Article pubs.acs.org/OPRD
Enzymatic Reduction of Adamantanones to Chiral Adamantanol Intermediates for the Synthesis of 11-β-Hydroxysteroid Dehydrogenase Inhibitors Ronald L. Hanson,*,† Steven L. Goldberg,† Zhiwei Guo,† Thomas P. Tully,† Animesh Goswami,† Xiang-Yang Ye,‡ Jeffrey A. Robl,‡ and Ramesh N. Patel† †
Early Phase Chemical Development, Research and Development, Bristol-Myers Squibb, One Squibb Drive, New Brunswick, New Jersey 08903, United States ‡ Department of Chemistry, Research and Development, Bristol-Myers Squibb, P.O. Box 5400, Princeton, New Jersey 08543, United States ABSTRACT: An enzymatic reduction process was developed to convert the ketone 2-(6-oxo-2-phenyladamantan-2-yl)acetic acid to the chiral alcohol 2-((2S, 6S)-6-hydroxy-2-phenyladamantan-2-yl)acetic acid and to convert the fluoro ketone 2-(2-(4fluorophenyl)-6-oxoadamantan-2-yl)acetic acid to the chiral alcohol 2-((2S,6S)-2-(4-fluorophenyl)-6-hydroxyadamantan-2yl)acetic acid. These chiral adamantanols are intermediates for the 11-β-hydroxysteroid dehydrogenase inhibitors 2-((2S,6S)-6hydroxy-2-phenyladamantan-2-yl)-1-(3-hydroxyazetidin-1-yl)ethanone and 2-((2S,6S)-2-(4-fluorophenyl)-6-hydroxyadamantan2-yl)-1-(3-hydroxyazetidin-1-yl)ethanone, respectively. Initial batches of both intermediates were prepared with a commercial ketoreductase giving yields near 100% with 96% ee. A more selective ketoreductase was purified 1800-fold from Candida utilis ATCC 42181 and then cloned and expressed in Escherichia coli. The reaction requires the cofactor NADPH which was regenerated during initial batches using a commercial glucose dehydrogenase. In later work a glucose dehydrogenase from Gluconobacter oxydans was cloned and expressed in the same E. coli strain together with the ketoreductase. To allow easy storage and shipment of the two enzymes, the E. coli cell paste was lyophilized to produce a stable form of the enzymes.
■
INTRODUCTION 11-β-Hydroxysteroid dehydrogenase type 1 (11-β-hsd1) catalyzes the reduction of inactive cortisone to the active form, cortisol, in human tissues that are targets for glucocorticoids. Inhibitors of 11-β-hsd1 are under development as possible therapy for type 2 diabetes and metabolic syndrome and are also under investigation as possible therapy for several other medical disorders.1−3 Compounds 7 and 8 (Scheme 1) are two 11-β-hsd1 inhibitors synthesized and investigated at Bristol-Myers Squibb.4 The synthesis of both 7 and 8 required the introduction of the adamantanol hydroxyl group with the desired stereochemistry. The syntheses of chiral alcohols 7 and 8 by conventional chemical processes are difficult. Ketoreductases are useful enzymes for preparation of chiral secondary alcohols from prochiral ketones.5 Many ketoreductases are commercially available, and this activity is very common in microbial strains.6 This paper describes screening to identify ketoreductases with the desired stereospecificity, initial preparation of the chiral alcohols with a commercial ketoreductase, and purification, cloning and expression of a more effective ketoreductase from Candida utilis ATCC 42181. Coexpression of a glucose dehydrogenase for NADPH regeneration and lyophilization of the E. coli cell paste to produce a stable form of the two enzymes is also described.
