ighly Stereoselective Reductions of α-alkyl-1,3-diketones and α- Alkyl-β-keto esters Catalyzed by Isolated NADP-dependent Ketoreductases Dimitris Kalaitzakis, a David J. Rozzell b, Spiros Kambourakis *b and Ioulia Smonou* a a Department of Chemistry, University of Crete, Iraklio 7149, Crete, Greece and b BioCatalytics Inc, 129 North ill Ave, Suite 13, Pasadena, CA 9116, USA Supporting Information Experimental Section General Considerations a-alkyl-1,3-diones 1-6 were prepared from commercially available 2,4-pentadione (acetylacetone) by alkylation with the proper alkyl halide. a-alkyl-β-keto esters 7, 8, were prepared from commercially available ethyl acetoacetate by alkylation with the proper alkyl halide. Racemic keto alcohols were prepared from pentan-2-one by aldol condensation with the corresponding aldehyde. Flash chromatography was carried out with 6 Å silica gel according to the procedure of Still (Still, W. C.; Kahn, M.; Mitra, A. J. rg. Chem. 1978, 14, 2923-2925.). The progress of the enzymatic reactions and the selectivities were determined by gas chromatography (P589II gas chromatograph equipped with an FID detector; column: 3m x.25mm x.25µm chiral capillary column, 2% permethylated cyclodextrin). MS were taken on a GC-MS (Simatzu GCMS-QP55 equipped with a S1
SPB-5 column and CI mass detector). 1 NMR and 13 C NMR spectra were recorded on a 3 Mz Bruker spectrometers in CDCl 3 solutions, by using Me 4 Si as internal standard. Chemical shifts are reported in ppm downfield from Me 4 Si. Yields refer to isolated and spectroscopically pure materials. Enzymatic Reductions Twenty different ketoreductases (KRED-11-12; BioCatalytics, Inc. Pasadena, CA USA) were screened to determine the best enzymes for the selective reduction of substrates 1-6. 3-methyl-2,4-pentanedione was used as the standard substrate. In addition to the ketoreductases, both NADP and glucose dehydrogenase (GLD) are products available from BioCatalytics. The screening was based in small-scale reactions (1 ml) where each substrate (25 mm) was mixed with NADP (2.5 mm, 2 mg), each ketoreductase (2 mg/ml), glucose (1 mm, 18 mg), glucose dehydrogenase (GLD, 2 mg/ml) for cofactor recycling, NaCl (1 mm, 6 mg) and sodium phosphate buffer (1 ml, 2 mm, p 6.9). The reactions were incubated at 37 ºC and reaction aliquots were taken every one hour and after extraction with ethyl acetate they were analyzed by GC chromatography. All the small-scale enzymatic reductions were accomplished according to the above screening reactions except for 3-methyl-2,4-pentanedione and 3-methyl-2,4-hexanedione which were reduced in p 6.5 and the reactions were completed in 24 hours. Larger-scale enzymatic reductions were prepared according to the following protocol. A phosphate-buffered solution (1 ml, p 6.9, 2 mm) containing 5 mm substrate, NaCl (2 mm), glucose (12 mm), NADP (.5 mm), glucose S2
