Better than Nature: nicotinamide biomimetics that outperform natural ...
Supporting Information for:
Better than Nature: nicotinamide biomimetics that outperform natural coenzymes Tanja Knaus,a,‡ Caroline E. Paul,b,‡ Colin W. Levy a, Simon de Vries,b Francesco G. Mutti a, Frank Hollmann,*,b and Nigel S. Scrutton*,a a
BBSRC/EPSRC Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, Faculty of Life Sciences, 131 Princess Street, Manchester M1 7DN, United Kingdom, [email protected]
b
Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628BL Delft (The Netherlands) [email protected]
Table of Contents 1.
General information
2
2.
Synthesis of the nicotinamide coenzyme analogues (mNADHs, mNAD+s and tetrahydro‐mNADHs)
2
2.1. Reduction of mNADH nicotinamide biomimetics to 1,4,5,6 tetrahydro‐mNADH derivatives 2 (mNADH4s) 3.
Expression and purification of the ERs
16
4.
Determination of the molar extinction coefficient for mNADHs (1‐5)
16
5.
Reductive half‐reaction
18
6. Steady‐state kinetics for the conversion of 2‐cyclohexen‐1‐one by ERs using varied concentration of coenzyme 23 6.1.
Determination of the saturation concentration of substrate 2‐cyclohexen‐1‐one for ERs
23
6.2.
Steady‐state kinetics
24
7.
Biocatalytic transformations
28
7.1.
Reaction conditions
28
7.2.
Analytical methods
28
7.3.
GC chromatograms
28
7.4.
Enantiomeric excess of 6 reduced to (R)‐6a by various ERs with different coenzymes
29
8.
mNAD+s recycling with the rhodium complex
31
9.
X‐ray crystallography
33
9.1. 10.
Data Collection and Refinement Beyond the ER family: Enzymes that accept the biomimetic analogues
34 35
10.1. non flavin‐dependent double bond reductase from Nicotiana tabacum
35
10.2. NADPH‐dependent oxidase
37
11.
38
References
S1
1. General information All commercial reagents and solvents were purchased from Sigma‐Aldrich, Fluka, Acros Organics and Alfa Aesar with the highest purity available and used as received. NMR spectra were recorded on a Bruker AV‐ 300 spectrometer at 300 (1H) and 75 (13C) MHz or on a Bruker Spectrospin 400 Ultrashield spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) relative to Me4Si (δ 0.00) using deuterated solvent (DMSO‐d6, D2O or CDCl3) as an internal standard.
2. Synthesis of the nicotinamide coenzyme analogues (mNADHs, mNAD+s and tetrahydro‐ mNADHs) The mNADHs (1‐5) and mNAD+s (1‐5)a were synthesised and characterised as previously described.1 NMR data obtained corresponded to the previously reported characterisation. Once synthesised, the compounds were kept at ‐20 C for storage. 2.1. Reduction of mNADH nicotinamide biomimetics to 1,4,5,6 tetrahydro‐mNADH derivatives (mNADH4s) The aim was to generate a further reduced form of the reduced mNADHs (1‐5). The 1,4,5,6 tetrahydro‐ mNADHs (mNADH4 (1‐5)b) were synthesised to bind in the active site of the ERs generating a “dead‐end” complex which should be suitable for crystallisation and structural biochemical characterisation. The general structure of the tetrahydro‐biomimetics is as follows: O X N R
Synthesis of 1‐benzyl‐1,4,5,6‐tetrahydropyridine‐3‐carboxamide (1b) O
O NH2
N
H2 , 1 atm ethanol
1
NH2
Pd 10% on activated carbon N
1b
Pd catalyst on carbon (25 mg; extent of labelling 10% w w‐1 loading matrix activated carbon support, Sigma Cat. 20, 569.9) was added into a Schlenk tube containing dried ethanol (6 mL). Then, biomimetic 1 (100 mg, 0.467 mmol) was added under inert atmosphere (no stirring). The solution became yellow.