■
collection, and 118 microbial strains (mainly yeasts) were screened for the reduction of ketone 2 to alcohol 4. Enzymes and yeasts able to produce either enantiomer were identified. The ketoreductases giving the highest ee in the initial screen were tested again at two enzyme concentrations (Table 1). From the initial screen Codexis KRED-102 was selected as the most effective commercially available ketoreductase. KRED-102 gave the best ee (95−96%) and fastest conversion (97% conversion of 2 mg/mL ketone by 0.1 mg/mL ketoreductase in 18 h). The reaction with KRED-102 was tried at pH 6, 7, and 8 and 15 °C as well as 28 °C. The ee was not affected by the variations in conditions, but the reaction rate was influenced by pH (rate at pH 8 > pH 7 > pH 6), and the rate was decreased at 15 °C compared to 28 °C as expected. When combined with glucose dehydrogenase for regeneration of NADPH, KRED102 gave both the fluoro-product 4 from reduction of 2 and the fluoro-product 3 from reduction of 1 with 96% ee and close to 100% solution yield. Preparation of Alcohol 3 and Alcohol 4 Using KRED102. Alcohol 3 was prepared using 40 mg/mL ketone 1 and 4 mg/mL KRED-102 with glucose and Amano glucose dehydrogenase for NADPH regeneration. The product alcohol was isolated from the acidified reaction mixture by extraction with ethyl acetate and precipitation from the concentrated extract with heptane. Two recrystallizations from acetone/water improved the ee from 96.2% to 99.1% with an isolated yield
RESULTS AND DISCUSSION
Screening. A total of 101 commercially available ketoreductases, six recombinant ketoreductases from our internal © 2014 American Chemical Society
Received: June 26, 2014 Published: July 25, 2014 960
dx.doi.org/10.1021/op5002098 | Org. Process Res. Dev. 2014, 18, 960−968
Organic Process Research & Development
Article
Scheme 1. Scheme for the reduction of adamantanone acid and amide intermediates
Therefore, some other strains were investigated as sources of a soluble enzyme for purification to determine sufficient amino sequences to design the primers needed for cloning. A soluble extract of Candida utilis ATCC 42181 after centrifugation at 108 800× g gave alcohol 4 with 96% ee. After chromatography on Q sepharose the main peak eluted at 0.2 M NaCl and also reduced ketone 2 to alcohol 4 with 96% ee. The ketoreductase was purified 1800-fold from a centrifuged extract of Candida utilis ATCC 42181 cells by sequential chromatography on Q Sepharose, Phenyl Sepharose, Sephacryl S-200, and UNO Q (Table 2) to give a single band on an SDS
Table 1. Conversion of 2 mg/mL ketone 2 to alcohol 4 by commercial ketoreductases in 18 h enzyme
mg/mL
conversion
ee of 4
KRED-102
0.1 1 0.1 1 0.1 1 0.1 1 0.1 1
97.5 96.7 3.9 32.4 15.8 39.1 5.1 43.8 1.4 12.8
95.8 95.9 80.4 87.0 88.3 88.8 85.6 89.9 83.8 84.2
KRED-106 KRED-125 KRED-A1K KRED-A1Y
Table 2. Purification of ketoreductase from Candida utilis ATCC 42181
(corrected for the input purity) of 86%. Alcohol 4 was prepared from ketone 2 with 98% isolated yield, 97.1% ee using a similar procedure. Purification of Ketoreductase. Although KRED-102 was sufficient for preparation of initial supplies of the two adamantanols, the selectivity of the enzyme was low. The ee of the alcohol obtained after enzymatic reduction was improved by crystallization which, however, resulted in some loss of yield. Further screening of microbial strains and previously cloned ketoreductases was carried out to try to find a more enantioselective enzyme. Of the yeasts screened, Hansenula fabianii ATCC 58045 gave the most conversion and highest ee for alcohol 4 in the initial screening experiments. When a microfluidized, centrifuged cell extract was passed through a 1 mL column of Q-sepharose, the fraction which did not bind to the column gave an ee of 98%, whereas a fraction eluting with 0.2 M NaCl gave an ee of 70%. However, the enzyme giving the high ee was found to form an insoluble pellet when centrifuged at 108 800× g. Attempts were made to solubilize the enzyme by treatment with Y-PER (yeast protein extraction reagent), BPER (bacteria protein extraction reagent), lysing enzymes from Rhizoctonia solani, or by vortexing with glass beads. In all cases the only soluble enzyme obtained gave a low ee (74−83%).