dehydrogenase (5 mg) and the appropriate ketoreductase (5 mg) was stirred at 37 ºC for 24 hours, until GC analysis of crude extracts showed complete reaction. Periodically the p was readjusted to 6.9 with Na (2 M). Product was isolated by extracting the crude reaction mixture with EtAc (7 ml x 2). Sometimes centrifugation (6 rpm, 1 min) was required for the aqueous and the organic layers to separate as clear solutions. The combined organic layers were then extracted with saturated NaCl solution, dried over MgS 4 and evaporated to dryness. Pure, optically active alcohols were obtained in 9% to 91 % yield. The products were analyzed by NMR spectroscopy. Their optical purity was determined by chiral GC chromatography using 2% permethylated cyclodextrin column. According to this protocol diketone 1 (57 mg, 5 mm) was reduced with KRED12. Isolated yield 513 mg, 9%. 1 NMR: Ketoester 7 (72 mg, 5 mm) was reduced also with KRED12. Isolated yield 656 mg, 91%. Preparation of MPA-esters To a solution of the corresponding alcohol (.1 mmol) in dry C 2 Cl 2 were added 1.1 equiv. of DCC (.11 mmol) and 1.1 eqiv. f the corresponding (R) or (S) MPA (.11 mmol) and the reaction mixture was stirred at o C for 4-6 hr. After completion of the reaction the produced urea was filtered, the filtrate was evaporated and then chromatographed with 5/1 ex/etac and the produced corresponding MPA-ester was isolated. S3
Analytical and Spectral Data 1) GC analysis of racemic keto alcohol-1 with chiral capillary column, 2% permethylated cyclodextrin 3 2 44.612 45.799 47.272 65.598 Sig. 1 in C:\PCEM\...\KALAITZ\123.D 1 4 5 6 7 Time (min.) S4
2) 1 NMR (3 Mz, CDCl 3 ) of the racemic keto alcohol-1 6 5 4 3 2 1 7. 6. 5. 4. 3. 2. 1. S5
3) GC analysis of the product, after enzymatic reduction of diketone-1 with KRED12 (chiral capillary column, 2% permethylated cyclodextrin) 5 4 3 2 44.214 12 65.92 Sig. 1 in C:\PCEM\...\KALAITZ\122.D 1 4 5 6 7 Time (min.) S6
4) 1 NMR (3 Mz, CDCl 3 ) of the produced keto alcohol-1 after enzymatic reduction with KRED12 5 4 12 3 2 1 7. 6. 5. 4. 3. 2. 1. S7
5) GC analysis of racemic keto alcohol-2 with chiral capillary column, 2% permethylated cyclodextrin 12 11 Sig. 1 in C:\PCEM\...\KALAITZ\144.D 1 39.131 4.754 41.683 42.584 9 3 4 5 6 Time (min.) S8
6) 1 NMR (3 Mz, CDCl 3 ) of the racemic keto alcohol-2 4 3 2 1 7. 6. 5. 4. 3. 2. 1.. S9
7) GC analysis of the product, after enzymatic reduction of diketone-2 with KRED12 (chiral capillary column, 2% permethylated cyclodextrin) 17 16 15 14 13 12 12 39.8 Sig. 1 in C:\PCEM\...\KALAITZ\145.D 11 1 9 8 28 3 32 34 36 38 4 42 44 Time (min.) S1
8) 1 NMR (3 Mz, CDCl 3 ) of the produced keto alcohol-2 after enzymatic reduction with KRED12 5 4 12 3 2 1 7. 6. 5. 4. 3. 2. 1. S11
9) GC analysis of the product, after enzymatic reduction of diketone-2 with KRED17 (chiral capillary column, 2% permethylated cyclodextrin) 4 3 2 17 41.67 42.58 Sig. 1 in C:\PCEM\...\KALAITZ\1262.D 1 3 35 4 45 Time (min.) S12
