X88R, X98F, X98T, X114N, X140L, X151A, X151H, X186G, X188G, and X205V.

6. The engineered polypeptide of claim 1, which converts substrate compound (2), (2S)-piperidine-2-carboxylic acid, ##STR00050## to product compound (1), (2S,5S)-5-hydroxypiperidine-2-carboxylic acid, ##STR00051## under suitable reaction conditions.

7. The engineered polypeptide of claim 6 in which the polypeptide converts substrate compound (2) to product compound (1) with at least 2 fold the activity of SEQ ID NO:2, wherein the amino acid sequence comprises one or more residue differences selected from the group consisting of: X3S; X4Q; X4L; X5I; X5L; X24S; X25R; X30P; X66Q; X86S; X92V; X103L; X103Q; X113E; X115E; X150S; X166Q; X1515; X225L; and X270E.

8. The engineered polypeptide of claim 1, which converts substrate compound (2) to product compound (1) in excess of product compound (1a), (2S,3R)-3-hydroxypiperidine-2-carboxylic acid, ##STR00052## wherein the amino acid sequence comprises one or more residue differences selected from the group consisting of: X103L; X115E; X131Y and X166Q.

9. The engineered polypeptide of claim 1, which forms product compound (1), ##STR00053## in diastereomeric excess of product compound (1R), (2S,5R)-5-hydroxypiperidine-2-carboxylic acid, ##STR00054##

10. The engineered polypeptide of claim 1 having proline hydroxylase activity in which the amino acid sequence comprises a sequence selected from the group consisting of SEQ ID NO: 24, 8, 10, 12, 14, 16, 18, 20, 22, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, and 228.

11. A polynucleotide encoding the engineered polypeptide of claim 1, the polynucleotide optionally comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, and 227.

12. A polynucleotide encoding the polypeptide of SEQ ID NO: 2, comprising a nucleic acid sequence optimized for expression in E. coli, optionally comprising a sequence having at least 80% or more identity to a nucleic acid sequence selected from SEQ ID NO: 1, 3, and 5.

13. An expression vector comprising the polynucleotide of claim 11, further comprising a control sequence.

14. A host cell comprising the polynucleotide of claim 11, wherein the host cell is Escherichia coli.

15. A method of preparing an engineered polypeptide, comprising culturing the host cell of claim 14 under conditions suitable for expression of the polypeptide, the method further comprises a step of isolating the engineered polypeptide.

Description

6. EXAMPLES

Example 1: Synthesis, Optimization, and Screening Engineered Proline Hydroxylase Polypeptides

[0386] Gene Synthesis and Optimization:

[0387] The polynucleotide sequence encoding the reported wild-type cis-4-proline hydroxylase polypeptide from Sinorhizobium meliloti, as represented by SEQ ID NO: 2, was synthesized as the gene of SEQ ID NO: 1. The synthetic gene of SEQ ID NO: 1 was cloned into a pCK110900 vector system (see e.g., US Patent Application Publication 20060195947, which is hereby incorporated by reference herein) and subsequently expressed in E. coli W3110fhuA. The E. coli W3110 expresses the proline hydroxylase polypeptides under the control of the lac promoter. Based on sequence comparisons with other proline hydroxylases and computer modeling of the enzyme structure docked to the substrate proline, residue positions associated with the active site, peptide loops, solution/substrate interface, and potential stability positions were identified and subjected to mutagenesis. These first round variants were screened under HTP Assay conditions with (2S)-piperidine-2-carboxylic acid as substrate. Variants with increased enzymatic activity and/or expression were identified. Two additional codon optimized polynucleotides encoding the amino acid sequence of the naturally occurring enzyme was also generated (i.e., SEQ ID NO:3 and 5) for comparison purposes. The codon optimized polynucleotides 3 and 5 expressing the naturally occurring cis-4-proline hydroxylase showed increased expression relative to the polynucleotide of SEQ ID NO:1. The residue differences from the first round screening were combined in various permutations and screened for improved properties under HTP Assay, SFP Assay, and DSP Assay conditions. The engineered proline hydroxylase polypeptide sequences and specific mutations and relative activities obtained from the screens are listed in Table 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H.