S2
Finally, the Schlenk tube was evacuated and filled with hydrogen at atmospheric pressure. The reaction was run for approximately 20 h. A small aliquot of the reaction mixture was analysed by GC‐MS as well as TLC. After removal of the Pd/C, the solution was colourless, indicating that all the starting material was reduced. The work‐up was performed by filtering over celite and extensive washing with methanol. The clear solution was evaporated to afford a colourless oil (80 mg of crude product). Judging from GC‐MS analysis, the conversion into the desired reduced biomimetic 1b was 50% whereas the rest of the material were side‐products generated by further reduction and/or cleavage of 1b. Therefore, the reaction was repeated as previously described using biomimetic 1 (100 mg) and Pd/C (25 mg) in dried ethanol (6 mL). The reaction time was shortened to 2h and 30 min to determine whether a better chemoselectivity could be obtained. Reducing the reaction time led to the formation of higher amounts of the desired product 1b. The composition of the product mixture (determined by GC‐MS) was: 82% of 1b, 13% of the fully reduced by‐product (i.e. 1‐benzylpiperidine‐3‐carboxamide) and 4% of cleaved derivatives. The aliquots of the crude mixture from the first and the second reaction were combined and purified together by column chromatography on silica. Conditions for column chromatography: Column diameter: 1 cm, Elution: CH2Cl2, 50 mL, fractions 1 – 7; CH2Cl2 / CH3OH, 98:2, v v‐1, 50 mL, fractions 8 – 13; CH2Cl2 / CH3OH, 96:4, v v‐1, 50 mL, fractions 14 – 19; CH2Cl2 / CH3OH, 94:6, v v‐1, 50 mL, fractions 20 – 25 1b was obtained as a yellow oil: m = 147 mg (0.680 mmol), total yield: 73%
GC‐MS characterisation
O NH2 N
S3
NMR characterisation 1H‐NMR (CDCl , 400 MHz, δ (ppm), J (Hz)): 1.77 (2H, CH , m), 2.15 (2H, CH , t, 3J= 6.4 Hz), 2.89 (2H, CH , t, 3 2 2 2 3J= 5.6 Hz), 4.20 (2H, CH , s), 5.37 (2H, NH , broad), 7.12 – 7.15 (2H, CH aromatic, m), 7.20 – 7.27 (3H, CH, 2 2
aromatic). 13C‐NMR (CDCl
3, 400 MHz, δ (ppm): 19.1, 20.0, 43.6, 58.4, 94.2, 126.2, 126.3, 127.3, 135.7, 143.4, 169.7.
1H‐NMR:
S4
13C‐NMR
DEPT‐135
S5
Synthesis of 1‐butyl‐1,4,5,6‐tetrahydropyridine‐3‐carboxamide (2b) O
O NH2
N
NH2
Pd 10% on activated carbon H2, 1 atm ethanol
N
2
2b
Pd catalyst on carbon (30 mg; extent of labelling 10% w w‐1 loading matrix activated carbon support, Sigma Cat. 20, 569.9) was added into a Schlenk tube containing dried ethanol (6 mL). Then, biomimetic 2 (150 mg, 0.832 mmol) was added under inert atmosphere. Finally, the Schlenk tube was evacuated and filled with hydrogen at atmospheric pressure. The reaction was run for approximately 16 h. A small aliquot of the reaction mixture was analysed by GC‐MS as well as TLC. GC‐MS analysis showed that the “mono”‐reduced desired product 2b was formed with 31% conversion. The remaining was the “double”‐reduced compound (i.e. 1‐butylpiperidine‐3‐carboxamide). The work‐up was performed by filtering over celite and extensive washing with methanol. The clear solution was evaporated to afford a colourless crude oil that was purified by column chromatography. Conditions for column chromatography: The crude mixture was purified by column chromatography on silica. Column diameter: 1 cm; Elution: CH2Cl2 / CH3OH, 98:2, v v‐1, 200 mL, fractions 1 – 24; CH2Cl2 / CH3OH, 95:5, v v‐1, 200 mL, fractions 25 – 48 A green oil was obtained. The amount of the product 2b was: m = 48 mg (0.2634 mmol), yield: 32%.
