Development of an R-selective amine oxidase with broad substrate specificity and high enantioselectivity

. General chemicals and methods.........................................................................................................5

.1 Chemicals ......................................................................................................................................5

.2 Analysis methods ..........................................................................................................................5

. Synthesis of substrates .......................................................................................................................6

.1 (R )-Nicotine (4)..............................................................................................................................6

.2 6-Hydroxy-nicotine (1) ..................................................................................................................6

.3 6-Amino-nicotine (8).....................................................................................................................6

.4 N-Ethyl nornicotine (9)..................................................................................................................7

.5 N-Isopropyl nornicotine (10).........................................................................................................7

.6 1-Propyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-2-ium (22)....................................................8

.7 3-(1-Methylpiperidin-2-yl)pyridine (33)........................................................................................8

. Mechanism experiments.....................................................................................................................8

.1 Study of the stereoinversion of (R )-nicotine (4) using wildtype 6-HDNO.....................................8

.1.1 Preparative procedure...........................................................................................................8

.1.2 Deuteration study (A): using a deuterated reducing agent.................................................10

.1.3 Deuteration study (B): using a deuterated reducing agent and deuterium oxide ..............12

.2 Study of the deracemisation of 6-hydroxy-nicotine (1) using wildtype 6-HDNO .......................14

.2.1 Preparative procedure.........................................................................................................14

.2.2 Deuteration study: using a deuterated reducing agent.......................................................15

. Biotransformations – procedures and analyses ...............................................................................16

.1 General procedure for biotransformations ................................................................................16

.2 Analysis of biotransformations carried out with wildtype 6-HDNO ...........................................16

.2.1 (S )-6-Hydroxy nicotine (1) ....................................................................................................16

.2.2 (S )-Nicotine (4) .....................................................................................................................17

.2.3 (S )-6-Amino nicotine (8).......................................................................................................18

.2.4 (S )-N-Ethyl nornicotine (9) ...................................................................................................19

.2.5 (S )-N-Isopropyl nornicotine (10) ..........................................................................................19

.2.6 (S )-Crispine A (26) ................................................................................................................20

.2.7 (S )- 3-(1-Methylpiperidin-2-yl)pyridine (33) ........................................................................21

.3 Analysis of biotransformations carried out with variant E350L/E352D .....................................21

S2

.3.1 (S )-6-Hydroxy nicotine (1) ....................................................................................................21

.3.2 (S )-Nornicotine (7)...............................................................................................................22

.3.3 (S )-6-Amino nicotine (8).......................................................................................................23

.3.4 (S )-2-Phenylpyrrolidine (12).................................................................................................23

.3.5 (S )-2-(p-Methoxyphenyl)-pyrrolidine (13) ...........................................................................24

.3.6 (S )-2-Cyclohexylpyrrolidine (14)...........................................................................................25

.3.7 (S )-6,7-Dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (15) ......................................26

.3.8 (S )-2-Isobutylazepane (18)...................................................................................................26

.3.9 (R )-1-Phenyl-1,2,3,4-tetrahydroisoquinoline (19) ...............................................................27

.3.10 (S )-1-(2-Bromophenyl)-1,2,3,4-tetrahydroisoquinoline (20) (oxidation only)...................28

.3.11 (S )-1-(Naphthalen-1-yl)-1,2,3,4-tetrahydroisoquinoline (21) (oxidation only)..................28

.3.12 (S )-1-Propyl-4,9-tetrahydro-3H-β -carboline (22)...............................................................29

.3.13 (S )-Crispine A (26) ..............................................................................................................30

.3.14 (S )-Anabasine (32)..............................................................................................................31

.3.15 (S )-3-(1-Methylpiperidin-2-yl)pyridine (33) .......................................................................31

.3.16 (S )-2-(Phenylethyl) piperidine (34).....................................................................................32

.3.17 Example comparison of opposite selectivities of MAO-N D5 and 6-HDNO.......................33

.3.18 Example of solvent compatability of 6-HDNO E350L/E352D.............................................33

.4 General preparative method ......................................................................................................33

.4.1 (S )-6-Hydroxy nicotine (1) ....................................................................................................34

.4.2 (S )-Nicotine ..........................................................................................................................34

.4.3 (S )-Nornicotine (7)...............................................................................................................34

.4.4 (S )-2-Cyclohexylpyrrolidine (14)...........................................................................................34

.4.5 (S )-2-Isobutylazepane (18)...................................................................................................34

.4.6 (R )-1-Phenyl-1,2,3,4-tetrahydroisoquinoline (19) ...............................................................35

.4.7 (S )-Anabasine (32)................................................................................................................35

.4.8 (S )-3-(1-Methylpiperidin-2-yl)pyridine (33) (wildtype)........................................................35

.4.9 (S )-3-(1-Methylpiperidin-2-yl)pyridine (33) (E350L/E352D variant)....................................36

.4.10 (S )-2-(Phenylethyl) piperidine (34).....................................................................................36

. DNA and protein methods ................................................................................................................36

.1 Cloning ........................................................................................................................................36

.2 Expression ...................................................................................................................................36

.3 Purification..................................................................................................................................37

.4 Solid phase screen ......................................................................................................................37

S3

.5 Libraries.......................................................................................................................................38

.6 Kinetics........................................................................................................................................38

.7 Protein structure examination....................................................................................................40

6

. References ........................................................................................................................................40

S4

1

. General chemicals and methods

1

.1 Chemicals

Potassium monophosphate, dipotassium phosphate, sodium chloride, imidazole, lysozyme, ,

-aminoantipyrine and 2,4,6-Tribromo-3-hydroxybenzoic acid were from Sigma or Fisher.

S)-Nicotine, 2-Isobutylazepane, and Anabasine were purchased from Sigma Aldrich, 2-

Phenyl pyrrolidine from Apollo Scientific, 6,7-Dimethoxy-1-methyl-3,4-

4

(

tetrahydroisoquinoline, 6,7-Dimethoxy-1,2-dimethyl-1,2,3,4-tetrahydroisoquinoline and 6,7-

Diethoxy-1,2,3,4-tetrahydroisoquinoline were purchased from Acros Organics, while

Nornicotine was from Alfa Aesar. 1-(Naphthalen-1-yl)-1,2,3,4-tetrahydroisoquinoline and 1-

(2-bromophenyl)-1,2,3,4-tetrahydroisoquinoline were kindly donated by J. M. Rosello and

Professor J. Clayden, Department of Chemistry, University of Manchester.