Example 2: Production of Engineered Proline Hydroxylases

[0388] The engineered proline hydroxylase polypeptides were produced in E. coli W3110 under the control of the lac promoter. Enzyme preparations for HTP, DSP, and SFP assays were made as follows.

[0389] High-Throughput (HTP) Growth, Expression, and Lysate Preparation.

[0390] Cells were picked and grown overnight in LB media containing 1% glucose and 30 g/mL chloramphenicol (CAM), 30 C., 200 rpm, 85% humidity. A 20 L aliquot of overnight growth was transferred to a deep well plate containing 380 L 2TB growth media containing 30 g/mL CAM, 1 mM IPTG, and incubated for 18 h at 30 C., 200 rpm, 85% humidity. Cell cultures were centrifuged at 4000 rpm, 4 C. for 10 mM., and the media discarded. Cell pellets were resuspended in 100 L Lysis Buffer (50 mM phosphate buffer, pH 6.3, containing 100 M Mohr's salt (i.e., (NH.sub.4).sub.2Fe(SO.sub.4).sub.2), 0.5 mg/mL PMBS (polymyxin B sulfate) and 1 mg/mL Lysozyme). Lysis Buffer was prepared fresh by adding to 60 mL of 50 mM phosphate buffer, pH 6.3, 60 mg Lysozyme and 30 mg of PMBS. After mixing the Lysozyme solution, 0.6 mL of 10 mM Mohr's salt solution (in H.sub.2O) was added.

[0391] Production of Shake Flask Powders (SFP):

[0392] A shake-flask procedure was used to generate engineered proline hydroxylase polypeptide powders used in secondary screening assays or in the biocatalytic processes disclosed herein. Shake flask powder (SFP) provides a more purified preparation (e.g., up to 30% of total protein) of the engineered enzyme as compared to the cell lysate used in HTP assays. A single colony of E. coli containing a plasmid encoding an engineered polypeptide of interest is inoculated into 50 mL Luria Bertani broth containing 30 g/ml chloramphenicol and 1% glucose. Cells are grown overnight (at least 16 hours) in an incubator at 30 C. with shaking at 250 rpm. The culture is diluted into 250 mL Terrific Broth (12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65 mM potassium phosphate, pH 7, 1 mM MgSO.sub.4) containing 30 ng/ml chloramphenicol, in a 1 liter flask to an optical density of 600 nm (OD600) of 0.2 and allowed to grow at 30 C. Expression of the proline hydroxylase gene is induced by addition of isopropyl--D-thiogalactoside (IPTG) to a final concentration of 1 mM when the OD600 of the culture is 0.6 to 0.8. Incubation is then continued overnight (at least 16 hours). Cells are harvested by centrifugation (5000 rpm, 15 mM, 4 C.) and the supernatant discarded. The cell pellet is resuspended with an equal volume of cold (4 C.) 50 mM potassium phosphate buffer, pH 6.3, and harvested by centrifugation as above. The washed cells are resuspended in two volumes of the cold 50 mM potassium phosphate buffer, pH 6.3 and passed through a French Press twice at 12,000 psi while maintained at 4 C. Cell debris is removed by centrifugation (9000 rpm, 45 minutes, 4 C.). The clear lysate supernatant is collected and stored at 20 C. Lyophilization of frozen clear lysate provides a dry shake-flask powder of crude engineered polypeptide. Alternatively, the cell pellet (before or after washing) can be stored at 4 C. or 80 C.

[0393] Production of Downstream Process Powders (DSP):

[0394] DSP powders provide a more purified preparation of the engineered proline hydroxylase enzyme as compared to the cell lysate used in the HTP or SFP assays. Larger-scale fermentation of the engineered proline hydroxylase for production of DSP powders (100-120 g from 10L) can be carried out as a short batch followed by a fed batch process according to standard bioprocess methods. Briefly, proline hydroxylase expression is induced by addition of IPTG to a final concentration of 1 mM. Following fermentation, the cells are harvested and resuspended in 50 mM phosphate buffer, then mechanically disrupted by homogenization. The cell debris and nucleic acid are flocculated with polyethylenimine (PEI) and the suspension clarified by centrifugation. The resulting clear supernatant is concentrated using a tangential cross-flow ultrafiltration membrane to remove salts and water. The concentrated and partially purified enzyme concentrate can then be dried in a lyophilizer and packaged (e.g., in polyethylene containers).