1) GC analysis of the product, after enzymatic reduction of diketone-2 with KRED118 (chiral capillary column, 2% permethylated cyclodextrin) 7 6 5 4 118 4.98 Sig. 1 in C:\PCEM\...\KALAITZ\626.D 3 2 1 38.513 32 34 36 38 4 42 44 46 Time (min.) S13
11) 1 NMR (3 Mz, CDCl 3 ) of the produced keto alcohol-2 after enzymatic reduction with KRED118 4 118 3 2 1 7. 6. 5. 4. 3. 2. 1. S14
12) GC analysis of racemic keto alcohol-3 (chiral capillary column, 2% permethylated cyclodextrin) 4 3 2 25.738 26.383 28.654 29.51 Sig. 1 in C:\PCEM\...\KALAITZ\452.D 1 2 25 3 35 4 Time (min.) S15
13) 1 NMR (3 Mz, CDCl 3 ) of racemic keto alcohol-3 4 3 2 1 7. 6. 5. 4. 3. 2. 1. S16
14) GC analysis of the product, after enzymatic reduction of diketone-3 with KRED12 (chiral capillary column, 2% permethylated cyclodextrin) 2 18 16 14 12 25.593 Sig. 1 in C:\PCEM\...\KALAITZ\451.D 12 1 8 2 22 24 26 28 3 Time (min.) S17
15) 1 NMR (3 Mz, CDCl 3 ) of the produced keto alcohol-3 after enzymatic reduction with KRED12 35 3 25 2 12 15 1 5 7. 6. 5. 4. 3. 2. 1. S18
16) GC analysis of the product, after enzymatic reduction of diketone-3 with KRED114 (chiral capillary column, 2% permethylated cyclodextrin) 1.6e4 1.4e4 1.2e4 1.e4 8 6 4 2 114 24.53 27.665 Sig. 1 in C:\PCEM\...\KALAITZ\125.D 18 2 22 24 26 28 3 Time (min.) S19
17) GC analysis of racemic keto alcohol-4 (chiral capillary column, 2% permethylated cyclodextrin) 7 6 5 4 28.657 29.561 Sig. 1 in C:\PCEM\...\KALAITZ\1167.D 3 26.317 27.65 2 1 2 22 24 26 28 3 Time (min.) S2
18) 1 NMR (3 Mz, CDCl 3 ) of racemic keto alcohol-4 3 25 2 15 1 5 7. 6. 5. 4. 3. 2. 1. S21
19) GC analysis of the product, after enzymatic reduction of diketone-4 with KRED12 (chiral capillary column, 2% permethylated cyclodextrin) 14 13 12 11 12 28.869 Sig. 1 in C:\PCEM\...\KALAITZ\416.D 1 9 8 2 22 24 26 28 3 32 34 Time (min.) S22
2) GC analysis of the product, after enzymatic reduction of diketone-4 with KRED17 (chiral capillary column, 2% permethylated cyclodextrin) 4 3 17 29.731 Sig. 1 in C:\PCEM\...\KALAITZ\1182.D 2 3.891 1 2 22 24 26 28 3 32 Time (min.) S23
21) 1 NMR (3 Mz, CDCl 3 ) of the produced keto alcohol-4 after enzymatic reduction with KRED17 6 5 17 4 3 2 1 7. 6. 5. 4. 3. 2. 1. S24
22) GC analysis of the product, after enzymatic reduction of diketone-4 with KRED12 (chiral capillary column, 2% permethylated cyclodextrin) 2.e4 1.8e4 1.6e4 1.4e4 1.2e4 1.e4 8 12 3.264 Sig. 1 in C:\PCEM\...\KALAITZ\124.D 6 4 2 27.9 24 26 28 3 32 Time (min.) S25
23) 1 NMR (3 Mz, CDCl 3 ) of the produced keto alcohol-4 after enzymatic reduction with KRED12 4 3 12 2 1 7. 6. 5. 4. 3. 2. 1. S26
24) GC analysis of racemic keto alcohol-5 (chiral capillary column, 2% permethylated cyclodextrin) 15 14 13 12 11 96.636 98.575 11.96 13.19 Sig. 1 in C:\PCEM\...\KALAITZ\223.D 1 9 8 9 1 11 Time (min.) S27
25) 1 NMR (3 Mz, CDCl 3 ) of racemic keto alcohol-5 3 25 2 15 1 5 7. 6. 5. 4. 3. 2. 1. S28
26) GC analysis of the product, after enzymatic reduction of diketone-5 with KRED12 (chiral capillary column, 2% permethylated cyclodextrin) 2 18 16 14 96.513 12 Sig. 1 in C:\PCEM\...\KALAITZ\228.D 12 1 8 9 1 11 12 Time (min.) S29