1

-Phenyltetrahydroisoquinoline was obtained by reduction of the corresponding lactam

with NaBH /I instead of LiAlH according to Amat et al. N-Methyl-2-phenylpyrrolidine and

4

2

4

-(Methylamino)-1-(3’-pyridyl)-1-butanone dihydrochloride (pseudoxynicotine) were

prepared according to Dunsmore et al. 6-Amino-nicotine and 6-Hydroxy nicotine were

synthetized according to procedures published by Tachitschibabin and Kirssanow; Crispine

A was synthesized as in Rowles et al. Methyl anabasine was obtained by methylation of

anabasine with methyl iodide/sodium hydride.

Solvents were analytical or HPLC grade or were purchased and dried over molecular sieves

where necessary.

1

.2 Analysis methods

Column chromatography was performed on silica gel (Sigma-Aldrich, 220-440 mesh).

1

13

H and C NMR spectra were recorded on a Bruker Avance 400 (400.1 MHz or 399.9 MHz

1

13

for H and 100.6 MHz for C) without additional internal standard. Chemical shifts are

reported in δ values (ppm) and are calibrated against residual solvent signal. The following

abbreviations were used to define the multiplicities: s, singlet; d, doublet; t, triplet; q,

quartet; m, multiplet; b, broad.

HPLC analysis was performed on an Agilent system equipped with a G1379A degasser,

G1312A binary pump, a G1329 autosampler unit, a G1315B diode array detector and a

G1316A temperature controlled column compartment. The columns used were CHIRALPAK®

IC (5 µm particle size, 4.6 mm diameter x 250 mm), CHIRALPAK® IE (5 µm particle size, 4.6

mm diameter x 250 mm), CHIRALPAK® IA (5 µm particle size, 4.6 mm diameter x 250 mm)

and CHIRALCEL® DAICEL OD-H (5 µm particle size, 4.6 mm diameter x 250 mm); conditions

are indicated separately for each compound. GC analysis was performed on Agilent 6850

GCs equipped with a Gerstel Multipurposesampler MPS2L; Measurements were carried out

using Varian Chirasil-Dex CB column 25 m × 0.25 mm × 0.26 μm film thickness or Supelco β-

DEX 325 column, 30 m × 0.25 mm × 0.25 μm film thickness; conditions are indicated

separately for each compound.

Optical rotation was measured at 28 °C on a Perkin–Elmer Polarimeter 341 against the

sodium D-line.

S5

2. Synthesis of substrates

2

.1 (R)-Nicotine (4)

(

S)-nicotine (500 mg, 3 mmol) and BH NH (360 mg, 12 mmol) were

3

dissolved in potassium phosphate buffer (30 mL, 1M, pH = 7.8). Cell pellet (5

g) from E. coli cultures containing MAO-N D5 was added to the solution. The

bottle was placed in a shaking incubator and shaken at 37 °C and 250 rpm.

When HPLC analysis (CHIRALPAK® IC column; flowrate 1 mL / min; UV 280 nM; eluent=

hexane/iPrOH 97:3 + 0.1% DEA; Rt [(S)] = 18.7 min, Rt [(R)] = 20.5 min) showed the

conversion reached > 98 % and the amount of imine < 2% aqueous, aqueous NaOH (2 mL,

1

0 M) and CH Cl (200 mL) were added. The mixture was transferred to Falcon tubes and

2

layers were separated by centrifugation (4000 rpm, 5 min) and the aqueous phase was

extracted with CH Cl (2 × 100 mL). The combined organic phases were dried with MgSO

2

4

and concentrated under reduced pressure to give the product (R)-nicotine (429 mg, 86%

yield) as a yellow liquid.

2

5

20

[

α]_D= + 133.0 (c = 1.0, CHCl ). (lit. (S)-nicotine [α] = -138.0 (c= 1, CHCl )).

3

D

3

1

H NMR (400 MHz, CDCl ) δ 8.53 (brd, J=2.1 Hz, 1H), 8.50 (dd, J=4.7 Hz, 1.7 Hz, 1H), 7.72 (dt,

3

J=7.8, 1.9 Hz, 1H), 7.26 (app q, J=5.1 Hz, 1H), 3.26 (t, J=8.0 Hz, 1H), 3.10 (t, J=8.3 Hz, 1H),

.33 (app q, J=8.0 Hz, 1H), 2.26 – 2.19 (m, 1H), 2.18 (s, 3H), 2.06 – 1.93 (m, 1H), 1.88 – 1.70

2

1

3

(m, 2H); C NMR (100 MHz, CDCl ) δ: 149.8, 148.9, 138.9, 135.1, 123.8, 69.1, 57.2, 40.5,

3

5.3, 22.8.

2

.2 6-Hydroxy-nicotine (1)

Under nitrogen atmosphere, to a solution of 6-amino-nicotine∙HCl (2.5

g, 11.7 mmol) in sulphuric acid (5% in water, 20 mL), a solution of

sodium nitrite (8.3 g, 120 mmol) was added and the mixture was stirred

for an additional hour. The reaction was neutralised with aqueous

NaOH (2 mL, 5 M) and then extracted with CH Cl (3 x 80 mL). The combined organic phases

2

were dried over MgSO and concentrated under reduced pressure. Purification by column

4

chromatography on silica gel (CH Cl /MeOH 9:1) gave the product 6-hydroxy nicotine (500

2

mg, 24% yield) as a brown solid.

