JOURNAL TRANSCRIPT

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Developing a Biocascade Process: Concurrent Ketone Reduction-Nitrile Hydrolysis of 2-Oxocycloalkanecarbonitriles Elisa Liardo,a Nicolás Ríos-Lombardía,b Francisco Morís,b Javier González-Sabín,*,b and Francisca Rebolledo*,a a

Departamento de Química Orgánica e Inorgánica, Universidad de Oviedo, 33006 Oviedo, Spain. b EntreChem, SL, Edificio Científico Tecnológico, Campus El Cristo, E-33006 (Oviedo), Spain.

SUPPORTING INFORMATION (page 1 of 41 pages)

Table of Contents 1.

General information (p. S2)

2.

Analytical scale biotransformations (p. S3)

3.

Enzymatic screening (p. S5)

4.

Preparative scale reactions and characterization of optically active compounds 2a-c, 3a-c and 4b (p. S10)

5.

Synthesis of racemic cis/trans 2a-c, 3a-c, cis-4b and their derivatives for ee and dr determinations (p. S14)

6.

GC analytical data for ee and dr determinations (p. S18)

7.

Assignment of the absolute configuration (p. S20)

8.

Copy of GC chromatograms (p. S22)

9.

Copy of NMR spectra (p. S34)

1

1. General information Enzymes Codex® KRED Screening Kit was purchased from Codexis. Cultures of Rhodococcus rhodochrous IFO 15564 were prepared according to previously reported procedures.1

General methods 1

H-NMR and proton-decoupled

13

DPX-300 (1H, 300.13 MHz and

13

C-NMR spectra (CDCl3, CD3OD) were obtained using a Bruker C, 75.5 MHz) spectrometer using the δ scale (ppm) for chemical

shifts. The 1H-NMR spectra of the Mosher’s derivative were obtained using a Bruker AV-600 (1H, 600. MHz). Calibration was made on the signal of the solvent (13C: CDCl3, 76.95; 1H: CDCl3, 7.26;

13

C:

1

CD3OD, 49.00; H: CD3OD, 3.31). High resolution mass spectra were recorded on a Bruker Impact II instrument. Optical rotations were measured using a Perkin-Elmer 241 polarimeter and are quoted in units of 10-1deg cm2 g-1. Gas chromatography (GC) analyses were performed on a Hewlett Packard 6890 Series II chromatograph, with the following columns: CP-ChiraSil-DEX CB 25 m × 0.25 mm × 0.25 µm or Rt®-bDEXse 30 m × 0.25 mm × 0.25 µm.

Racemic substrates Racemic 2-oxocycloalkanecarbonitriles 1a-c were prepared as previously described for 1a-b2 and 1c.3 Obtained yields: 1a (92%), 1b (93%), and 1c (80%).

1

Morán-Ramallal, R.; Liz, R.; Gotor, V. Org. Lett., 2007, 9, 521. Dehli, J. R.; Gotor, V. J. Org. Chem., 2002, 67, 6816. 3 Hsing-Jang, L.; Tai, W. L.; Chia-Liang, T.; Jen-Dar, W.; Jinn-Kwei, L.; Jiunn-Cheh, G.; Nai-Wen, T.; Kak-Shan, S. Tetrahedron, 2003, 59, 1209. 2

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2. Analytical scale biotransformations 2.1. General procedure for the bioreduction of 1a-c at pH 7.0. In a 2.0 mL eppendorf tube, KRED (2 mg), ketone (20 mM) and IPA (190 µL) were added to 900 µL of 125 mM potassium phosphate buffer, pH 7.0. This buffer also contains MgSO4 (1.25 mM) and the cofactor NADP+ (1.0 mM). The resulting reaction mixture was shaken at 250 rpm and 30 ºC for 24 h. Then, the mixture was extracted with ethyl acetate (2 × 500 µL), the organic layers were separated by centrifugation (90 s, 13000 rpm), combined, and finally dried over Na2SO4. The enantiomeric excess and the diastereomeric ratio of the resulting optically active 2-hydroxycycloalkanecarbonitriles 2a-c were determined by GC equipped with a chiral column, after conversion into the corresponding Otrimethylsilyl derivative (see Sections 5 and 6 for details). In all the cases, the GC analysis revealed the absence of the starting material.

2.2. General procedure for the bioreduction of 1a-b at pH ≠ 7.0. The general procedure 2.1 was followed, but the pH of the buffer solution was previously changed from 7.0 to the required one by addition of 3 N aq NaOH or 3 N aq HCl. In both cases, once the reaction was completed, the pH of the resulting mixture was adjusted to pH 7.0 before the extraction with ethyl acetate.

