Supporting Information

Transcription

1 Supporting Information Wiley-VCH Weinheim, Germany

2 S1 Deracemisation of sec-alcohols via a concurrently tandem biocatalytic oxidation-reduction sequence Constance V. Voss, Christian C. Gruber, and Wolfgang Kroutil* Supporting information Contents 1. Substrates and reference compounds 2. Enzymes and micro-organisms 3. Results for deracemisation with strains from literature 4. Procedures 5. Analytics

3 S2 1. Substrates and reference compounds The following chemicals were purchased with highest obtainable purity and used as received: Sigma Aldrich: rac-2-octanol (1a), (R)- and (S)-2-octanol, rac-1-octen-3-ol (1f), rac-1.5- hexanediol (1e), rac-1-phenylethanol (1h), rac-ethyl 3-hydroxybutyrate (1j), rac-hydroxy tetrahydrofuran (1g), 2-octanone (2a), 6-methyl-5-hepten-2-one (2b), 1-octen-3-one (2f). ethyl acetoacetate (2j); Acros: rac-6-methyl-5-hepten-2-ol (1b); Lancaster: rac-2-decanol (1c), rac-4-phenyl-2-butanol (1i); Fluka: rac-2-dodecanol (1d), 2-decanone (2c), 2-dodecanone (2d), acetophenone (2h), 4-phenyl-2-butanone (2i). 2. Enzymes and micro-organisms Enzymes Enzymes were purchased from: Jülich Fine Chemicals: alcohol dehydrogenase from Rhodococcus erythropolis (RE-ADH, Nr. 4.11, 33.8 U/ g); glucose dehydrogenase BS (Nr. 29.1, U ml -1 ); Fluka: alcohol dehydrogenase from Lactobacillus kefir (LK-ADH, Nr. 6543,.4 U mg -1 ). Alcohol dehydrogenase from Rhodococcus ruber DSM (ADH- A ) is commercially available from BioCatalytics Inc. For the experiments described here we used a cell-free preparation (7 U ml -1 ), which was prepared as described previously. [ 1] Micro-organisms Alcaligenes faecalis DSM 139 was obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DMSZ, Braunschweig, Germany, Strain 91B was provided by Dr. Carel Weijers, Laboratory for Organic Chemistry, Wageningen (NL) and identified by DSMZ as Rhodococcus erythropolis DSM Geotrichum candidum NBRC 5767, Candida parapsilosis NBRC 78 (or ATCC 733) and Sphingomonas paucimobilis NBRC (= ATCC 1829, NCIB 8195, NRRL B-54) were obtained from Nite Biological Resource Center (NBRC, Japan, Strain Maintenance Bacteria were maintained in frozen stock solutions and long-term storage was at -8 C. Media Alcaligenes faecalis DSM 139 was grown in M1 medium, Rhodococcus erythropolis DSM 4366 T in Streptomyces medium.