1

H NMR (400 MHz, CDCl ) δ 13.34 (bs, 1H), 7.53 (dd, J=9.4, 2.1 Hz, 1H), 7.25 (s, 1H), 6.56 (d,

3

J=9.4 Hz, 1H), 3.20 – 3.12 (m, 1H), 2.82 (t, J=8.3 Hz, 1H), 2.28 – 2.15 (m, 1H), 2.11 (s, 3H),

1

3

2

1

.10 – 2.00 (m, 1H), 1.98 – 1.82 (m, 1H), 1.82 – 1.58 (m, 2H). C NMR (100 MHz, CDCl ) δ

3

+

65.4, 141.5, 132.8, 121.6, 120.4, 67.6, 56.6, 40.0, 33.7, 22.3. TOF-Ms (m/z) = 179.2 [M+H] .

2

.3 6-Amino-nicotine (8)

Under nitrogen atmosphere, to a solution of (S)-nicotine (25 g, 154

mmol) in xylene (50 mL), sodium amide (12 g, 307 mmol) was

added portionwise and the mixture was stirred and heated under

S6

reflux. After 5 hours, the reaction mixture was cooled to rt and poured onto ice;

concentrated hydrochloric acid was then added unti a pH of 2 was reached and the two

phases separated. The aqueous phase was neutralised with 1 M NaOH and then extracted

with CH Cl (3 x 100 mL). The combined organic phases were dried over MgSO and

2

4

concentrated under reduced pressure. Purification by column chromatography on silica gel

(

CH Cl /MeOH 95:5) afforded the 6-amino-nicotine (19.4 g, 71% yield) as a brown oil.

2

1

H NMR (400 MHz, CDCl ) δ 7.89 (d, J=2.1 Hz, 1H), 7.41 (dd, J=8.5, 2.3 Hz, 1H), 6.45 (t, J=8.4

3

Hz, 1H), 4.53 (bs, 2H), 3.19 – 3.13 (m, 1H), 2.89-2.82 (m, 1H), 2.24 – 2.14 (m, 1H), 2.09 (s,

1

3

1

H), 2.09 – 2.00 (m, 1H), 1.95 – 1.81 (m, 1H), 1.79-1.59 (m, 2H); C NMR (100 MHz, CDCl ) δ

3

+

58.0, 147.5, 136.9, 127.9, 108.9, 68.6, 56.9, 40.2, 34.5, 22.3. TOF-MS (m/z) = 178.1 [M+H] .

2

.4 N-Ethyl nornicotine (9)

Nornicotine (105 mg, 0.71 mmol) was dissolved in DMF (4 mL) and NaH (0.71

mmol) 60 % dispersion in oil was added. Ethyl iodide (0.85 mmol) was added

and the reaction was stirred at rt for 3 hours. TLC of the reaction mixture

revealed that the starting material was still present, consequently ethyl

iodide (0.85 mmol) was added and the reaction temperature was increased to 60 °C for 2

hours. The reaction was quenched by the addition of water (2 mL) and the mixture

extracted with toluene (3 x 5 mL). The combined organic phases were dried over MgSO₄,

filtered and concentrated under reduced pressure. The crude product was purified by

column chromatography on silica gel (ethyl acetate/MeOH 9:1 + 1% Et N), to give the

3

product N-Ethyl nornicotine (75 mg, 60 % yield) as yellow oil.

1

13

H NMR, C NMR and Mass analysis for N-Ethyl nornicotine were consistent with those

6

reported in the literature.

2

.5 N-Isopropyl nornicotine (10)

Nornicotine (100 mg, 0.675 mmol) was dissolved in acetone (3 mL) and a

catalytic amount of acetic acid (0.050 mL) was added. The reaction was

stirred at rt for 1 hour. Sodium triacetoxyborohydride (0.945 mmol) was

added and the reaction was stirred at room temperature for an additional 2

hours. The reaction was then quenched by the addition of water (2 mL) and aqueous NaOH

0 % w/w (2 mL), and extracted with CH Cl (2 mL), and the organic phases were dried over

5

2

MgSO , filtered and concentrated under reduced pressure. The crude product was purified

4

by column chromatography on silica gel (ethyl acetate/MeOH 9:1, 1% Et N), to give the

3

product N-Isopropyl nornicotine (90 mg, 70 % yield) as a yellow oil.

1

H NMR (400 MHz, CDCl ) δ 8.54 (d, J=2.0 Hz, 1H), 8.44 (dd, J=4.7 and 1.7 Hz, 1H), 7.71 (d,

3

J=7.7 Hz, 1H), 7.19 – 7.22 (m, 1H), 3.67 (t, J=7.6 Hz, 1H), 3.06 – 3.11 (m, 1H), 2.69 –2.76 (m,

1

H), 2.55 – 2.63 (m, 1H), 2.10 – 2.18 (m, 1H), 1.81 – 1.90 (m, 1H), 1.70 – 1.79 (m, 1H), 1.57 –

1

3

.66 (m, 1H), 0.98 (d, J=6.7 Hz, 3H), 0.90 (d, J=6.4 Hz, 3H); C NMR (100 MHz, CDCl ) δ

3

S7

1

49.3, 148.1, 141.2, 134.8, 123.4, 62.4, 49.0, 46.7, 35.7, 23.0, 22.4, 15.4. TOF-Ms (m/z) =

91.1 [M+H] .

+

2

.6 1-Propyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-2-ium (22)

Tryptamine hydrochloride (197 mg, 1 mmol) and butyraldehyde (90

µL, 1 mmol) were added to aqueous maleic acid buffer (10 mM, pH

2

°

.0, total volume 2 mL) and the solution was stirred overnight at 60

C. The salt precipitate and was filtered and washed with water (2

mL) to afford the product 1-Propyl-2,3,4,9-tetrahydro-1H-

pyrido[3,4-b]indol-2-ium (187 mg, 75% yield).

1

H NMR (400 MHz, CD OD) δ 7.49 (d, J=7.8 Hz, 1H), 7.38 (d, J=8.1 Hz, 1H), 7.17 (t, J=7.6 Hz,

3

1

9

2

1

H), 7.07 (t, J=7.4 Hz, 1H), 4.70 (dd, J=5.4, 3.5 Hz, 1H), 3.81 – 3.70 (m, 1H), 3.45 (ddd, J=12.6,

.3, 5.8 Hz, 1H), 3.20 – 2.98 (m, 2H), 2.34 – 2.19 (m, 1H), 2.01 – 1.87 (m, 1H), 1.71 – 1.55 (m,

1

3

H), 1.12 (t, J=7.3 Hz, 3H). C NMR (100 MHz, CD OD) δ 138.3, 130.3, 127.4, 123.5, 120.6,

3

19.1, 112.3, 107.1, 54.9, 43.1, 35.4, 28.7 19.5, 14.2.