Example 3: Analytical Procedures

[0395] Method 1HPLC Analysis of HTP Assay Reactions:

[0396] In a 96 deep well format assay block, 10 L of reaction solution was diluted with 230 L of 5% sodium bicarbonate solution followed by 160 L of dansyl chloride solution (6 mg/mL dansyl chloride in MeCN). The plate was heat sealed, centrifuged, and placed in an incubator at 55 C. for 45 minutes. The reaction solution turns from yellow to light yellow when derivatization with dansyl chloride is complete. In cases where the solution remained yellow, the plate was heated for another 15 mM. After incubation, the plate was centrifuged for 5 min at 4000 rpm. A 200 L aliquot of supernatant was transferred into a 96 Corning plate for HPLC analysis. The final concentration of the substrate was below 0.25 g/L.

[0397] The quenched reaction was subject to HPLC analysis under the following conditions.

TABLE-US-00011 Column Agilent Poroshell120 SB-C18 50 4.6 mm (2.7 11 m) with guard column Temperature 30 C. Mobile Phase Solution A: 2 mM Ammonium Acetate pH 3.3 Solution B: Acetonitrile Mobile Phase Profile Time: Flow rate: min A % B % mL/min 0.00 80 20 2.0 2.00 30 70 2.0 3.00 30 70 2.0 Postime 1 min Detection Wavelength 250 nm Column Temperature 25 C. Injection Volume 10 L Total Runtime 4 min Response Factor N.A. (Substrate area/ Product area)

[0398] Conversion of compound (2) to compound (1) was determined from the resulting chromatograms as follows:

% Conversion={(RFProduct Area)/[(RFProduct Area)+Substrate Area]}100

where

Response Factor (RF)=Substrate Area/Product Area.

[0399] This method was used for rapid identification for conversion of (2S)-piperidine-2-carboxylic acid to hydroxypiperidine-2-carboxylic acid.

[0400] The chromatographic elution profiles, denoted as Response time, are as follows:

TABLE-US-00012 (2S,5S)-1-(5- (dimethylamino) naphthalene-1- ylsulfonyl)-5- hydroxypiperidine- 2-carboxylic acid [00035] Response time: 1.59 min (2S,3R)-1-(5- (dimethylamino) naphthalene-1- ylsulfonyl)-3- hydroxypiperidine- 2-carboxylic acid [00036] Response time: 1.76 min (5)-1-(5- (dimethylamino) naphthalene-1- ylsulfonyl) piperidine-2- carboxylic acid [00037] Response time: 2.23 min

[0401] Method 2HPLC Analysis of DSP and SFP Reactions:

[0402] A 10 L of reaction solution from a DSP or SFP reaction was pipetted into a 1.5 ml Eppendorf tube and diluted with 230 L of 5% sodium bicarbonate. A 160 L aliquot of dansyl chloride solution (6 mg/ml dansyl chloride in MeCN) was then added. The tube was heated with open cover at 55 C. for at least 30 minutes in a heating block to ensure full derivatization, as indicated by change in color of the derivatization mixture from a yellow to a light yellow color. The tube was vortexed and then centrifuged at 12,000 rpm for 5 minutes. A 200 L aliquot of supernatant was transferred into a 2 ml HPLC vial with insert. The vial was submitted to reverse phase HPLC-UV for analysis, as described below. The final concentration of the substrate was below 0.25 g/1.

[0403] The quenched reaction was subject to HPLC analysis under the following conditions.