S6
GC‐MS characterisation: O NH2 N
NMR characterisation 1H‐NMR (CDCl
3, 400 MHz, δ (ppm), J (Hz)): 0.84 (3H, CH3,
3J= 7.2 Hz), 1.22 (2H, CH , m), 1.44 (2H, CH , m), 2 2
1.81 (2H, CH2, m), 2.15 (2H, CH2, 3J= 6.4 Hz), 3.02 (4H, CH2, m), 5.32 (2H, NH2, broad), 7.31 (1H, CH, s). 13C‐NMR (CDCl
3, 400 MHz, δ (ppm): 12.8, 18.8, 19.6, 20.5, 29.6, 44.3, 54.6, 93.2, 143.7, 170.0.
S7
1H‐NMR:
13C‐NMR:
S8
13C‐DEPT135‐NMR:
Synthesis of 1‐(1‐benzyl‐1,4,5,6‐tetrahydropyridin‐3‐yl) ethanone (3b) O
O Pd 10% on activated carbon
N
H2 , 1 atm ethanol
N
3
3b
Pd catalyst on carbon (30 mg; extent of labelling 10% w w‐1 loading matrix activated carbon support, Sigma Cat. 20, 569.9) was added into a Schlenk tube containing dried ethanol (6 mL). Then, biomimetic 3 (150 mg, 0.704 mmol) was added under inert atmosphere. Finally, the Schlenk tube was evacuated and filled with hydrogen at atmospheric pressure. The reaction was run for approximately 16 h. A small aliquot of the reaction mixture was analysed by GC‐MS as well as TLC. GC‐MS showed that the desired product 3b was formed with quantitative conversion (> 99%). Therefore, in this case, the hydrogenation was completely chemo‐ and regioselective, even when the reaction time was prolonged to 16 h. S9
The work‐up was performed by filtering over celite and extensive washing with methanol. The clear solution was evaporated to afford a red oil (150 mg, 0.697 mmol). The total yield was 99%. GC‐MS characterisation O
N
NMR characterisation 1H‐NMR (CDCl , 400 MHz, δ (ppm), J (Hz)): 1.71 (2H, CH ,m), 2.09 (3H, CH , s), 2.25 (2H, CH , t, 3J= 6.4 Hz), 3 2 3 2
2.97 (2H, CH2, t, 3J= 5.6 Hz), 4.28 (2H, CH2, s), 7.14 – 7.16 (2H, CH aromatic), 7.21 – 7.32 (3H, CH aromatic), 7.39 (1H, CH, s). 13C‐NMR (CDCl
3, 400 MHz, δ (ppm): 19.1, 22.0, 43.9, 58.2, 125.4, 126.1, 127.0, 134.6, 146.2, 191.5.
S10
1H‐NMR:
13C‐NMR:
S11
13C‐DEP135‐NMR:
Synthesis of 1‐benzyl‐1,4,5,6‐tetrahydropyridine‐3‐carbonitrile (5b) N
N Pd(OH)2 10 - 20% on activated charcoal
N
H2 , 1 atm EtOAc
5
N
5b
Pd(OH)2 (60 mg on activated charcoal, moistened with water, 10‐20% Pd basis based on dry substance; Sigma Cat. 76063‐5G) was added into a Schlenk tube containing dried ethyl acetate (12 mL). Then, biomimetic 5 (150 mg, 0.764 mmol) was added under inert atmosphere. Finally, the Schlenk tube was evacuated and filled with hydrogen at atmospheric pressure. The reaction was monitored over time (3 h – 6h – 9 h) by GC‐MS. The reaction was complete after 9 h, yielding the desired product 5b. The work‐up was performed by filtering over celite and extensive washing with EtOAc. The clear solution was evaporated to afford a red oil: m = 151 mg (0.761 mmol). The total yield was >99%.