4 S3 M1 medium: Bacteriological peptone (5 g L -1, Oxoid L37), Meat extract (3 g L -1, Oxoid L29). The medium was adjusted to ph 7 with sodium hydroxide solution. Streptomyces medium: Glucose (2 g L -1, Fluka 491), Yeast extract (5 g L -1, Oxoid L21), (NH 4 ) 2 SO 4 (5 g L -1, Roth 6314) and MgSO 4 *7H 2 O (2.5 g L -1, Fluka 6314), ph adjusted to ph 7. Geotrichum candidum NBRC 5767 was grown in PDA medium, Candida parapsilosis NBRC 78 in M186 medium and Sphingomonas paucimobilis NBRC in standard-complex medium. PDA medium: Potato Dextrose Broth (24g L -1, Difco), ph adjusted to ph 5.6. M186 medium: Yeast extract (3 g L -1, Oxoid L21), Malt extract (3g L -1, Oxoid L39), Peptone (5g L -1, Oxoid L37), Glucose (1 g L -1, Fluka 491). Standard-complex medium: components of the Standard-komplex medium were sterilised in four separate groups: group I: Yeast extract (1 g L -1, Oxoid L21), bacteriological peptone (1 g L -1, Oxoid L37); group II: glucose (1 g L -1, Fluka 491); group III: sodium chloride (2 g L -1, Roth ), MgSO 4 *7H 2 O (.15g L -1, Fluka 6314); group IV: NaH 2 PO 4 (1.3 g L -1, Fluka 71496), KH 2 PO 4 (4.4 g L -1, Merck 511). Cultivation of micro-organisms Alcaligenes faecalis DSM 139 was cultivated in M1 medium (see above) in baffled shake flasks (1 L) and 12 rpm for one day at 3 C. Shaking was continued at 12 rpm for further 2 days at 37 C. Rhodococcus erythropolis DSM 4366 T was grown in Streptomyces medium (see above) in baffled shake flasks (1 L) and 12 rpm at 3 C for three days. The cells were harvested by centrifugation (18 g) and washed twice with sodium/ phosphate buffer ( mm, ph 7.5). Geotrichum candidum NBRC 5767 was cultivated in PDA medium (see above) in shake flasks (1 L) and 1 rpm at C for three days. Candida parapsilosis NBRC 78 was cultivated in M186 medium (see above) in baffled shake flasks (1 L) and 12 rpm at C for three days. Sphingomonas paucimobilis NBRC was grown in standard-complex mediun (see above) in baffled shake flasks (1 L) and 12 rpm at 3 C for three days. The cells were harvested by centrifugation (18 g) and washed twice with sodium/ phosphate buffer ( mm, ph 7.5).

5 S4 3. Results for deracemisation with strains from literature Micro-organism Substrate [a] t [h] Alcohol [%] Ketone [%] ee [%] Geotrichum Candidum NBRC 5767 rac-1b (R) rac-1b (R) rac-1c (R) rac-1c (R) rac-1h (R) rac-1h (R) Candida Parapsilosis NBRC 78 rac-1b 24 >99 2 (R) rac-1b (R) rac-1c (R) rac-1c (R) rac-1e 24 >99 12 (R) rac-1e 92 >99 13 (R) rac-1h 24 >99 4 (R) rac-1h (R) Sphingomonas paucimobilis NBRC13934 rac-1b (R) rac-1b (R) rac-1c 24 >99 2 (R) rac-1c 72 >99 24 (R) rac-1h 24 >99 rac rac-1h 72 >99 rac Table S1: Investigated micro-organisms, described for deracemisation in literature. Whole cells of the respective strain (-2 mg, wet cell weight) were washed and resuspended in TRIS-buffer (.5 ml, ph 7.5, mm). The racemic substrate (5 mg) was added and the mixture was shaken at 3 C and 1 rpm for 24h, 72 or 92h. [a] 6-8 mm substrate concentration. 4. Procedures 1.1 Sreening for micro-organisms with deracemisation activity Lyophilised cells (4 mg) were rehydrated in TRIS-buffer (.5 ml, mm, ph 7.5) for 1 h at C shaking at 17 rpm. Racemic substrate (6-8 mm) was added and the mixture was shaken at 3 C and 1 rpm for 24 h, 72 h and 92 h. The biotransformation was stopped by the addition of ethyl acetate (8 mm). After centrifugation of the solution at 13 rpm for 5 min, the organic phase was transferred in a new Eppendorf tube. For determination of the ee, the alcohols were derivatised by the addition of acetic anhydrid (2 μl, 2.5 mm) and a catalytic amount of DMAP (4-Dimethylaminopyridine,.2 mm). This reaction mixture was shaken for 1 hour at 17 rpm and room temperature ( C). Afterwards H 2 O dest. (3 μl) and an internal standard (1 mg, 2 μl of a standard solution with mg/ml 1-decanol in ethyl acatate) was added. The solution was centrifuged (2 min) and the organic phase was dried (Na 2 SO 4 ) and analysed using chiral GC.