27) 1 NMR (3 Mz, CDCl 3 ) of the produced keto alcohol-5 after enzymatic reduction with KRED12 5 4 12 3 2 1 7. 6. 5. 4. 3. 2. 1. S3
28) GC analysis of the product, after enzymatic reduction of diketone-5 with KRED17 (chiral capillary column, 2% permethylated cyclodextrin) 16 15 14 13 12 11 17 97.758 Sig. 1 in C:\PCEM\...\KALAITZ\735.D 1 9 12.13 8 85 9 95 1 Time (min.) S31
29) 1 NMR (3 Mz, CDCl 3 ) of the produced keto alcohol-5 after enzymatic reduction with KRED17 35 3 17 25 2 15 1 5 7. 6. 5. 4. 3. 2. 1. S32
3) GC analysis of the product, after enzymatic reduction of diketone-5 with KRED119 (chiral capillary column, 2% permethylated cyclodextrin) 3 25 2 119 98.475 Sig. 1 in C:\PCEM\...\KALAITZ\1199.D 15 94.219 1 8 85 9 95 1 15 Time (min.) S33
31) 1 NMR (5 Mz, CDCl 3 ) of the produced keto alcohol-5 after enzymatic reduction with KRED119 1 119 5 7. 6. 5. 4. 3. 2. 1. S34
32) GC analysis of racemic keto alcohol-6 (chiral capillary column, 2% permethylated cyclodextrin) 18 16 14 12 1 98 96 34.251 35.178 36.218 36.995 Sig. 1 in C:\PCEM\...\KALAITZ\161.D 94 92 28 3 32 34 36 38 4 Time (min.) S35
33) 1 NMR (3 Mz, CDCl 3 ) of racemic keto alcohol-6 1 5 7. 6. 5. 4. 3. 2. 1. S36
34) GC analysis of the product, after enzymatic reduction of diketone-6 with KRED12 (chiral capillary column, 2% permethylated cyclodextrin) 14 13 12 11 1 34.221 12 Sig. 1 in C:\PCEM\...\KALAITZ\166.D 9 28 3 32 34 36 38 4 42 Time (min.) S37
35) 1 NMR (3 Mz, CDCl 3 ) of the produced keto alcohol-6 after enzymatic reduction with KRED12 6 5 4 12 3 2 1 7. 6. 5. 4. 3. 2. 1. S38
36) GC analysis of racemic hydroxy ester-7 (chiral capillary column, 2% permethylated cyclodextrin) 3 2 29.8 3.492 31.9 33.61 Sig. 1 in C:\PCEM\...\KALAITZ\316.D 1 2 25 3 35 4 Time (min.) S39
37) 1 NMR (3 Mz, CDCl 3 ) of racemic hydroxy ester-7 25 2 15 1 5 7. 6. 5. 4. 3. 2. 1. S4
38) GC analysis of the product, after enzymatic reduction of keto ester-7 with KRED12 (chiral capillary column, 2% permethylated cyclodextrin) 26 24 22 2 18 16 29.84 12 Sig. 1 in C:\PCEM\...\KALAITZ\322.D 14 12 1 8 2 25 3 35 4 Time (min.) S41
39) 1 NMR (3 Mz, CDCl 3 ) of the produced hydroxy ester-7 after enzymatic reduction with KRED12 4 3 12 2 1 7. 6. 5. 4. 3. 2. 1. S42
4) GC analysis of the product, after enzymatic reduction of keto ester-7 with KRED17 (chiral capillary column, 2% permethylated cyclodextrin) 16 15 14 13 12 11 31.186 17 34.255 Sig. 1 in C:\PCEM\...\KALAITZ\338.D 1 9 26 28 3 32 34 36 Time (min.) S43
41) 1 NMR (3 Mz, CDCl 3 ) of the produced hydroxy ester-7 after enzymatic reduction with KRED17 25 2 17 15 1 5 7. 6. 5. 4. 3. 2. 1. S44
42) GC analysis of racemic hydroxy ester-78(chiral capillary column, 2% permethylated cyclodextrin) 28 26 24 22 2 18 16 14 63.748 65.6 72.6 75.381 Sig. 1 in C:\PCEM\...\KALAITZ\326.D 12 1 8 6 55 6 65 7 75 8 Time (min.) S45