S12
GC‐MS characterisation
N
N
NMR characterisation 1H‐NMR (CDCl , 400 MHz, δ (ppm), J (Hz)): 1.73 (2H, CH ,m), 2.12 (2H, CH , t, 3J= 6.4 Hz), 2.93 (2H, CH , t, 3 2 2 2 3J= 5.6 Hz), 4.14 (2H, CH , s), 6.84 (1H, CH, s, broad), 7.11 – 7.13 (2H, CH aromatic), 7.21 – 7.31 (3H, CH 2
aromatic). 13C‐NMR (CDCl
3, 400 MHz, δ (ppm): 19.8, 20.8, 44.0, 58.5, 72.0, 122.5, 126.5, 127.0, 127.8, 135.4, 146.4.
S13
1H‐NMR:
13C‐NMR:
S14
13C‐DEPT135‐NMR:
S15
3. Expression and purification of the ERs All the enzymes used during this study were expressed and purified as described previously.2‐4 TsOYE was additionally purified by anion exchange chromatography using a HiPrepQ HP 16/10 column (GE Healthcare). The heat‐shock purified protein was dissolved in start buffer (20 mM Tris/HCl, pH 8.0 buffer) and loaded onto the column. The elution of the protein was performed with a gradient between start buffer and elution buffer (20 mM Tris/HCl, 1M NaCl, pH 8.0 buffer). All proteins were stored as concentrated stocks in 50 mM MOPS‐NaOH, pH 7.0 buffer, supplemented with 5 mM CaCl2 at ‐20 °C.
4. Determination of the molar extinction coefficient for mNADHs (1‐5) 24‐32 mg of mNADHs (1‐5) were dissolved in a certain amount of DMSO (600 µL for 1 and 2, 2.5 mL for 3 and 4 and 50 mL for 5) and filled up with buffer (50 mM MOPS‐NaOH, pH 7.0, supplemented with 5 mM CaCl2) to 500 mL using a volumetric flask. Then, 4 dilutions were prepared by pipetting 5, 10, 20 and 40 mL in 100 mL volumetric flasks. UV‐vis absorbance spectra were recorded and the absorbance of selected wavelengths was plotted against the concentration of the biomimetic‐solution. The slope of the linear fit represents the molar extinction coefficient.
A summary of the calculated molar extinction coefficients can be found in Table S1 Table S1. Calculated extinction coefficients for mNADHs (1‐5). mNADH
wavelength [nm]
ε [M‐1∙cm‐1]
1
360
7254
1
380
4784
1
395
1965
2
364
6450
2
390
3277
3
378
10831
3
409
5264
3
424
1876
4
283
8988
5
342
5925
Figure S1 shows the UV‐vis absorbance spectra of the mNADHs (1‐5).
S16
Figure S1. UV‐vis absorbance spectra of mNADHs (1‐5) at 4 different concentrations. The insets represent the absorbance at the peak‐maximum plotted against the biomimetic concentration for obtaining the molar extinction coefficient.
S17
5. Reductive half‐reaction The reductive half‐reactions between ERs, NAD(P)H and mNADHs were investigated using a High‐Tech stopped‐flow spectrophotometer (TgK Scientific Limited). The experiments were prepared and run under anaerobic conditions in N2‐environment as previously described. All measurements were acquired in MOPS‐NaOH buffer (50 mM, pH 7) supplemented with CaCl2 (5 mM) at 30 °C. After mixing of the two reactant solutions the enzyme concentration was between 8 and 10 µM. Measurements were taken with various coenzyme concentrations (25 µM‐20 mM). The reduction of FMN was monitored continuously by measuring the decrease in absorbance at the corresponding wavelength (464 nm for PETNR and TOYE, 458 nm for XenA and 457 nm for TsOYE). Initial rates were calculated by fitting the curves with Kinetic Studio (TgK Scientific Limited) using a single exponential equation. 5‐6 Shots were made for each experimental condition and reaction transients averaged. The reductive half‐reaction was modelled as shown in (1) (1)
ER(ox) + NADPH
k1 k‐1
[ER(ox)
NAD(P)H]
kred k‐red
ER(red) + NAD(P)+
where ER(ox) is the enzyme bound flavin in its oxidised state, [ER(ox)—NAD(P)H] is the enzyme‐coenzyme complex and ER(red) is the enzyme bound flavin in its reduced state upon immediate NAD(P)+ release after the reduction process. The dissociation constant KD is therefore determined according to equation (2): (2)
Figure S2 to Figure S5 show the hyperbolic fits for the reductive half‐reaction of ERs with NAD(P)H and mNADHs.