step extract Q Sepharose phenyl Sepharose Sephacryl S-200 UnoQ
total protein (mg)
total activity (U)
specific activity (U/mg)
purification (fold)
2080 89.4 2.74
1.743 0.733 0.403
0.0008 0.0082 0.147
1 10.2 184
0.575
0.234
0.406
507
0.016
0.024
1.455
1819
gel with an estimated molecular weight of 28 000. A PVDF blot of the 28 kDa band was submitted for determination of the Nterminal sequence and a gel slice containing this band was used for determination of internal sequences. The purified enzyme reduced ketone 1 to alcohol 3 with >99% ee. Sequences of three internal peptides from a trypsin digest of the ketoreductase from Candida utilis ATCC 42181 were obtained from the Keck sequencing facility at Yale University. No N-terminal sequences could be obtained suggesting that the N-terminus is blocked. The internal sequences were: ATALEYLEASN VHVAQLDITQAEK NSGDIVNL. A BLAST search found all three sequences in a short-chain alcohol dehydrogenase with the sequence shown in Figure 1 961
dx.doi.org/10.1021/op5002098 | Org. Process Res. Dev. 2014, 18, 960−968
Organic Process Research & Development
Article
Figure 1. Amino acid sequence of Saccharomyces cerevisiae dehydrogenase/ketoreductase.
from Saccharomyces cereviseae as well as homologies to dehydrogenases from other organsisms. The S. cerevisiae dehydrogenase/reductase has been characterized7 and used for reduction of a few ketones to chiral alcohols.8,9 Cloning and Expression of C. utilis Ketoreductase (CUKR) Gene. The CUKR protein is produced at low levels in C. utilis. Moreover, yeasts often contain several ketoreductases, some of which can decrease the enantioselectivity of the desired reaction. Therefore, for subsequent bioconversion studies the production of CUKR was carried out in the bacterium Escherichia coli. This organism has a high growth rate, a low level of background ketoreductases, and is very amenable to genetic and fermentative manipulation to achieve high specific productivity. The gene sequence encoding the CUKR protein was amplified from the C. utilis genome using the polymerase chain reaction (PCR). Sequencing of the CUKR gene confirmed that its amino acid sequence was identical to a S. cerevisiae dehydrogenase with the exception of a single amino acid change (E instead of Q at amino acid position 62). The CUKR gene is 801 base pairs in length and encodes a protein of 31 500 Da. The CUKR gene was transferred to a plasmid (pBMS2004) capable of replicating in E. coli. Production of the recombinant CUKR protein is dependent upon addition of the inducer isopropyl-β-D-1-thiogalactopyranoside (IPTG) to E. coli strain BL21(pBMS2004-CUKR). Although CUKR activity was present in the cells, only very low expression of the ketoreductase was obtained. The first CUKR extracts from cells induced with 0.05 or 1 mM IPTG had specific activities of 2.2 and 3.7 U/g of wet cells, respectively, using an HPLC assay for product formation. Subsequently, both whole recombinant cells as well as cell extracts were assayed for conversion of ketone 1 to alcohol 3 by following the decrease in NADPH in a spectrophotometer. Cells gave alcohol 3 with 97% ee and extract gave alcohol 3 approaching 100% ee. However, there is some endogenous reduction activity from the E. coli expression host which gives nearly racemic alcohol, and expression levels needed to be increased to make this background reduction insignificant. Low expression levels in recombinant E. coli are due to a variety of factors. Two common reasons are (1) secondary structure in the mRNA, which prevents ribosomes from initiating translation, and (2) the presence of rare codons in the mRNA, for which there is a limited supply of the appropriately charged tRNAs. Therefore, we attempted to improve expression by (1) adding a translational enhancer10 (TE) to the gene (i.e., a short DNA sequence at the 5′ end of the gene which is less susceptible to forming secondary structure), and (2) transforming this plasmid into E. coli BL21 Codon Plus-RIPL, which contains a plasmid encoding additional rare arginine tRNAs (corresponding to one of the codons used by the CUKR gene). For further development, a synthetic CUKR gene that eliminated any rare codons was used