43) 1 NMR (3 Mz, CDCl 3 ) of racemic hydroxy ester-8 35 3 25 2 15 1 5-5 7. 6. 5. 4. 3. 2. 1. S46
44) GC analysis of the product, after enzymatic reduction of keto ester-8 with KRED12 (chiral capillary column, 2% permethylated cyclodextrin) 2 18 16 14 64.19 12 Sig. 1 in C:\PCEM\...\KALAITZ\33.D 12 1 8 5 6 7 8 Time (min.) S47
45) 1 NMR (3 Mz, CDCl 3 ) of the produced hydroxy ester-8 after enzymatic reduction with KRED12 3 25 12 2 15 1 5 7. 6. 5. 4. 3. 2. 1. S48
46) GC analysis of the product, after enzymatic reduction of keto ester-8 with KRED116 (chiral capillary column, 2% permethylated cyclodextrin) 2 18 16 14 12 1 63.366 116 71.414 Sig. 1 in C:\PCEM\...\KALAITZ\58.D 8 6 6 65 7 75 Time (min.) S49
47) 1 NMR (5 Mz, CDCl 3 ) of the produced hydroxy ester-8 after enzymatic reduction with KRED12 25 2 116 15 1 5 7. 6. 5. 4. 3. 2. 1. S5
Determination of absolute configuration of hydroxy compounds* MPA R 1 Me R 2 δ RS < δ RS > R 2 R 1 S Me Me R 1 Me R R 2 1 R R 2 2 R 1 or R2 R 1 Syn Me R 1 Me R R R 2 2 2 R 1 R 1 or R2 R 1 Anti J vic (anti) > J vic (syn) *Seco, J. M.; Quinoa, E.; Riguera, R. Chem. Rev. 24, 14, 17-117. Stiles, M.; Winkler, R. R.; Chang, Y.-L.; Traynor, L. J. Am. Chem. Soc.1964, 86, 3337-3342. ouse,..; Crumrine, D. S.; Teranishi, A. Y.; lmstead,. D. J. Am. Chem. Soc.1973, 95, 331-3324. S51
48) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of keto alcohol-1 produced after enzymatic reduction with KRED12 2.728 2.74 2.684 2.661 2.638 2.17 1.65 1.44 1.22 4 3 From 12 Me C C (R) 2 1 7. 6. 5. 4. 3. 2. 1. S52
49) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of keto alcohol-1 produced after enzymatic reduction with KRED12 2.6 2.576 2.554 2.532 2.59 1.853 1.22 1.181.867.844 3 25 From 12 Me C C (S) 2 15 1 5 1. 5.. S53
5) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of keto alcohol-2 produced after enzymatic reduction with KRED12 5 From 12 Me C C (R) 4 3 2 1 7. 6. 5. 4. 3. 2. 1. 2.7 2.686 2.677 2.669 2.663 2.655 2.647 2.633 2.97 1.691 1.686 1.666 1.66 1.642 1.635 1.62 1.611 1.595 1.589 1.571 1.565 1.54 1.525 1.495 1.48 1.47 1.455 1.444 1.43 1.423 1.49 1.398 1.384 1.374 1.36 1.37 1.15.89.784.759 6 5 From 12 Me C C (R) 4 3 2 1 2.5 2. S54 1.5 1..5
51) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of keto alcohol-2 produced after enzymatic reduction with KRED12 5 4 From 12 Me C C (S) 3 2 1 7. 6. 5. 4. 3. 2. 1. 2.565 2.551 2.541 2.535 2.528 2.521 2.511 2.497 1.856 1.469 1.444 1.419 1.414 1.398 1.389 1.373 1.367 1.343 1.318 1.31 1.262 1.247 1.236 1.222 1.211 1.197 1.19 1.166 1.145.665.64.615 5 4 From 12 Me C C (S) 3 2 1 2.5 2. S55 1.5 1..5
52) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of keto alcohol-2 produced after enzymatic reduction with KRED118 35 3 From 118 Me C C (R) 25 2 15 1 5-5 7. 6. 5. 4. 3. 2. 1. 2.63 2.587 2.571 2.561 2.555 2.545 2.529 2.43 1.645 1.639 1.621 1.613 1.597 1.589 1.568 1.55 1.543 1.519 1.511 1.495 1.486 1.471 1.446 1.44 1.425 1.416 1.4 1.78 1.57.842.817.792 3 25 2 From 118 Me C C (R) 15 1 5 2.5 2. S56 1.5 1.
53) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of keto alcohol-2 produced after enzymatic reduction with KRED118 3 25 From 118 Me C C (S) 2 15 1 5 7. 6. 5. 4. 3. 2. 1. 2.484 2.469 2.458 2.452 2.442 2.437 2.426 2.41 1.73 1.465 1.457 1.441 1.433 1.419 1.49 1.395 1.387 1.371 1.362 1.347 1.331 1.322 1.36 1.282 1.276 1.261 1.251 1.213 1.192.727.72.677 25 2 From 118 Me C C (S) 15 1 5 2.5 2. 1.5 1..5 S57
54) 1 NMR (5 Mz, CDCl 3 ) of keto alcohol-2 produced after enzymatic reduction with KRED12 (decoupling at 1.2ppm) 4.3814 4.2829 1 From 12 5 4.5 4. 3.5 3. 2.5 2. 1.5 1..5 J vic (syn) = 4,9 z S58
55) 1 NMR (5 Mz, CDCl 3 ) of keto alcohol-2 produced after enzymatic reduction with KRED118 (decoupling at 1.24ppm) 3.9837 3.9725 3 From 118 2 1 4.5 4. 3.5 3. 2.5 2. 1.5 1..5 J vic (anti) = 6.7 z S59
56) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of keto alcohol-4 produced after enzymatic reduction with KRED12 3 25 From 12 Me C C (R) 2 15 1 5 7. 6. 5. 4. 3. 2. 1. 5.79 5.691 5.668 5.655 5.644 5.633 5.613 5.6 5.59 5.577 5.554 5.537 4.984 4.952 4.925 2.855 2.838 2.825 2.88 2.787 2.375 2.345 2.326 2.32 2.273 2.184 2.167 2.145 2.87 1.56 1.35 3 25 2 From 12 Me C C (R) 15 1 5 6. 5. 4. S6 3. 2. 1.
57) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of keto alcohol-4 produced after enzymatic reduction with KRED12 25 From 12 Me C C (S) 2 15 1 5 7. 6. 5. 4. 3. 2. 1. 5.553 5.53 5.52 5.57 5.496 5.473 5.463 5.45 5.439 5.416 4.897 4.863 4.832 4.781 2.74 2.688 2.681 2.674 2.665 2.658 2.635 2.158 2.133 2.19 2.82 2.55 1.943 1.926 1.93 1.873 1.837 1.174 1.152 3 25 From 12 Me C C (S) 2 15 1 5 6. 5. 4. S61 3. 2. 1.
58) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of keto alcohol-4 produced after enzymatic reduction with KRED12 7 6 From 12 Me C C (R) 5 4 3 2 1-1 7. 6. 5. 4. 3. 2. 1. 5.679 5.656 5.641 5.626 5.598 5.565 5.542 5.6 4.968 4.952 2.778 2.761 2.746 2.73 2.722 2.75 2.335 2.31 2.288 2.257 2.232 2.219 2.22 2.18 2.154 2.133 2.46 1.11 1.8 6 5 From 12 Me C C (R) 4 3 2 1 6. 5. 4. S62 3. 2. 1.
59) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of keto alcohol-4 produced after enzymatic reduction with KRED12 From 12 Me C C (S) 5 7. 6. 5. 4. 3. 2. 1. 5.545 5.521 5.511 5.499 5.489 5.465 5.455 5.442 5.432 5.48 4.92 4.886 4.87 4.813 2.647 2.63 2.615 2.66 2.599 2.591 2.574 2.16 2.135 2.113 2.87 2.57 2.27 2.6 1.989 1.959 1.942 1.767 1.223 1.21 8 7 6 From 12 Me C C (S) 5 4 3 2 1 6. 5. 4. S63 3. 2. 1.
6) 1 NMR (5 Mz, CDCl 3 ) of keto alcohol-4 produced after enzymatic reduction with KRED12 (decoupling at 1.21ppm) 4.7525 4.6579 3 25 2 From 12 15 1 5 6. 5. 4. 3. 2. 1. J vic (syn) = 4.7 z S64
61) 1 NMR (5 Mz, CDCl 3 ) of keto alcohol-4 produced after enzymatic reduction with KRED12 (decoupling at 1.25ppm) 3.97989 3.96713 3 25 2 From 12 15 1 5 6. 5. 4. 3. 2. 1. J vic (anti) = 6.4 z S65
62) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of keto alcohol-5 produced after enzymatic reduction with KRED12 3 From 12 Me C C (R) 25 2 15 1 5 7. 6. 5. 4. 3. 2. 1. 4.965 4.941 4.921 4.916 2.785 2.768 2.763 2.755 2.746 2.738 2.733 2.716 2.39 2.279 2.259 2.233 2.25 2.139 2.119 2.95 2.68 1.634 1.532 1.45 1.23 35 3 25 From 12 Me C C (R) 2 15 1 5 5. 4. 3. S66 2. 1.
63) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of keto alcohol-5 produced after enzymatic reduction with KRED12 4 From 12 Me C C (S) 3 2 1 7. 6. 5. 4. 3. 2. 1. 4.857 4.837 4.832 4.813 2.653 2.635 2.622 2.613 2.64 2.583 2.116 2.9 2.68 2.44 2.14 1.935 1.914 1.893 1.865 1.823 1.591 1.439 1.18 1.158 4 3 From 12 Me C C (S) 2 1 5. 4. 3. S67 2. 1.
64) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of keto alcohol-5 produced after enzymatic reduction with KRED119 4 3 From 119 Me C C (R) 2 1 7. 6. 5. 4. 3. 2. 1. 35 4.951 4.929 4.93 2.694 2.678 2.669 2.663 2.652 2.646 2.638 2.621 2.283 2.255 2.234 2.28 2.179 2.169 2.145 2.138 2.133 2.119 2.112 2.97 2.83 2.16 1.63 1.525 1.95 1.74 3 25 From 119 Me C C (R) 2 15 1 5-5 5. 4. 3. S68 2. 1.
65) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of keto alcohol-5 produced after enzymatic reduction with KRED119 8 7 6 From 119 Me C C (S) 5 4 3 2 1-1 7. 6. 5. 4. 3. 2. 7 4.846 4.841 4.824 4.82 2.58 2.563 2.554 2.547 2.537 2.531 2.522 2.55 2.151 2.121 2.12 2.92 2.73 2.44 2.27 2.15 1.987 1.969 1.949 1.922 1.711 1.586 1.436 1.229 1.28 6 5 From 119 Me C C (S) 4 3 2 1-1 5. 4. 3. S69 2. 1.
66) 1 NMR (5 Mz, CDCl 3 ) of keto alcohol-5 produced after enzymatic reduction with KRED12 (decoupling at 1.2ppm) 4.6342 4.5436 5 From 12 5. 4. 3. 2. 1. J vic (syn) = 4.5 z S7
67) 1 NMR (5 Mz, CDCl 3 ) of keto alcohol-5 produced after enzymatic reduction with KRED119 (decoupling at 1.24ppm) 3.95937 3.9468 6 5 4 From 119 3 2 1 5. 4.5 4. 3.5 3. 2.5 2. 1.5 J vic (anti) = 6.3 z S71
68) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of keto alcohol-6 produced after enzymatic reduction with KRED12 7 6 5 From 12 Me C C (R) 4 3 2 1 7. 6. 5. 4. 3. 2. 1. 6 2.752 2.729 2.78 2.685 2.661 2.452 2.441 2.43 2.416 2.46 2.392 2.382 2.368 2.358 2.346 1.5 1.31 1.8 1.1.977.953 5 4 From 12 Me C C (R) 3 2 1 2.5 2. S72 1.5 1.
69) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of keto alcohol-6 produced after enzymatic reduction with KRED12 35 3 From 12 Me C C (S) 25 2 15 1 5 7. 6. 5. 4. 3. 2. 1. 2.628 2.64 2.583 2.56 2.537 2.172 2.16 2.148 2.135 2.111 2.9 2.67 2.54 1.187 1.166.858.836.813 3 25 2 From 12 Me C C (S) 15 1 5 2.5 2. S73 1.5 1.
7) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of hydroxy ester-7 produced after enzymatic reduction with KRED12 7 6 From 12 Me C C (R) 5 4 3 2 1 7. 6. 5. 4. 3. 2. 1. 4.113 4.89 4.66 4.42 2.637 2.614 2.593 2.57 2.546 1.223 1.2 1.176 1.128 1.15 1.98 1.77 7 6 5 From 12 Me C C (R) 4 3 2 1 4. 3.5 3. 2.5 S74 2. 1.5 1.
71) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of hydroxy ester-7 produced after enzymatic reduction with KRED12 4 From 12 Me C C (S) 3 2 1 7. 6. 5. 4. 3. 2. 1. 3.994 3.971 3.958 3.947 3.935 3.923 3.91 3.887 3.864 3.851 3.84 3.828 3.816 3.84 3.781 2.52 2.479 2.457 2.434 2.411 1.25 1.229 1.19 1.85 1.62.93.96 4 3 From 12 Me C C (S) 2 1 4.5 4. 3.5 3. 2.5 S75 2. 1.5 1.
72) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of hydroxy ester-7 produced after enzymatic reduction with KRED17 4 3 From 17 Me C C (R) 2 1 7. 6. 5. 4. 3. 2. 1. 4.81 4.69 4.57 4.45 4.33 4.22 4.1 3.997 3.986 3.974 2.73 2.679 2.655 2.631 2.66 1.2 1.177 1.153 1.117 1.93 1.8 1.59 5 4 From 17 Me C C (R) 3 2 1 4. 3.5 3. 2.5 S76 2. 1.5 1.
73) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of hydroxy ester-7 produced after enzymatic reduction with KRED17 1 From 17 Me C C (S) 5 7. 6. 5. 4. 3. 2. 1. 3.867 3.844 3.831 3.82 3.88 3.796 3.784 3.764 3.74 3.728 3.716 3.74 3.692 3.681 3.656 2.6 2.576 2.551 2.527 2.52 1.235 1.214 1.79 1.55 1.31.983.959 1 From 17 Me C C (S) 5 4.5 4. 3.5 3. 2.5 S77 2. 1.5 1..5
74) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of hydroxy ester-8 produced after enzymatic reduction with KRED12 6 5 From 12 Me C C (R) 4 3 2 1 7. 6. 5. 4. 3. 2. 1. 4.154 4.13 4.16 4.82 2.531 2.516 2.56 2.498 2.491 2.484 2.474 2.459 1.647 1.639 1.623 1.615 1.62 1.59 1.577 1.569 1.551 1.544 1.525 1.515 1.5 1.494 1.476 1.47 1.455 1.25 1.226 1.22 1.87 1.66.87.845.82 5 4 From 12 Me C C (R) 3 2 1 4. 3. 2. S78 1..
75) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of hydroxy ester-8 produced after enzymatic reduction with KRED12 4 3 From 12 Me C C (S) 2 1 7. 6. 5. 4. 3. 2. 1. 5 4.111 4.86 4.74 4.63 4.5 4.39 4.27 4.22 4.9 4.3 3.998 3.985 3.974 3.962 3.938 2.389 2.376 2.362 2.357 2.349 2.343 2.33 2.316 1.387 1.362 1.354 1.342 1.33 1.317 1.39 1.292 1.284 1.241 1.22 1.189 1.165 1.142.696.672.647 4 3 From 12 Me C C (S) 2 1 4. 3. S79 2. 1..
76) 1 NMR (3 Mz, CDCl 3 ) of the (R)-MPA ester of hydroxy ester-8 produced after enzymatic reduction with KRED116 5 4 From 116 Me C C (R) 3 2 1 7. 6. 5. 4. 3. 2. 1. 4.76 4.68 4.53 4.45 4.28 4.21 4.5 3.997 2.493 2.485 2.476 2.461 2.45 2.445 2.435 2.418 1.594 1.588 1.582 1.569 1.562 1.556 1.545 1.515 1.212 1.189 1.164 1.94 1.73.92.877.853 4 From 116 Me C C (R) 3 2 1 4.5 4. 3.5 3. 2.5 S8 2. 1.5 1..5
77) 1 NMR (3 Mz, CDCl 3 ) of the (S)-MPA ester of hydroxy ester-8 produced after enzymatic reduction with KRED116 2 From 116 Me C C (S) 15 1 5 7. 6. 5. 4. 3. 2. 1. 3.819 3.795 3.783 3.771 3.759 3.747 3.735 3.725 3.712 3.72 3.69 3.678 3.665 3.654 3.642 3.618 2.42 2.386 2.375 2.358 2.344 2.327 2.317 1.494 1.469 1.449 1.442 1.424 1.417 1.399 1.392 1.375 1.367 1.35 1.33 1.255 1.233 1.7 1.46 1.23.788.763.739 25 2 From 116 Me C C (S) 15 1 5 4. 3. 2. S81 1..
78) 1 NMR (5 Mz, CDCl 3 ) of hydroxy ester-8 produced after enzymatic reduction with KRED12 (decoupling at 1.21ppm) 4.1515 4.54 2 15 From 12 1 5 4.5 4. 3.5 3. 2.5 2. 1.5 1. J vic (syn) = 5 z S82
79) 1 NMR (5 Mz, CDCl 3 ) of hydroxy ester-8 produced after enzymatic reduction with KRED116 (decoupling at 1.24ppm) 3.95427 3.94232 8 7 6 From 116 5 4 3 2 1 5. 4. 3. 2. 1. J vic (anti) = 6 z S83