S18
Figure S2. Hyperbolic fit for the reductive half‐reaction of PETNR with NAD(P)H and mNADHs.
S19
Figure S3. Hyperbolic fit for the reductive half‐reaction of TOYE with NAD(P)H and mNADHs.
S20
Figure S4. Hyperbolic and linear fits for the reductive half‐reaction of XenA with NAD(P)H and mNADHs.
S21
Figure S5. Hyperbolic and linear fits for the reductive half‐reaction of TsER with NAD(P)H and mNADHs.
S22
6. Steady‐state kinetics for the conversion of 2‐cyclohexen‐1‐one by ERs using varied concentration of coenzyme 6.1. Determination of the saturation concentration of substrate 2‐cyclohexen‐1‐one for ERs The saturation concentration of the substrate 2‐cyclohexen‐1‐one for the ERs PETNR, TOYE and XenA was determined by following the oxidation of NADPH (ε340 = 6220 M‐1∙cm‐1) under anaerobic conditions in N2‐ environment at 30 °C using a Varian Cary 50 Bio UV‐visible spectrophotometer. Measurements were carried out at 30 °C in 50 mM MOPS‐NaOH, pH 7.0 buffer, supplemented with 5 mM CaCl2. Variable concentrations of 2‐cyclohexen‐1‐one were added as a stock solution (500 mM, dissolved in DMSO) to a fixed concentration of enzyme (100 nM), and the reaction was started by the addition of NADPH. The path length of the cuvette was chosen so that the initial absorbance was always below 1. The initial velocity was calculated from the linear range of the fitted trend line of the progress curve and plotted against the substrate concentration to obtain the kinetic parameter. Table S2 summarises the reaction conditions for the assay. Table S2. Kinetic assay for the determination of the saturation concentration of 2‐cyclohexen‐1‐one for ERs. ER
enzyme [nM]
substrate [mM]
NADPH [µM]
kcat [sec‐1]
KM [mM]
KI [mM]
conc. substrate employed for steady state kinetics
PETNR
100
0‐10
250
6.9
1.8
18.9
5.5 mM
TOYE
100
0‐45
50
9.8
6.6
‐
35 mM
XenA
100
0‐2
150
24.6
0.145
23.1
1.5 mM
Figure S6. Michaelis‐Menten plots for the determination of the saturation concentration of 2‐cyclohexen‐1‐one for ERs. S23
6.2. Steady‐state kinetics Steady‐state kinetic data for the conversion of 2‐cyclohexen‐1‐one by ERs using different coenzymes were performed under anaerobic conditions in N2‐environment by continuously following the oxidation of the reduced coenzymes on a Cary 50 Bio UV‐visible spectrophotometer (Varian) or on a Hi‐Tech stopped flow device (TgK Scientific Limited). Measurements were carried out at 30 °C in 50 mM MOPS‐NaOH, pH 7.0 buffer, supplemented with 5 mM CaCl2. The reaction was started by the addition of varied concentrations of the coenzyme to a constant concentration of substrate which was determined in the previous experiment and enzyme. Initial rates were obtained from the slope of the fitted trend line from the linear range of the progress curve. For spectrophotometric detection the path length of the cuvette (1 cm, 0.4 cm or 0.2 cm) as well as the wavelength for monitoring coenzyme oxidation were chosen and varied in a way that the initial absorbance was always below 1. The extinction coefficients for the mNADHs (1‐5) used for calculating kobs and KM are summarised in paragraph 4. Due to an overlap between the UV‐vis absorbance of mNADH 4 and 2‐cyclohexen‐1‐one the kinetic parameters for this coenzyme have not been determined. The experimental conditions for the single enzyme‐coenzyme combinations are summarised below:
(I) PETNR, 2‐cyclohexen‐1‐one (5.5 mM, added as a 1 M DMSO stock solution): spectrophotometric: NADPH: PETNR (100 nM), NADPH (0‐1 mM), 340 nm. 1: PETNR (100 nM), 1 (0‐700 µM, added as 500 mM stock solution in DMSO), 360 nm. 2: PETNR (100 nM), 2 (0‐700 µM, added as 500 mM stock solution in DMSO), 364 nm. 3: PETNR (100 nM), 3 (0‐2.3 mM, added as 150 mM stock solution in DMSO), 409 or 424 nm. NADH: PETNR (450 nM), NADH (0‐2.6 mM), 340 nm. (II) TOYE, 2‐cyclohexen‐1‐one (35 mM, added as a DMSO stock solution of 3.5 M): spectrophotometric: 2: TOYE (100 nM), 2 (0‐1.2 mM, added as DMSO stock solution 500 mM), 390 nm. 1: TOYE (100 nM), 1 (0‐2.2 mM, added as DMSO stock solution 500 mM), 380 or 395 nm. 3: TOYE (100 nM), 3 (0‐2.3 mM, added as DMSO stock solution 150 mM), 409 or 424 nm. Stopped flow (concentrations after mixing of the two syringes): NADPH: TOYE (100 nM), NADPH (5‐60 µM), 340 nm. NADH: TOYE (500 nM), NADH (5‐70 µM), 340 nm. (III) XenA, 2‐cyclohexen‐1‐one (1.5 mM, added as a DMSO stock solution of 750 mM): spectrophotometric: NADH: XenA (250 nM), NADH (0‐2.6 mM), 340 nm. Stopped flow (concentrations after mixing of the two syringes): NADPH: XenA (100 nM), NADPH (7‐150 µM), 340 nm. 3: XenA (100 nM), 3 (5‐70 µM, added as a 150 mM DMSO stock solution), 378 nm. 1: XenA (100 nM), 1 (5‐120 µM, added as a 150 mM DMSO stock solution), 360 nm. 2: XenA (100 nM), 2 (7‐110 µM, added as a 150 mM DMSO stock solution), 364 nm.
S24
Figure S7. Steady‐state kinetics for PETNR with a fixed concentration of 2‐cyclohexen‐1‐one (5.5 mM) and varied concentrations of NAD(P)H and mNADHs.
S25
Figure S8. Steady‐state kinetics for TOYE with a fixed concentration of 2‐cyclohexen‐1‐one (35 mM) and varied concentrations of NAD(P)H and mNADHs.
S26
Figure S9. Steady‐state kinetics for XenA with a fixed concentration of 2‐cyclohexen‐1‐one (1.5 mM) and varied concentrations of NAD(P)H and mNADHs.
S27
7. Biocatalytic transformations 7.1. Reaction conditions Standard reactions for screening of the ERs with ketoisophorone 6 were performed using the following procedure: in a 2 mL microcentrifuge plastic tube, MOPS buffer (50 mM, pH 7) supplemented with CaCl2 (5 mM) was deoxygenized and ketoisophorone (10 mM), coenzyme (11 mM), enzyme (3‐5 µM) were consecutively added, to a final volume of 1 mL. The reactions were run at 30 °C, 800 rpm, for 4 h unless otherwise stated. Then, the product was extracted with EtOAc (2 × 500 µL), dried over MgSO4 and analysed by GC using a calibration curve, with 2 mM dodecane as an internal standard. Reactions with a NAD(P)H recycling system were performed using GDH with the following concentrations: [NAD(P)H] = 20 μM, [glucose] = 30 mM, [GDH] = 20 U.