TABLE-US-00013 Column Supelco Ascentis Express C18 100 4.6 mm (2.7 11 m), attached Mobile Phase Solution A: 2 mM Ammonium Acetate, pH 3.3 Solution B: Acetonitrile Mobile Phase Profile Time: Flow rate: min A % B % mL/min 0 80 20 1.0 9.0 27.5 72.5 1.0 9.1 0 100 1.0 12.0 0 100 1.0 12.1 80 20 1.0 Postime 2.90 min Detection Wavelength 250 nm Column Temperature 25 C. Injection Volume 10 L Total Runtime 15.0 min Response Factor (RF) N.A. (Substrate area/ Product area)

[0404] The chromatographic elution profiles are as follows:

TABLE-US-00014 (2S,5R)-1-(5- (dimethylamino) naphthalene-1- ylsulfonyl)-5- hydroxypiperidine- 2-carboxylic acid [00038] Response time: 5.16 min (2S,5S)-1-(5- (dimethylamino) naphthalene-1- ylsulfonyl)-5- hydroxypiperidine- 2-carboxylic acid [00039] Response time: 5.57 min (2S,3R)-1-(5- (dimethylamino) naphthalene-1- ylsulfonyl)-3- hydroxypiperidine- 2-carboxylic acid [00040] Response time: 6.02 min (5)-1-(5- (dimethylamino) naphthalene-1- ylsulfonyl) piperidine-2- carboxylic acid [00041] Response time: 8.17 min

Example 4: High Throughput (HTP) Screening of Proline Hydroxylases for Conversion of Compound (2) to Compound (1)

[0405] HTP Screening Assays: High-throughput screening used to guide primary selection of variants was carried out in 96-well plates using cell lysates. Two conditions, Condition A and Condition B, were used.

[0406] Condition A reactions were carried out as follows. Cells were grown in 96-well plates as described above and lysates prepared by dispensing 100 L Lysis Buffer into each well. Lysis buffer was prepared by dissolving 30 mg of lysozyme and 15 mg of PMBS in 30 mL of 50 mM phosphate buffer, pH 6.3. A 600 L volume of 10 mM Mohr's salt, freshly prepared in sterile water, was added to the lysozyme solution. The plate was heat sealed and then shaken on a titre-plate shaker at Speed #8 for 2 h at room temperature. Subsequently, the plate was quick-spun to settle the lysate at the bottom of the plate. This crude lysate was to be used for the reaction.

[0407] The Condition A reactions in 200 L scale were carried out in 98 well plates. A premix stock solution was prepared by dissolving 1.33 g of -ketoglutaric acid and 1.47 g of L-ascorbic acid in 31.5 mL of 50 mM phosphate buffer, pH 6.3 (pH adjusted by using KOH). After mixing, the pH was adjusted to 6.3 with KOH. To the pH adjusted premix, 41.16 mg of Mohr's salt was added. The solution turns cloudy due to the low solubility of Mohr's salt in aqueous solvents.

[0408] To each 100 L of crude lysates prepared above, 90 L of the premix stock solution was added into each well, followed immediately by 10 uL/well of a 200 g/L substrate stock solution, i.e., compound (2) prepared in 50 mM phosphate buffer, pH 6.3. The plate was sealed with an AirPore seal (Qiagen) and the reaction left to proceed overnight on a titre-shaker, with shaking speed of #2.5 at room temperature.

[0409] Condition A has the following final reaction parameters: (a) 10 g/L substrate loading; (b) 19 g/L -ketoglutaric acid; (c) 21 g/L ascorbic acid; (d) 1.5 mM Mohr's salt; (e) 50 mM phosphate buffer, pH 6.3 (pH adjusted with KOH); (f) ambient temperature (20 C. to 25 C.); and (g) reaction time of about c.a. 24 h.

[0410] Following the overnight incubation, the plates were centrifuged at 4000 rpm for 5 mM at room temperature. The reaction samples were derivatized and quenched by aliquoting 10 L of the clear reaction mix into a 96 deep well plate containing 2304/well of 5% sodium bicarbonate (aq). A 160 L volume of 6 mg/mL of dansyl chloride in MeCN was added to each well, the plate heat sealed, and then quick spun to settle the reaction solution to the bottom of the well. The plate was then heated at 55 C. for at least 45 mM without shaking, and centrifuged at 4000 rpm for 10 min at room temperature. A 200 L volume of the derivatized solution was transferred into a 96 round bottom plate and submitted for HPLC analysis.