6 S Typical procedure for the biocatalytic deracemisation of secondary alcohols on 5 mg scale using 2-propanol for cofactor recycling Resting cells of Alcaligenes faecalis DSM 139 ( mg) were resuspended in TRIS-buffer (.5 ml, mm, ph 7.5). The racemic alcohol (5 mg, 6-8 mm), ADH- A (1 μl,.7 U), NAD + (.17 mg,.5 mm) and 2-propanol (5% (v/v) =.6 M) were added. The mixture was shaken at 3 C and 3 rpm (Thermo-Shaker) for a specified time. The biotransformation was stopped and analysed as described above (section 1.1.) 1.3 Stereoinversion of (R)-2-octanol to (S)-2-octanol: Resting cells of Alcaligenes faecalis DSM 139 ( mg wet cell weight), resuspended in TRIS-buffer (.5 ml, mm, ph 7.5), were incubated with (R)-2-octanol (35 mm), NADHrecycling system [glucose dehydrogenase (5 μl, 2.5 U), glucose ( mm),.5 mm NAD + ] and RE-ADH (.17 U) at 3 C and 3 rpm (Thermo-Shaker) for a specified time. The biotransformation was stopped and analysed as described above (section 1.1.)

7 S6 5. Analytics GC analysis Chiral column: Chirasil-Dex CB-cyclodextrin (CP73, WCOT FUSED SILICA m x.32 mm); Table S2. Retention times for GC analysis with chiral columns. Substrate [a] Conditions [b] (S) [min] rac-1a (R) [min] Ketone [min] A rac-1b A rac-1c rac-1d A A rac-1e rac-1f A A rac-1g B O rac-1h A rac-1i A rac-1j O O B [a] measured as its acetate derivative [b] Conditions: A: temperature program: /1/2.5/12/1/1/1 (start temperature C/ holding time [min]/ heating rate [ C/ min]/ plateau temperature [ C]/ holding time [min]/ heating rate [ C/ min]/ final temperature [ C]/ holding time [min]). B: Temperature program: 85/1/2.5/11/1/1/1.

8 c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac mit recyclingsystem \1.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac mit recyclingsystem \1.run Last recalc: : c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac mit recyclingsystem \2.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac mit recyclingsystem \2.run Last recalc: :1 X: Y: 4.9 X: Y: 1.72 S7 Chromatograms Deracemisation with Alcaligenes faecalis DSM 139 GC-analytics of preparative deracemisation experiment starting with µl rac-4-phenyl-2- butanol 1i. rac-1i (S)-1i Main picture: GC-chromatogram of deracemisation-product (S)-1i employing a chiral stationary phase. Neither ketone 2i nor (R)-1i nor any side product was detectable. The inserted chromatogram shows the racemate, the starting material ketone (S) (R) Top: Biocatalytic oxidation of rac-2-octanol (6 mm) for 4 hours employing Alcaligenes faecalis DSM 139. Bottom: Deracemisation of rac-2-octanol (6 mm) employing Alcaligenes faecalis DSM 139, GDH-NADHrecycling system and RE-ADH after 4 hours.

9 c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac mit recyclingsystem \3.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac mit recyclingsystem \3.run Last recalc: :2 c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac mit recyclingsystem \4.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac mit recyclingsystem \4.run Last recalc: :3 c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\substrate sreening15.1\8.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\substrate sreening15.1\8.run Last recalc: : c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\substrate sreening15.1\9.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\substrate sreening15.1\9.run Last recalc: :24 c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac-19.3\1-ko.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac-19.3\1-ko.run Last recalc: :2 c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac-19.3\1.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac-19.3\1.run Last recalc: :29 X: Y: 2.56 X: 3.95 Y: 1.37 X: Y:.497 X: Y: 4.7 X: 2.87 Y: 1.34 X: Y: 29.8 S Top: Biocatalytic oxidation of rac-2-octanol (6 mm) for 16 hours employing Alcaligenes faecalis DSM 139. Bottom: Deracemisation of rac-2-octanol (6 mm) employing Alcaligenes faecalis DSM 139, GDH-NADHrecyclingsystem and RE-ADH after 16 hours. 1 (S) FPketone (R) Top: Biocatalytic oxidation of rac-4-phenyl-2-butanol for 16 hours employing Alcaligenes faecalis DSM 139. Bottom: Deracemisation of rac-4-phenyl-2-butanol employing Alcaligenes faecalis DSM 139, GDH-NADHrecyclingsystem and RE-ADH after 16 hours (S) (R) Top: Biocatalytic oxidation of rac-sulcatol for 1 hours employing Alcaligenes faecalis DSM 139. Bottom: Deracemisation of rac-sulcatol employing Alcaligenes faecalis DSM 139, 2-propanol, catalytic amount of NAD + and ADH- A after 1 hours.