7.2. Analytical methods Data were analysed by GC with a flame ionization detector (FID) using a Chirasil Dex CB column (Agilent, 25 m × 0.32 mm × 0.25 µm); 250 °C injection, split ratio: 50, linear velocity: 25.4 cm/s. Temperature program: 80 °C hold 2 min; 5 °C/min to 90 °C hold 2 min; 5 °C/min to 100 °C hold 2 min; 5 °C/min to 110 °C hold 2 min; 5 °C/min to 120 °C hold 2 min; 40 °C/min to 200 °C hold 2 min. Retention times:
ketoisophorone 6 15.6 min
levodione 6a
16.9 min (R), 17.3 min (S)
7.3. GC chromatograms rac‐Levodione 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 15.00
15.25
15.50
15.75
16.00
16.25
16.50
16.75
17.00
17.25
16.00
16.25
16.50
16.75
17.00
17.25
17.50
17.75
18.00
18.25
18.50
18.75
min
(R)‐Levodione 0.30 0.25 0.20 0.15 0.10 0.05 0.00 -0.05 15.00
15.25
15.50
15.75
17.50
17.75
18.00
18.25
18.50
18.75
min
S28
7.4. Enantiomeric excess of 6 reduced to (R)‐6a by various ERs with different coenzymes The following table shows the enantiomeric excess for the conversion of ketoisophorone 6 to levodione (R)‐6a using 12 different ERs. The table with conversions can be found in the main paper (Table 3). Discussion of the results from biotransformations
The applicability of the mNAD biomimetics in organic synthesis as inexpensive alternatives to natural nicotinamide coenzymes was evaluated for the asymmetric reduction of an activated alkene as the test substrate. An extended panel of 12 ERs from the OYE family (PETNR, TOYE, OYE2, OYE3, XenA, XenB, LeOPR1, NerA, MR, TsOYE, DrOYE and RmOYE) was employed for the conversion of ketoisophorone 6 to levodione (R)‐6a (see Table 3, main paper). The results of the screening showed that the biomimetics were overall well accepted, the only exception being biomimetic 5. This finding is in line with the kinetic parameters previously described in Table 1Error! Reference source not found. and Table 2 (see main paper). The mNADH 5 stands out because it lacks the oxygen atom of the carbonyl moiety that may form a hydrogen bond with other residues within the enzyme pocket. Nevertheless, a conversion of 80% was still obtained with XenA suggesting that this ER might use a different binding mode for the coenzyme biomimetic. The biomimetics 1‐4 gave conversions up to >99%. Among the others, TsOYE, DrOYE and RmOYE afforded >99% conversion for the alkene reduction employing at least 2 biomimetics out of 5. We note that the conversions for alkene reduction catalysed by PETNR with biomimetic 3 (98%), TOYE with biomimetic 1 (90%), TsOYE with biomimetics 1‐4 (>99%), DrOYE with biomimetic 1‐4 (98 ‐ >99%) and RmOYE with biomimetic 1,4 (>99%) were equal or superseded the conversions for the same reactions using stoichiometric amounts of the natural coenzyme (i.e. 93% for PETNR, 56% for TOYE, >99% for TsOYE, 64% for DrOYE and 63% for RmOYE). In general, a minimum of 59% conversion was obtained with most of the ERs tested. The exceptions were the reductions catalysed by OYE2, OYE3 and MR, that generally afforded poor conversions (99
n.d.
NADH
79
n.d.
1
21
2
2
5
0.5
3
18
1
4
n.d.
n.d.
5
5
2
n.d. not detectable
Kinetic parameter for the conversion of cinnamaldehyde by NtDBR using NAD(P)H and mimics 1 and 3 Steady‐state kinetic data for the conversion of cinnamaldehyde by NtDBR using NADPH as coenzyme were performed spectrophotometrically at 30 °C in KPi buffer (50 mM, pH 7.5) by following the NADPH oxidation at 365 nm (ε = 3256 M‐1 cm‐1). The reaction was started by the addition of varied concentrations of the substrate (50 µM to 1 mM, dissolved in DMSO as a 50 mM stock solution) to a constant concentration of NADPH (150 µM) and enzyme (3 µM); the initial rates were plotted against the substrate concentration. After fitting of the curve, a kapp value of 40 min‐1 and a KM value of 91 µM could be obtained (Figure S11).
S35
Figure S11. Steady‐state kinetics for NtDBR with a fixed concentration of NADPH (150 µM) and varied concentrations of cinnamaldehyde.