[0411] Condition B reactions were carried out as follows. Cells were grown in 96-well plates as described above and lysates prepared by dispensing 100 L of Lysis Buffer into each well. Lysis buffer was freshly prepared by dissolving 30 mg of lysozyme and 15 mg of PMBS in 30 mL of 50 mM phosphate buffer, pH 6.3, followed by 600 L volume of 10 mM Mohr's salt, freshly prepared in sterile water. The lysis plate was heat sealed and then shaken on a titre-plate shaker at Speed #8 for 2 h at room temperature. Subsequently, the plate was quick-spun to settle the lysate at the bottom of the plate. This 100 L crude lysate was to be used for the reaction.

[0412] The Condition B reactions in 200 L scale were carried out in 98 well plates. A premix stock solution was prepared by dissolving 1.33 g of -ketoglutaric acid and 1.47 g of L-ascorbic acid in 31.5 mL of 50 mM phosphate buffer, pH 6.3 (pH adjusted by using KOH). After mixing, the pH was adjusted to 6.3 with KOH. To the pH adjusted premix, 41.16 mg of Mohr's salt was added.

[0413] To each 100 L of crude lysates prepared above, 90 L of the premix stock solution was added into each well, followed immediately by 10 uL/well of a 200 g/L substrate stock solution, i.e., compound (2) prepared in 50 mM phosphate buffer, pH 6.3. The plate was sealed with an AirPore seal (Qiagen) and the reaction left to proceed overnight on a titre-shaker, with shaking speed of #2.5 at room temperature.

[0414] Condition B has the following final reaction parameters: (a) 10 g/L substrate loading; (b) 19 g/L -ketoglutaric acid; (c) 21 g/L ascorbic acid; (d) 1.5 mM Mohr's salt; (e) 50 mM phosphate buffer pH 6.3 (pH adjusted with KOH); (f) ambient temperature (20 C. to 25 C.); and (g) reaction time of c.a. 24 h.

[0415] Following the overnight incubation, the plates were centrifuged at 4000 rpm for 5 mM at room temperature. The reaction samples were derivatized and quenched by aliquoting 10 L of the clear reaction mix into a 96 deep well plate containing 230 L/well of 5% sodium bicarbonate (aq). A 160 L volume of 6 mg/mL of dansyl chloride in MeCN was added to each well, the plate heat sealed, and then quick spun to settle the reaction solution to the bottom of the well. The plate was then heated at 55 C. for at least 45 mM with shaking on an Infors HT Microtron with a shaking speed of 500 rpm. The plates were centrifuged at 4000 rpm for 10 mM at room temperature. A 200 L volume of the derivatized solution was transferred into a 96 round bottom plate and submitted for HPLC for analysis.

Example 5: Process for Conversion of Compound (2) to Compound (1) Using Shake Flask Powder (SFP) Preparations

[0416] A 200 L scale reaction using SFP enzyme powder was carried out as follows. A premix stock solution was freshly prepared by dissolving 1.05 g of -ketoglutaric acid, 420 mg of L-ascorbic acid, and 600 mg of substrate (2S)-piperidine-2-carboxylic acid in 10 mL of 50 mM phosphate buffer, pH 6.3 (pH adjusted by using KOH). After thoroughly mixing the solution, the pH was adjusted to 6.3 using KOH. To the pH adjusted premix solution, 45 mg of Mohr's salt was added.

[0417] A stock solution of enzyme was prepared by dissolving 20 mg of SFP enzyme powder into 2 mL of 50 mM phosphate buffer, pH 6.3. To initiate the reaction, 100 L of enzyme solution was added into a plate followed by 100 L of premix stock solution for a final reaction volume of 200 L. The plate was sealed with an AirPore seal (Qiagen) and the reaction allowed to proceed overnight (c.a., 24 h) with shaking on a titre-plate shaker (speed #2.5) at room temperature.