to obviate the need for supplemental tRNA genes. The increases in activity seen by use of the translational enhancer, extra tRNA genes, and codon optimization are shown in Table 3. Finally, the optimized ketoreductase gene was ligated as a Table 3. Improvement of expression of the CUKR gene E. coli strain/protein expressed BL21/CUKR[TE] BL21/CUKR[TE] BL21 Codon Plus RIPL/ CUKR[TE] BL21 Codon Plus RIPL/ CUKR[TE] BL21/CUKR[TE]-CO BL21/GoGDH + CUKR[TE]CO
IPTG (mM)
KR (U/g cells)
0.05 1.00 0.05
11.16 13.95 40.02
1.00
86.34
0.05 0.05
166.67 93.61
GDH (U/g cells)
29623
transcriptional fusion to our expression plasmid that already possessed the Gluconobacter oxydans glucose dehydrogenase gene11 to provide an endogeneous means of recycling cofactor. Expression of both enzymes in a single cell was optimized by reducing IPTG concentration from 1.0 to 0.05 mM (Figure 2). Production of E. coli Expressing Ketoreductase and Glucose Dehydrogenase. Strain SC16620 [E. coli BL21 (pBMS2004-GoGDH-CUKR[TE]-CO)] was used to produce
Figure 2. Optimization of expression of ketoreductase and glucose dehydrogenase. Lane 1. SeeBlue plus 2 protein standard, 2 BL21(pBMS2004) + 50 μM IPTG, 3. BL21(pBMS2004-GoGDH) + 50 μM IPTG, 4. BL21(pBMS2004-GoGDH) + 1 mM IPTG, 5. BL21(pBMS2004-GoGDH−CUKR[TE]-CO) + 50 μM IPTG, 6. BL21(pBMS2004-GoGDH−CUKR[TE]-CO) + 1 mM IPTG. 962
dx.doi.org/10.1021/op5002098 | Org. Process Res. Dev. 2014, 18, 960−968
Organic Process Research & Development
Article
7 with this substrate was only 92.2%. The C. utilis ketoreductase reduced 6 to 8 with 83% ee. KRED-102 from Codexis converted 5 and 6 to 7 and 8, respectively, with 99% ee. A sample of 400 mg of ketone 5 (10 mg/mL) was converted to alcohol 7 (∼100% yield, 100% ee) using 40 mg of KRED-102. The low solubility of the amide substrates makes reduction at this step less attractive than reduction of the acids 1 and 2. Lyophilization of Cells Containing Ketoreductase and Glucose Dehydrogenase. Extracts of E. coli prepared from frozen cell paste containing cloned ketoreductase were used for the batches of alcohols 3 and 4 described to this point. Both frozen cells and cell extracts were not suitable for long-term storage and shipment. A stable dry form was needed for storage and shipment. E. coli SC16220 wet cell paste (3.57 kg) expressing both Candida utilis ketoreductase (46.9 U/g) and glucose dehydrogenase (20592 U/g) from Gluconobacter oxydans was lyophilized and ground with a mortar and pestle to give 1.03 kg of dried cells containing 164.7 U/g ketoreductase and 72684 U/g glucose dehydrogenase. After lyophilization 100% of both enzyme activities were recovered (Table 4). In a use test, the dried cells converted 400 mg
Gluconobacter oxydans glucose dehydrogenase and Candida utilis ketoreductase. A 380 L tank containing 250 L of medium was induced with 0.1 mM IPTG 4.5 h after inoculation then harvested at 27.5 h to produce 14.982 kg cell paste containing ketoreductase activity of 47 U/g cells and glucose dehydrogenase activity of 20 592 U/g cells. Reduction Process Using Cloned Ketoreductase and Glucose Dehydrogenase. For the development of an efficient enzymatic ketone reduction, it is convenient to use enzyme assays to determine the pH optimum and the Michaelis constant for cofactor and then run the reaction near the optimum pH with cofactor in excess of the Michaelis constant and at the highest possible ketone concentration, unless there are other overriding factors. The pH optimum was