2.3. Hydrolytic activity essay of 2a-c with R. rhodochrous IFO-15564. General method. In a 2.0 mL eppendorf tube, racemic cis- and/or trans-2a-c (2.0 mg) and IPA (50 µL) were added to 1000 µL of a suspension of the microorganism with A650 = 4.0 in 100 mM potassium phosphate buffer pH 7.0. The reaction was shaken at 200 rpm and 28 °C for 5-24 h. An aliquot was periodically analyzed by TLC (hexane:ethyl acetate 1:1). Once the starting material was consumed completely, the mixture was acidified until pH 2 and extracted with ethyl acetate (2 x 450 µL). The organic layers were separated by centrifugation (90 s, 13000 rpm), combined and finally dried over Na2SO4. The GC analysis of the resulting mixtures revealed the formation of the 2-hydroxycycloalkanecarboxylic acids almost racemic and with the same diastereomeric ratio of the starting 2a-c.

2.4. General procedure for the concurrent one-pot synthesis In a 2.0 mL eppendorf tube, a suspension of harvested cells of R. rhodochrous IFO 15564 in 125 mM potassium phosphate buffer pH 7.0 of a high absorbance value (A650 = 22-16) was mixed with the 3

adequate amount of 125 mM potassium phosphate buffer pH 7.0 containing MgSO4 (1.25 mM) and the cofactor NADP+ (1.0 mM) to get a final A650 = 4.0. Then, substrate 1a-c (20 mM), KRED (2 mg), and IPA (50 µL) were added. The reaction was shaken at 200 rpm and 28 °C until disappearance of the intermediate β-hydroxynitrile 2a-c (TLC control, 5-48h). Then, the procedure described above (section 2.3) was followed. The diastereomeric ratio and enantiomeric excess of each optically active 2hydroxycycloalkanecarboxylic acid were determined by GC equipped with a chiral column after the appropriate derivatization (see Sections 5 and 6 for details).

2.5. General procedure for the sequential one-pot synthesis: bioreduction at pH 5.0 and subsequent biohydrolysis at pH 7.0. First, the general procedure of the bioreduction at pH 5.0 (section 2.2) was followed. After 24 h of reaction, the pH was adjusted at 7.0 using 3 N aq NaOH and the adequate amount of a suspension of R. rhodochrous IFO 15564 of high A650 value was added to get a final A650 = 4.0. As a consequence, a 20-25% degree of dilution occurred. Then, the procedure described above (section 2.3) was followed.

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3. Enzymatic screening Table S1. Enzymatic reduction of 2-oxocyclopentanecarbonitrile (1a) using KREDs at pH 7.0 following the general procedure of section 2.1.

ee (%)a

diastereomeric ratio (%)

a

KRED

cis

trans

cis

trans

NADH-101

87

13

80 (1S,2S)

>99 (1R,2S)

NADH-110

29

71

22 (1S,2S)

88 (1R,2S)

P1-A04

45

55

99 (1R,2R)

>99 (1R,2S)

P1-H08

>99

20

80

>99 (1R,2R)

>99 (1R,2S)

P1-H10

>99

22

78

64 (1S,2S)

98 (1R,2S)

P2-B02

>99

51

49

70 (1S,2S)

>99 (1R,2S)

P2-C02

>99

50

50

99

90

10

84 (1S,2S)

>99 (1R,2S)

P2-D03

>99

28

72

>99 (1R,2R)

>99 (1R,2S)

P2-D11

>99

6

94

>99 (1R,2R)

>99 (1R,2S)

P2-D12

>99

11

89

>99 (1R,2R)

>99 (1R,2S)

P2-G03

>99

98

2

95 (1S,2S)

P2-H07

>99

76

24

96 (1R,2R)

95 (1R,2S)

P3-B03

>99

72

28

49 (1S,2S)

98 (1R,2S)

P3-G09

>99

66

34

52 (1S,2S)

43 (1S,2R)

a

The absolute configuration of the major stereoisomer is indicated between brackets. b This bioreduction reached complete conversion in the preparative scale reaction (Section 4.1) by adding the KRED gradually.

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Table S3. Enzymatic reduction of 2-oxocyclohexanecarbonitrile (1b) using KREDs at pH 7.0 following the general procedure of section 2.1.

ee (%)a

diastereomeric ratio (%)

a

KRED

cis

trans

cis

trans

P1-A04

>99

99 (1S,2S)

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P1-B05

57

43

80 (1R,2R)

54 (1S,2R)

P1-B10

94

6

>99 (1R,2R)

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P1-B12

>99

99 (1R,2R)

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P1-C01

65

35

48 (1R,2R)

71 (1R,2S)

P1-H08

81

19

41 (1S,2S)

>99 (1R,2S)

P1-H10

>99

99 (1R,2S)

P2-C02

54

46

99