2

.7 3-(1-Methylpiperidin-2-yl)pyridine (33)

Anabasine (100 mg, 0.62 mmol) was dissolved in DMF (4 mL) and NaH (1.5

mg, 0.62 mmol, 60 % dispersion in oil) was added. Methyl iodide (0.74

mmol) was added and the reaction was stirred at rt for 3 hours. TLC of the

reaction mixture revealed that the starting material was still present,

consequently the reaction temperature was increased to 60 °C for 2 hours. The reaction was

quenched by the addition of water (2 mL) and the mixture extracted with toluene (3 x 5 mL),

and the combined organic phases were dried over MgSO , filtered and concentrated under

4

reduced pressure. The crude product was purified by column chromatography on silica gel

(

ethyl acetate/MeOH 9:1 + 1% Et N), to give the product 3-(1-Methylpiperidin-2-yl)pyridine

3

(65 mg, 60 % yield) as a yellow oil.

1

13

H and C NMR data for 3-(1-methylpiperidin-2-yl)pyridine were consistent with those

7

reported in the literature.

3. Mechanism experiments

3

.1 Study of the stereoinversion of (R)-nicotine (4) using wildtype 6-HDNO

3

.1.1 Preparative procedure

In a 500 mL screw cap bottle, (R)-nicotine (100 mg, 0.62 mmol), BH NH (74

3

mg, 2.47 mmol) and whole cells wildtype 6-HDNO (2 g) were dissolved in

potassium phosphate buffer (30 mL, 1 M, pH = 7.8). The reaction was

performed at 37 °C with shaking at 250 rpm. After 16 hours HPLC analysis

(

CHIRALPAK® IC column; flowrate 1 mL / min; UV 280 nM; eluent= hexane/iPrOH 97:3 +

.1% DEA; Rt [(S)] = 18.7 min, Rt [(R)] = 20.5 min) showed full conversion to the (S)-

0

enantiomer and the reaction was worked up by adding aqueous 10 M NaOH. The aqueous

S8

phase was extracted with CH Cl (3 × 30 mL) and the combined organic phases were dried

2

over MgSO and concentrated under reduced pressure to give (S)-nicotine (93 mg, 93%

4

yield) as a yellow liquid.

2

8

20

[

α]_D= - 129.0 (c = 1.0, CHCl ). (lit. [α] = -138.0 (c= 1, CHCl )).

3

D

3

1

H NMR (400 MHz, CDCl ) δ 8.53 (b, J=2.1 Hz, 1H), 8.50 (dd, J=4.7, 1.7 Hz, 1H), 7.72 (dt,

3

J=7.8, 1.9 Hz, 1H), 7.26 (app q, J=5.1, 1H), 3.26 (t, J=8.0 Hz, 1H), 3.10 (t, J=8.3 Hz, 1H), 2.33

(

2

app q, J=8.0 Hz, 1H), 2.26 – 2.19 (m, 1H), 2.18 (s, 3H), 2.06 – 1.93 (m, 1H), 1.88 – 1.70 (m,

1

3

H); C NMR (100 MHz, CDCl ) δ: 149.8, 148.9, 138.9, 135.1, 123.8, 69.1, 57.2, 40.5, 35.3,

3

2.8.

1

Figure S1. H NMR (400 MHz, CDCl ) of the crude extracts of the product (S)-nicotine from the

3

biotransformation with wildtype 6-HDNO.

S9

13

Figure S2. C NMR (100 MHz, CDCl ) of the crude extracts of the product (S)-nicotine from the

3

biotransformation with wildtype 6-HDNO.

3

.1.2 Deuteration study (A): using a deuterated reducing agent

The reaction was set up using (R)-nicotine (50 mg, 0.31 mmol), whole cells

wildtype 6-HDNO (1.5 g) in potassium phosphate buffer (30 mL, 1 M, pH =

7

.8), performed at 37 °C with shaking at 250 rpm and monitored by HPLC

(CHIRALPAK® IC column; flowrate 1 mL / min; UV 280 nM; eluent=

hexane/iPrOH 97:3 + 0.1% DEA; Rt [(S)] = 18.7 min, Rt [(R)] = 20.5 min). After 16 hours the

disappearance of the substrate was detected, together with the absence of formation of any

other peaks in the HPLC profile (corresponding for example to pseudoxynicotine (open form

of nicotine)). NaBD CN (77.5 mg, 1.23 mmol) was added and the reaction mixture was left

3

overnight at 37 °C, with shaking at 250 rpm. After observing the full conversion to the (S)-

enantiomer by HPLC, adding aqueous NaOH (2 mL, 10 M) was added and the mixture

extracted with CH Cl (3 × 50 mL). The aqueous phase was then continuously extracted

2

overnight with CH Cl The combined organic phases were dried over MgSO₄and

2

2.

concentrated under reduced pressure to give (S)-nicotine (48 mg, 96% yield) and no

presence of 4‐(methylamino)‐1‐(3‐pyridyl)‐1‐butanol as by-product).

1

13

H NMR (Figure S3, 400 MHz, CDCl ) and C NMR (Figure S4, 100 MHz, CDCl ) differ from

3

1

the corresponding NMR spectrums of nicotine: the signal at 3.10 ppm (for the H spectrum)

and at 69.1 ppm (for the C NMR spectrum) corresponding to the CH are not present

1

3





S10

1

Figure S3. H NMR (400 MHz, CDCl ) of the crude extracts of the deuterated (S)-nicotine from the

3

biotransformation with wildtype 6-HDNO and NaBD CN.

3

13

Figure S4. C NMR (100 MHz, CDCl ) of the crude extracts of the deuterated (S)-nicotine from the

3

biotransformation with wildtype 6-HDNO and NaBD CN .