[0418] The SFP Assay condition (i.e., Condition C) has the following final parameters: (a) 5 g/L enzyme powder loading; (b) 30 g/L substrate loading; (c) 52.5 g/L -ketoglutaric acid; (d) 21 g/L L-ascorbic acid; (e) 2.25 mM Mohr's salt; (f) 50 mM potassium phosphate buffer, pH 6.3 (pH adjusted with KOH), (g) reaction temperature at ambient room temperature; and (h) reaction time of about c.a. 24 h. In some reactions, the reaction conditions further contained 1% (v/v) Y-30 antifoam (Dow Corning), and the reaction solution was sparged with 02 gas at 2 L/h.

[0419] The reactions were quenched with 400 L of 75% MeCN and 25% H.sub.2O. The plates were shaken for 10 mM at room temperature and centrifuged at 4000 rpm. Derivatization was carried out by transferring 20 L of quenched reaction to a 96 deep well plate containing 230 L/well of 5% sodium bicarbonate (aq). A 1504 aliquot of 21 mg/mL dansyl chloride in MeCN was added to each well. The plate was heat sealed and quick spun, and the plates incubated at 65 C. for at least 1 h with shaking on an Infors HT Microtron at a shaking speed of 500 rpm. The plates were then centrifuged at 4000 rpm for 10 mM at room temperature. A 200 L volume of the derivatized solution was transferred into a 96 round bottom plate and submitted for analysis by HPLC.

Example 6: Process for Conversion of Compound (2) to Compound (1) Using Downstream Process Powder (DSP) Preparations

[0420] Two reaction conditions were used for downstream process powder (DSP) preparations. The first reaction conditions, referred to as mini-DSP conditions (i.e., Condition D) were carried out on a 1 mL scale as follows. A premix stock solution was freshly prepared by dissolving 120 mg of (2S)-piperidine-2-carboxylic acid (i.e., L-pipecolic acid), 228 mg of -ketoglutaric acid and 252 mg of L-ascorbic acid in 11.88 mL of 50 mM phosphate buffer, pH 6.3. The pH of the premix solution was then adjusted to 6.3 using KOH. A 120 L volume of 150 mM Mohr's salt (in sterile water) was added to form the premix stock solution.

[0421] Reactions were run by weighing 20 mg of the DSP enzyme powder into a vial followed by 1 mL of premix stock solution. The solution was thoroughly mixed, and the vial left open overnight (24 h) at room temperature. The reaction solution was stirred at 1200 rpm during the course of the reaction.

[0422] The mini DSP reaction conditions have the following parameters: (a) 20 g/L substrate loading; (b) 34 g/L -ketoglutaric acid (1.5 equivalents of substrate); (c) 13.6 g/L ascorbic acid (0.5 equivalents of substrate); (d) 1.5 mM Mohr's salt; (e) 20 g/L protein of DSP enzyme preparation; (f) 50 mM phosphate buffer, pH 6.3 (pH adjusted with KOH); (f) ambient temperature; and (g) reaction time of 24 h. In some reactions, the reaction solution further contained 1% (v/v) of Y-30 antifoam (Dow Corning) and the reaction solution was sparged with O.sub.2 gas at 2 L/h during the course of the reaction.

[0423] To follow the course of the reaction, 10 L samples were taken and dissolved in 230 L of 5% sodium bicarbonate (aqueous). A 160 L volume of 6 mg/mL of dansyl chloride in MeCN was then added to the mixture, the tubes thoroughly mixed, and then heated, uncapped at 50 C. for 30 minutes. The samples were then centrifuged, and the clear supernatant analyzed by HPLC as described in Example 2.

[0424] The second reaction conditions, referred to as full DSP conditions, were carried out as follows. A premix stock solution was freshly prepared for 1 mL scale reactions by dissolving 240 mg of (2S)-piperidine-2-carboxylic acid (L-pipecolic acid), 228 mg of -ketoglutaric acid, and 252 mg of L-ascorbic acid in 11.88 mL of 50 mM phosphate buffer, pH 6.3. The pH of the premix solution was adjusted to 6.3 with KOH. A 120 L volume of 150 mM Mohr's salt (in sterile water) was added to form the premix stock solution.