10 c:\dokumente und einstellungen\vossco\desktop\derac-screening\weijers fcc42\27\derac-weijers-15.2\7.run c:\dokumente und einstellungen\vossco\desktop\derac-screening\weijers fcc42\27\derac-weijers-8.2.7\ run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\weijers fcc42\27\derac-weijers-15.2\7.run Last recalc: :48 X: Y: 6.44 File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\weijers fcc42\27\derac-weijers-8.2.7\ run Last recalc: :52 X: Y:.63 S9.Stereoinversion II+ II c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\stereoininversion24.5\stereoininversion24.5\r-2o.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\stereoininversion24.5\stereoininversion24.5\r-2o.run Last recalc: :1 Reference of (R)-2-octanol X: Y: II+ c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\stereoininversion24.5\stereoininversion24.5\r-2o-2h.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\stereoininversion24.5\stereoininversion24.5\r-2o-2h.run Last recalc: :42 Stereoinversion of (R)-2-octanol after 2 h X: Y: 3.46 II+ c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\stereoininversion24.5\stereoininversion24.5\r-2o-3h.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\stereoininversion24.5\stereoininversion24.5\r-2o-3h.run Last recalc: :42 Stereoinversion of (R)-2-octanol after 3 h X: Y: 3.44 II+ 1 c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\stereoininversion24.5\stereoininversion24.5\r-2o-4h.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\stereoininversion24.5\stereoininversion24.5\r-2o-4h.run Last recalc: :18 Stereoinversion of (R)-2-octanol after 4 h X: Y:. 1 II+ c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\stereoininversion24.5\stereoininversion24.5\r-2o-6h.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\stereoininversion24.5\stereoininversion24.5\r-2o-6h.run Stereoinversion Channel: of Front = (R)-2-octanol FID Results after 6 h Last recalc: :18 X: Y:.422 Volts Stereoinversion of (R)-2-octanol (R)-1a (35 mm) to (S)-2-octanol (S)-1a, employing Alcaligenes faecalis DSM 139, RE-ADH and cofactor recycling system (GDH, glucose and NAD + ). From top to bottom: Only oxidation, then stereoinversion experiments after 2, 3,4 and 6 hours. Deracemisation employing Rhodococcus erythropolis DSM Top: Biocatalytic oxidation of rac-1-octen-3-ol for 16 hours employing Rhodococcus erythropolis DSM Bottom: Deracemisation of rac-1-octen-3-ol employing Rhodococcus erythropolis DSM 4366, GDH-NADHrecyclingsystem and LK-ADH after 16 hours.

11 c:\dokumente und einstellungen\vossco\desktop\derac-screening \probe 47\27\derac-7.5\2.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac-7.5\2.run Last recalc: :45 c:\dokumente und einstellungen\vossco\desktop\derac-screening \probe 47\27\derac-7.5\5.run File: c:\dokumente und einstellungen\vossco\desktop\derac-screening\probe 47\27\derac-7.5\5.run Last recalc: : X: Y:.264 X: Y:.362 S Top: Biocatalytic oxidation of rac-4-phenyl-2-butanol for 16 hours employing Rhodococcus erythropolis DSM Bottom: Deracemisation of rac-4-phenyl-2-butanol employing Rhodococcus erythropolis DSM 4366, GDH- NADH-recyclingsystem and LK-ADH after 16 hours. References 1 G. de Gonzalo, I. Lavandera, K. Faber, W. Kroutil, Org. Lett. 27, 9,