3

S11

3

.1.3 Deuteration study (B): using a deuterated reducing agent and deuterium oxide

The reaction was set up using (R)-nicotine (50 mg, 0.31 mmol), freeze-dried

whole cells wildtype 6-HDNO (1.5 g) and NaBD CN (77.5 mg, 1.23 mmol) in

3

D₂O-potassium phosphate buffer (30 mL, 1 M, pH = 7.8) and performed at 37

°

C with shaking at 250 rpm. After 16 hours HPLC analysis (CHIRALPAK® IC

column; flowrate 1 mL / min; UV 280 nM; eluent= hexane/iPrOH 97:3 + 0.1% DEA; Rt [(S)] =

8.7 min, Rt [(R)] = 20.5 min) showed full conversion to product. Aqueous NaOH (2 mL, 10

M) was added and the mixture extracted with CH Cl (3 × 50 mL), then the aqueous phase

1

2

was continuously extracted overnight with CH Cl The combined organic phases were dried

2

2.

over MgSO and concentrated under reduced pressure to give (S)-nicotine (47 mg, 94%

4

yield) as a yellow liquid. There was no presence of 4‐(methylamino)‐1‐(3‐pyridyl)‐1‐butanol

as product or by-product.

1

13

H NMR (Figure S5, 400 MHz, CDCl ) and C NMR (Figure S6, 100 MHz, CDCl ) spectra again

3

differ from the corresponding NMR spectrumss of nicotine: the signal at 3.10 ppm (for the

1

3

protonic spectrum) and at 69.1 ppm (for the C NMR) corresponding to the CH are not



present

1

Figure S5. H NMR (400 MHz, CDCl ) of the crude extracts of the deuterated (S)-nicotine from the

3

biotransformation with wildtype 6-HDNO and NaBD CN in deuterium oxide .

3

S12

13

Figure S6. C NMR (100 MHz, CDCl ) of the crude extracts of the deuterated (S)-nicotine from the

3

biotransformation with wildtype 6-HDNO and NaBD CN in deuterium oxide.

3

Figure S7. HSQC spectrum of the crude extracts of the deuterated (S)-nicotine from the

biotransformation with wildtype 6-HDNO and NaBD CN in deuterium oxide.

3

S13

3

.2 Study of the deracemisation of 6-hydroxy-nicotine (1) using wildtype 6-HDNO

.2.1 Preparative procedure

In a 500 mL screw cap bottle, (rac)-6-hydroxy-nicotine (100 mg, 0.56

mmol), BH NH (67 mg, 2.2 mmol) and purified wildtype 6-HDNO (10

3

mg) were dissolved in potassium phosphate buffer (30 mL, 1 M, pH =

.8). The reaction was performed at 37 °C with shaking at 250 rpm.

7

After 6 hours HPLC analysis (CHIRALCEL® DAICEL IA column; flowrate 1.5 mL / min; UV 280

nM; eluent= hexane/iPrOH 95:5 + 0.1% DEA; Rt [(R)] = 19.8 min, Rt [(S)]) showed full

conversion to the (S)-enantiomer. Aqueous NaOH (2 mL, 10 M) was added and the mixture

extracted with CH Cl (3 × 40 mL) and the combined organic phases were dried over MgSO

2

4

and concentrated under reduced pressure. The residue was, purified by column

chromatographic on silica gel (CH Cl /MeOH 90:10) to give (S)-6-hydroxy-nicotine (55 mg,

2

5

5% yield) as a brown solid.

1

H NMR (400 MHz, CDCl ) δ 13.34 (bs, 1H), 7.53 (dd, J=9.4, 2.1 Hz, 1H), 7.25 (s, 1H), 6.56 (d,

3

J=9.4 Hz, 1H), 3.20 – 3.12 (m, 1H), 2.82 (t, J=8.3 Hz, 1H), 2.28 ‒ 2.15 (m, 1H), 2.11 (s, 3H),

1

3

2

1

.10 – 2.00 (m, 1H), 1.98 – 1.82 (m, 1H), 1.82 – 1.58 (m, 2H). C NMR (100 MHz, CDCl ) δ

65.4, 141.5, 132.8, 121.6, 120.4, 67.6, 56.6, 40.0, 33.7, 22.3.

3

1

Figure S8. H NMR (400 MHz, CDCl ) of the crude extracts of the product (S)-6-hydroxy-nicotine from

3

the biotransformation with wildtype 6-HDNO.

S14

13

Figure S9. C NMR (100 MHz, CDCl ) of the product (S)-6-hydroxy-nicotine from the

3

biotransformation with wildtype 6-HDNO.

3

.2.2 Deuteration study: using a deuterated reducing agent

The reaction was set up using (rac)-6-hydroxy-nicotine (50 mg, 0.28

mmol), NaBD CN (73.8 mg, 1.12 mmol) and whole cells wildtype 6-

3

HDNO (1.5 g) in potassium phosphate buffer (30 mL, 1 M, pH = 7.8). The

reaction was performed at 37 °C with shaking at 250 rpm and monitored

by HPLC (CHIRALCEL® DAICEL IA column; flowrate 1.5 mL/min; UV 280 nM; eluent=

hexane/iPrOH 95:5 + 0.1% DEA; Rt [(R)] = 19.8 min, Rt [(S)]). After 8 hours HPLC analysis

showed full conversion, aqueous 10 M NaOH (2 mL, 10 M) was addedand the reaction

mixture was extracted with CH Cl (3 × 50 mL). The aqueous phase was then continuously

2

extracted overnight with CH Cl The combined organic phases were dried over MgSO ,

2

2.

4

concentrated under reduced pressure and then purified on silica gel (CH Cl /MeOH 90:10)

2

to give (S)-6-hydroxy-nicotine (38 mg, 76% yield) as a brown solid.

1

H NMR spectrum (Figure S10, 400 MHz, CDCl ) differs for one signal’s integration from the

H NMR spectrum of racemic 6-hydroxy-nicotine. The substrate of this biotransformation is

3

1

the racemic 6-hydroxy-nicotine and, consequently, the product will have the deuterium in

the  position with ratio CH: CD 1:1.