[0425] Reactions were run by weighing 10 mg of the DSP enzyme powder and adding 1 mL of premix stock solution. After mixing, the vial was left open overnight (24 h) at room temperature. The reaction solution was stirred at 1200 rpm during the course of the reaction.

[0426] The full DSP reaction conditions has the following parameters; (a) 10 g/L substrate loading; (b) 38 g/L -ketoglutaric acid; (c) 21 g/L ascorbic acid; (d) 1.5 mM Mohr's salt; (e) 10 g/L DSP enzyme preparation; (f) 50 mM phosphate buffer, pH 6.3 (pH adjusted with KOH); (f) reaction temperature of 25 C.; and (g) reaction time of c.a. 24 h. In some reactions, the reaction solution contained 1% (v/v) Y-30 antifoam (Dow Corning), and the reaction solution was sparged with 02 gas at 2 L/h.

[0427] To follow the course of the reaction, 10 L samples were removed and mixed with 230 L of 5% sodium bicarbonate (aq). A 160 L volume of 6 mg/mL of dansyl chloride in MeCN was then added to the mixture. The tubes were thoroughly mixed and then heated, uncapped at 50 C. for 30 minutes. The samples were then centrifuged and the clear supernatant analyzed by HPLC as described in Example 2.

Example 7: Process for Conversion of Compounds of Formula (II) to Compounds of Formula (I) Using DSP Powders of Engineered Proline Hydroxylase Polypeptides

[0428] The ability of the engineered proline hydroxylases to recognize substrates other than proline or pipecolic acid were examined. The reaction conditions comprised (a) 20 g/L substrate loading; (b) 35 g/L -ketoglutaric acid; (c) 14 g/L ascorbic acid; (d) 1.5 mM Mohr's salt; (e) 10 g/L protein of DSP enzyme preparation of SEQ ID NO:108; (f) 50 mM phosphate buffer, pH 6.3 (pH adjusted with KOH); (f) reaction temperature of 25 C.; and (g) reaction time of 24 h. The negative controls used enzyme preparations obtained from cells transformed with expression vector that did not have a gene encoding a proline hydroxylase.

TABLE-US-00015 Neg Substrate Substrate Structure Control Rxn Product(s) Product Structure(s) L-pipecolic acid [00042] +++++ 2 regioisomers [00043] L-proline [00044] ++++ 1 isomer [00045] L-norvaline [00046] + ++ 2 isomers [00047] S-(1,2,3,4)- tetrahydro-3- isoquinoline carboxylic acid [00048] + ++ 3 major products [00049]

[0429] The reactions were quenched by diluting 2000-fold in 50:50 acetonitrile:H.sub.2O, and the reaction products analyzed by LC/MS/MS.

[0430] LC/MS/MS analysis for pipecolic acid, proline and norvaline was carried out under the following conditions:

TABLE-US-00016 Column ChiroBiotic TAG 250 4.6 mm, 5 m Mobile Phase Solution A: 0.1% formic acid Solution B: 0.1% formic acid in Acetonitrile A:B = 50:50 Postime 5.0 min MS conditions Source dependent parameters: CUR 30, IS 5500, TEM 590 C., GS1 60, GS2 60, DP30, EP10, CE 20 MRM: 130/84 (pipecolic acid RT 4.2 min), 146/100 and 146/82 (hydroxylated pipecolic acid 3.2 min and 3.7 min) MRM: 116/70 (proline RT 3.4 min), 132/86 and 132/68 (hydroxylproline RT 2.7 min) MRM: 118/72 (norvaline RT 2.7 min), 134/88, 134/74 and 134/70 (hydroxylated norvaline RT 2.6&2.7 min) Column Not controlled Temperature Injection 2 L Volume

[0431] The quenched reaction for tetrahydroisoquinoline carboxylic acid was subject to LC/MS/MS analysis under the following conditions:

TABLE-US-00017 Column Poroshell EC C18 100 4.6 mm, 2.7 m Mobile Phase Solution A: 0.5 mM perfluoroheptanoic acid Solution B: Acetonitrile Time Flow (min) (ml/min) A B 0-1.5 0.8 97 3 9 0.8 70 30 12 0.8 70 30 13 0.8 97 3 20 0.8 97 3 Postime 20 min MS conditions Source dependent parameters: CUR 30, IS 5500, TEM 600 C., GS1 60, GS2 60, DP30, EP10, CE 30 MRM: 194/148 and 194/146 (hydroxylated tetrahydroisoquinoline carboxylic acid RT 5.7 min, 7.26 min and 9.73 min) Column Not controlled Temperature Injection 2 L Volume

Example 8: Process for Conversion of L-Pipecolic Acid (Compound (2)) to (2S,5S)-5-Hydroxypiperidine-2-Carboxylic Acid (Compound (1)) Followed by Boc-Protection Step

[0432] Enzymatic Reaction:

[0433] A solution of L-pipecolic acid (15 g) dissolved in 138 ml of 50 mM potassium phosphate buffer, pH 6.3 was charged to a pre-mixed solution containing: (i) DSP preparation of the polypeptide of SEQ ID NO: 132 (5 g); (ii) Antifoam Y-30 emulsion (5 mL); (iii) Mohr's salt (1.08 g); (iv) -ketoglutaric acid (25.5 g); and (iv) ascorbic acid (10.2 g); all dissolved in 250 mL of 50 mM potassium phosphate buffer, pH 6.3. The resulting mixture was stirred and sparged with oxygen at a rate of 3 L/h and 25 C. The progress of the enzymatic reaction was monitored by HPLC using Method 2 of Example 3. The conversion of L-pipecolic acid to (2S,5S)-5-hydroxypiperidine-2-carboxylic acid followed a reaction course of 78% conversion at 25 h, 92% conversion at 45 h, and 94% conversion by 52 h, but did not reach higher conversion at 75h. The region purity of the (2S,5S)-5-hydroxypiperidine-2-carboxylic acid product after 52 h reaction was 6:1.

[0434] Boc-Protection:

[0435] Crude mixture from the enzymatic reaction was adjusted to pH 9.5 with KOH (50% w/w), heated to 60 C. for 1 h, and then cooled to room temperature. Thereafter, Celite filter-aid (15 g) was added with stirring (10 minutes) and the mixture was filtered through a 1 cm thick pad of Celite 545. The filter cake was washed with water (60 mL) and the filtrate charged with NaOH (38.5 mL at 10 M) and di-tert-butyl dicarbonate (Boc.sub.2-O) (42.2 g in 75 mL THF). Upon reaction completion (96% conversion in two days) the aqueous phase was washed twice with heptanes (2125 mL). The heptane washings were discarded and the aqueous phase was adjusted to pH 3.5 with 5 M HCl, and treated with NaCl (40 g). The aqueous phase was extracted with t-butyl methyl ether (TBME) (3125 mL), and the resulting organic phase extracts were combined, dried over MgSO.sub.4, filtered and concentrated to give crude (2S,5S)-1-(tert-butoxycarbonyl)-5-hydroxypiperidine-2-carboxylic acid.

[0436] Isolation:

[0437] A solution of the crude (2S,5S)-1-(tert-butyloxycarbonyl)-5-hydroxypyridine-2-carboxylic acid product (25 g) in TBME (250 mL) was agitated with a magnetic stirrer bar, insolubles were filtered, and rinsed with TBME (150 mL). The concentrated filtrate was dried under vacuum overnight and dissolved in isopropyl acetate (100 mL) and heptane (100 mL). The resulting mixture was heated at 80 C. for 20-25 minutes, insolubles were removed by hot filtration, filtrate was cooled and stirred for 24 h at room temperature. The solid was filtered and the cake washed with chilled (0 to 5 C.) heptanes-isopropyl acetate mixture (1:1 of 50 mL). Solid product was collected and dried under vacuum overnight to afford purified (2S,5S)-1-(tert-butyloxycarbonyl)-5-hydroxypyridine-2-carboxylic acid (8.6 g, 30% yield).

[0438] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

[0439] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).