S15

1

Figure S10. H NMR (400 MHz, CDCl ) of the product (S)-6-hydroxy-nicotine from the

3

biotransformation with wildtype 6-HDNO and NaBD CN.

3

4. Biotransformations – procedures and analyses

4

.1 General procedure for biotransformations

Typically, half a gram of E. coli cells containing the expressed HDNO protein were

resuspended in 1 M phosphate buffer (pH = 7.8) to a final volume of 5 mL. Substrates were

added for a final concentration of 10 mM, followed by 40 mM (final concentration) of

NH BH . The biotransformation was performed at 37 °C with shaking at 250 rpm. Samples

3

(300 µl) were basified with 10 M NaOH and extracted with 1 mL dichloromethane of which

typically 10 µL was injected onto the column for analysis by HPLC or GC methods. In the

analysis details below, this general procedure was followed to prepare the samples unless

otherwise stated. For samples analysed by OD-H column methods, dichloromethane was

evaporated and the product redissolved in tert-butyl methyl ether.

4

.2 Analysis of biotransformations carried out with wildtype 6-HDNO

4

.2.1 (S)-6-Hydroxy nicotine (1)

The biotransformation was carried out with (rac)-(1). After 1 hour HPLC

analysis showed 99 % e.e. Determination of the enantiomeric excess was

carried out by HPLC on a chiral phase column: CHIRALCEL® IA column;

flowrate 1.5 mL / min; UV 280 nM; eluent= hexane/iPrOH 95:5 + 0.1%

DEA, Rt [(R)] = 19.8 min, Rt [(S)] = 23.1 min.

S16

The absolute stereochemistry was assigned by previous reports in which the enzyme is only

8

,9

active on the (R)-enantiomer and not the (S)-enantiomer, and by comparison to nicotine.

(

R)

(

S)

Figure S11. Chiral phase HPLC trace of (rac)-(1) conversion with 6-HDNO showing the signals of (rac)

compound (1) (top) and the deracemization product (S)-(1) (bottom).

4

.2.2 (S)-Nicotine (4)

The biotransformation was carried out with (R)-(4) (made by deracemisation

of (S)-nicotine with MAO-N, see 2.1.). After 16 hours HPLC analysis showed

9 % e.e. Determination of the enantiomeric excess was carried out by HPLC

9

on a chiral phase column: CHIRALPAK® IC column; flowrate 1 mL / min; UV

2

80 nM; eluent= hexane/iPrOH 97:3 + 0.1% DEA; Rt [(S)] = 18.7 min, Rt [(R)] = 20.5 min. The

absolute configuration was assigned by comparison to a commercial standard of (S )-nicotine

0

and by comparison to the deracemisation product with MAO-N ((R)-enantiomer).

S17

(

R)

(

S)

Figure S12. Chiral phase HPLC trace of (R)-(4) conversion with 6-HDNO showing the signals of (R)-(4)

substrate (top) and the deracemization product (S)-(4) (bottom).

4

.2.3 (S)-6-Amino nicotine (8)

The biotransformation was carried out with (rac)-(8). After 4 hours

HPLC analysis showed 99 % e.e. Determination of the enantiomeric

excess was carried out by HPLC on a chiral phase column: CHIRALPAK®

IC column; flowrate 1 mL / min; UV 280 nM; eluent= hexane/iPrOH 98:2

+

0.1% DEA; Rt [(S)] = 86.2 min, Rt [(R)] = 91.9 min. The absolute stereochemistry was

assigned by analogy to 6-hydroxy nicotine and comparison to MAO-N (unpublished data).

(

S)

(

R)

Figure S13. Chiral phase HPLC trace of (rac)-(8) conversion with 6-HDNO showing the signals of

racemic compound (8) (top) and the deracemization product (S)-(8) (bottom).

S18

4

.2.4 (S)-N-Ethyl nornicotine (9)

The biotransformation was carried out with (rac)-(9). After 93 hours HPLC

analysis showed 90 % e.e. Determination of the enantiomeric excess was

carried out by HPLC on a chiral phase column: CHIRALPAK® IC column;

flowrate 1 mL / min; UV 265 nM; eluent= hexane/iPrOH 95:5 + 0.1% DEA; Rt

[

(R)] = 12.8 min, Rt [(S)] = 12.1 min. The absolute stereochemistry was assigned by analogy

to nicotine.

(

S)

(R)

Figure S14. Chiral phase HPLC trace of (rac)-(9) conversion with 6-HDNO showing the signals of

racemic compound (9) (top) and the deracemization product (S)-(9) (bottom).

4

.2.5 (S)-N-Isopropyl nornicotine (10)

The biotransformation was carried out with (rac)-(10). After 96 hours HPLC

analysis showed 54 % e.e. Determination of the enantiomeric excess was

carried out by HPLC on a chiral phase column: CHIRALPAK® IC column;

flowrate 1 mL / min; UV 265 nM; eluent = hexane/iPrOH 99:1 + 0.1% DEA; Rt

[

(R)] = 25.1 min, Rt [(S)] = 23.6 min. The absolute stereochemistry was assigned by analogy

to nicotine.

S19

(

S)

(R)

Figure S15. Chiral phase HPLC trace of (rac)-(10) conversion with 6-HDNO showing the signals of

racemic compound (10) (top) and the deracemization product (S)-(10) (bottom).

4

.2.6 (S)-Crispine A (26)

The biotransformation was carried out with (rac)-(26). After 4 hours

HPLC analysis showed 99 % e.e. Determination of the enantiomeric

excess was carried out by HPLC on a chiral phase column: CHIRALCEL®

DAICEL OD-H column; flowrate 1 mL / min; UV 254 nM; eluent=

hexane/iPrOH 90:10 + 0.1% DEA; Rt [(R)] = 15.8 min, Rt [(S)] = 10.3 min. The absolute

stereochemistry was assigned by comparison of HPLC traces with the MAO-N

biotransformation product ((R)-enantiomer).

(

S)

(

R)

Figure S16. Chiral phase HPLC trace of (rac)-(26) conversion with 6-HDNO showing the signals of

racemic compound (26) (top) and the deracemization product (S)-(26) (bottom).

S20

4

.2.7 (S)- 3-(1-Methylpiperidin-2-yl)pyridine (33)

The biotransformation was carried out with (rac)-(33). After 48 hours HPLC

analysis showed 84 % e.e. Determination of the enantiomeric excess was

carried out by HPLC on a chiral phase column: CHIRALCEL® DAICEL OD-H

column; flowrate 1 mL/min; UV 254 nM; eluent= hexane/iPrOH 95:5 + 0.1%

DEA; Rt [(S)] = 6.5 min, Rt [(R)] = 8.1 min. The absolute stereochemistry was assigned by

comparison of its optical rotation with the literature (see preparative procedure).

(

S)

(

R)

Figure S17. Chiral phase HPLC trace of (rac)-(33) conversion with 6-HDNO showing the signals of

racemic compound (33) (top) and the deracemisation product (S)-(33) (bottom).

4

.3 Analysis of biotransformations carried out with variant E350L/E352D

4

.3.1 (S)-6-Hydroxy nicotine (1)

The biotransformation was carried out with (rac)-(1). After 1 hour HPLC

analysis showed 99 % e.e. Determination of the enantiomeric excess was

carried out by HPLC on a chiral phase column: CHIRALCEL® DAICEL IA

column; flowrate 1.5 mL / min; UV 280 nM; eluent= hexane/iPrOH 95:5

+

0.1% DEA; Rt [(R)] = 19.8 min, Rt [(S)] = 23.1 min. The absolute stereochemistry was

assigned by previous reports in which the enzyme is only active on the (R)-enantiomer and

8,9

not the (S)-enantiomer.

S21

(

R)

(S)

Figure S18. Chiral phase HPLC trace of (rac)-(1) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (1) (top) and the deracemisation product (S)-(1) (bottom).

4

.3.2 (S)-Nornicotine (7)

The biotransformation was carried out with (rac)-(7). After 28 hours HPLC

analysis showed 95 % e.e. Determination of the enantiomeric excess was

carried out by HPLC on a chiral phase column: CHIRALCEL® DAICEL OD-H

column; flowrate 1 mL / min; UV 254 nM; eluent= hexane/iPrOH 95:5 + 0.1%

DEA; Rt [(R)] = 26.9 min, Rt [(S)] = 31.1 min.

The absolute stereochemistry was assigned by comparison of HPLC traces with the MAO-N

biotransformation product ((R)-enantiomer) (unpublished data) and analogy with 2-phenyl

pyrrolidine.

(

R)

(

S)

Figure S19. Chiral phase HPLC trace of (rac)-(7) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (7) (top) and the deracemisation product (S)-(7) (bottom).

S22

4

.3.3 (S)-6-Amino nicotine (8)

The biotransformation was carried out with (rac)-(8). After 3 hours

HPLC analysis showed 99 % e.e. Determination of the enantiomeric

excess was carried out by HPLC on a chiral phase column: CHIRALPAK®

IC column; flowrate 1 mL / min; UV 280 nM; eluent= hexane/iPrOH

-

1

9

8:2 + 0.1% DEA, flow rate 1.5 mL / min ; Rt [(S)] = 86.2 min, Rt [(R)] = 91.9 min. The

absolute stereochemistry was assigned by analogy to 6-hydroxy nicotine.

(

S)

(

R)

Figure S20. Chiral phase HPLC trace of (rac)-(8) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (8) (top) and the deracemisation product (S)-(8) (bottom).

4

.3.4 (S)-2-Phenylpyrrolidine (12)

The biotransformation was carried out with (rac)-(12). After 48 hours HPLC

analysis showed 99 % e.e. Determination of the enantiomeric excess was

®

carried out by HPLC on a chiral phase column: CHIRALPAK® IC column;

flowrate 1 mL / min; UV 265 nM; eluent= hexane/iPrOH 90:10 + 0.1% DEA; Rt [(R)] = 7.5 min,

Rt [(S)] = 8.2 min. The absolute stereochemistry was assigned by comparison of HPLC traces

2

,11

with the MAO-N biotransformation product ((R)-enantiomer).

S23

(

R)

(S)

Figure S21. Chiral phase HPLC trace of (rac)-(12) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (12) (top) and the deracemisation product (S)-(12) (bottom).

4

.3.5 (S)-2-(p-Methoxyphenyl)-pyrrolidine (13)

The biotransformation was carried out with (rac)-(13). After 44 hours

HPLC analysis showed 99 % e.e. Determination of the enantiomeric

excess was carried out by HPLC on a chiral phase column: CHIRALPAK®

IE column; flowrate 1 mL / min; UV 265 nM; eluent= hexane/iPrOH

9

0:10 + 0.1% DEA; Rt [(R)] = 12.6 min, Rt [(S)] = 13.9 min. The absolute stereochemistry was

assigned by comparison of HPLC traces with the MAO-N biotransformation product ((R)-

enantiomer) (unpublished data) and analogy with 2-phenyl pyrrolidine.

S24

(

R)

(S)

Figure S22. Chiral phase GC trace of (rac)-(13) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (13) (top) and the deracemisation product (S)-(13) (bottom).

4

.3.6 (S)-2-Cyclohexylpyrrolidine (14)

The biotransformation was carried out with (rac)-(14). After 48 hours GC

analysis showed 99 % e.e. Determination of the enantiomeric excess was by

GC on a chiral phase column. (14) was acetylated for GC analysis using

trifluoracetic anhydride. Column used: Supelco β-DEX 325; carrier gas= Helium; injector

-

1

temperature 250 ˚C; detector temperature (FID) 250 ˚C; flow 1.7 mL·min ; temperature

program = isothermal 115 °C, Rt [(S)] = 48.6 min, Rt [(R)] = 50.5 min. The absolute

stereochemistry was assigned by comparison of GC traces with the MAO-N

0

biotransformation product ((R)-enantiomer).

(

S)

(

R)

S25

Figure S23. Chiral phase GC trace of (rac)-(14) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (14) (top) and the deracemisation product (S)-(14) (bottom).

4

.3.7 (S)-6,7-Dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (15)

The biotransformation was carried out with (rac)-(15). After 48 hours

HPLC analysis showed 81 % e.e. Determination of the enantiomeric

excess was carried out by HPLC on a chiral phase column: CHIRALCEL®

DAICEL OD-H column; flowrate 1 mL / min; UV 280 nM; eluent=

hexane/iPrOH 90:10 + 0.1% DEA; Rt [(R)] = 17.3 min, Rt [(S)] = 12.9 min. The absolute

stereochemistry was assigned by comparison of HPLC traces with the MAO-N

biotransformation product ((R)-enantiomer).

(

S)

(R)

Figure S24. Chiral phase HPLC trace of (rac)-(15) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (15) (top) and the deracemisation products, predominantly (S)-(15)

(

bottom).

4

.3.8 (S)-2-Isobutylazepane (18)

The biotransformation was carried out with (rac)-(18). After 24 hours GC

analysis showed 99 % e.e. Determination of the enantiomeric excess was by

GC on a chiral phase column: (18) was acetylated for GC analysis using

S26

acetic anhydride and triethylamine. Column used: Varian Chirasil-dex CB; carrier gas=

-

1

Helium; injector temperature 200 ˚C; detector temperature (FID) 250 ˚C; flow 1.0 mL·min ;

-

1

temperature program = from 90 °C to 150 °C with slope 10 °C·min and from 150 °C to 200

-

1

°

C with slope 1 °C·min ; Rt [(R)] = 13.1 min, Rt [(S)] = 13.6 min. The absolute

stereochemistry was assigned by comparison of HPLC traces with the MAO-N

biotransformation product ((R)-enantiomer) (unpublished data).

(

R)

(S)

Figure S25. Chiral phase GC trace of (rac)-(18) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (18) (top) and the deracemisation product (S)-(18) (bottom).

4

.3.9 (R)-1-Phenyl-1,2,3,4-tetrahydroisoquinoline (19)

The biotransformation was carried out with (rac)-(19). After 120 hours HPLC

analysis showed 84 % e.e. Determination of the enantiomeric excess was

carried out by HPLC on a chiral phase column: CHIRALCEL® DAICEL OD-H

column; flowrate 1 mL / min; UV 265 nM; eluent = hexane/iPrOH 97:3 + 0.1%

DEA; Rt [(R)] = 17.6 min, Rt [(S)] = 10.2 min. The absolute stereochemistry

was assigned by comparison of HPLC traces with the MAO-N biotransformation product ((S)-

enantiomer). The apparent change in selectivity is merely due to the Cahn-Ingold-Prelog

naming convention.

(

S)

(

R)

S27

Figure S26. Chiral phase HPLC trace of (rac)-(19) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (19) (top) and the predominant deracemization product (R)-(15)

(

bottom).

4

.3.10 (S)-1-(2-Bromophenyl)-1,2,3,4-tetrahydroisoquinoline (20) (oxidation only)

The biotransformation was carried out with (rac)-(20). After 48 hours HPLC

analysis showed 99 % e.e. Determination of the enantiomeric excess was

carried out by HPLC on a chiral phase column: CHIRALCEL® DAICEL OD-H

column; flowrate 1 mL / min; UV 265 nM; eluent= hexane/iPrOH 80:20 +

0

.1% DEA; Rt [(R)] = 6.2 min, Rt [(S)] = 6.8 min. The absolute

stereochemistry was assigned by analogy to 1-phenyl-1,2,3,4-tetrahydroisoquinoline. (N.B.

The imine of this compound is difficult to reduce under the conditions used in this work

hence only the oxidation was studied).

(

S)

(

R)

Figure S27. Chiral phase HPLC trace of (rac)-(20) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (20) (top) and the oxidation products (S)-(20) and imine (bottom).

4

.3.11 (S)-1-(Naphthalen-1-yl)-1,2,3,4-tetrahydroisoquinoline (21) (oxidation only)

The biotransformation was carried out with (rac)-(21). After 24 hours HPLC

analysis showed that the enzyme had some selectivity for the (R)

enantiomer. Analysis was carried out by HPLC on a chiral phase column:

CHIRALCEL® DAICEL OD-H column; flow rate 1 mL / min; UV 265 nM; eluent=

S28

hexane/iPrOH 80:20 + 0.1% DEA; Rt [(R)] = 5.7 min, Rt [(S)] = 7.4 min. The absolute

stereochemistry was assigned by analogy to 1-phenyl-1,2,3,4-tetrahydroisoquinoline. (N.B.

The imine of this compound is difficult to reduce under the conditions used in this work

hence only the oxidation was studied).

(

R)

(S)

Figure S28. Chiral phase HPLC trace of (rac)-(21) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (21) (top) and the oxidation products, predominantly (S)-(21) and imine

(

bottom).

4

.3.12 (S)-1-Propyl-4,9-tetrahydro-3H-β-carboline (22)

The biotransformation was carried out with (rac)-(22). After 28 hours

HPLC analysis showed 29 % e.e. Determination of the enantiomeric

excess was carried out by HPLC on a chiral phase column: CHIRALPAK® IC

column; flowrate 1 mL / min; UV 254 nM; eluent= hexane/iPrOH 95:5 +

0

.1% DEA; Rt [(R)] = 22.9 min, Rt [(S)] = 14.1 min.

The absolute stereochemistry was assigned by comparison of HPLC traces with the MAO-N

biotransformation product ((S)-enantiomer).

S29

(

S)

(

R)

Figure S29. Chiral phase HPLC trace of (rac)-(22) conversion with 6-HDNO E350L/E352D showing the

signals of racemic compound (22) (top) and the predominant product, (S)-(22) (bottom).

4

.3.13 (S)-Crispine A (26)

The biotransformation was carried out with (rac)-(26). After 4 hours

HPLC analysis showed 99 % e.e. Determination of the enantiomeric

excess was carried out by HPLC on a chiral phase column: CHIRALCEL®

DAICEL OD-H column; flowrate 1 mL / min; UV 254 nM; eluent=

hexane/iPrOH 90:10 + 0.1% DEA; Rt [(R)] = 15.8 min, Rt [(S)] = 10.3 min. The absolute

stereochemistry was assigned by comparison of HPLC traces with the MAO-N