The reaction rate for NtDBR with cinnamaldehyde and NADH, mNADH 1 and mNADH 3 were determined using biocatalytic reactions in KPi buffer (50 mM, pH 7.5). The reactions consisted of a constant amount of enzyme (10 µM) and coenzyme (6 mM) and various concentrations of substrate (1 to 5 mM) in 500 µL of total volume. The reactions were run at 30 °C, 800 rpm and, for each substrate concentration, the initial rate was determined by quenching the reactions at different time points. Then, the product was extracted with EtOAc (1 × 500 µL), dried over MgSO4 and analysed by GC as described before. For a comparison of the coenzymes the observed rates at 5 mM substrate concentrations are summarized in Table S7
Figure S12. Reaction rates for NtDBR with a fixed concentration of coenzyme (6 mM) and varied concentrations of cinnamaldehyde. Table S7. Reaction rates for NADH and mNADHs 1 and 3 at 5 mM substrate concentration. coenzyme
kapp [min‐1]
NADH
4
1
0.15
3
0.08
S36
10.2. NADPH‐dependent oxidase YcnD was expressed and purified as described previously. 14 The activity for YcnD with NAD(P)H and biomimetics (1‐5) was tested spectrophotometrically. First, the UV‐vis absorbance spectrum of the coenzyme was recorded. Then, enzyme (final concentration 400 nM) was added and after an incubation time of 5 min the spectrum was recorded again. Immediately after the addition of the coenzyme, the solution containing the flavin‐dependent enzyme became colourless, indicating the reduction of the flavin. Then, the flavin underwent slowly reoxidation by molecular oxygen. A bleach of the UV‐vis absorbance spectra of the coenzyme showed activity for YcnD towards NAD(P)H and biomimetics 1, 2, 3 and 5 which can be seen in Figure S13.
Figure S13. UV‐vis absorbance spectra of YcnD (dotted line), reduced coenzymes (solid line) and coenzyme after incubation with YcnD for 5 min (dashed line). S37
11. References (1) Paul, C. E.; Gargiulo, S.; Opperman, D. J.; Lavandera, I.; Gotor‐Fernandez, V.; Gotor, V.; Taglieber, A.; Arends, I. W. C. E.; Hollmann, F. Org. Lett. 2013, 15, 180. (2) Opperman, D. J.; Sewell, B. T.; Litthauer, D.; Isupov, M. N.; Littlechild, J. A.; van Heerden, E. Biochem. Biophys. Res. Commun. 2010, 393, 426. (3) Knaus, T.; Mutti, F. G.; Humphreys, L. D.; Turner, N. J.; Scrutton, N. S. Org. Biomol. Chem. 2015, 13, 223. (4) Litthauer, S.; Gargiulo, S.; van Heerden, E.; Hollmann, F.; Opperman, D. J. J. Mol. Catal. B: Enzym. 2014, 99, 89. (5) Fryszkowska, A.; Toogood, H.; Sakuma, M.; Gardiner, J. M.; Stephens, G. M.; Scrutton, N. S. Adv. Synth. Catal. 2009, 351, 2976. (6) Poizat, M.; Arends, I. W. C. E.; Hollmann, F. J. Mol. Catal. B: Enzym. 2010, 63, 149. (7) Bernard, J.; van Heerden, E.; Arends, I. W. C. E.; Opperman, D. J.; Hollmann, F. ChemCatChem 2012, 4, 196. (8) Winter, G. J. Appl. Crystallogr. 2010, 43, 186. (9) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Acta Crystallogr. Sect. D Biol. Crystallography 2010, 66, 486. (10) Adams, P. D.; Afonine, P. V.; Bunkóczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse‐Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. Acta Crystallogr. Sect. D Biol. Crystallography 2010, 66, 213. (11) Chen, V. B.; Arendall, W. B.; Headd, J. J.; Keedy, D. A.; Immormino, R. M.; Kapral, G. J.; Murray, L. W.; Richardson, J. S.; Richardson, D. C. Acta Crystallogr. Sect. D Biol. Crystallography 2010, 66, 12. (12) The {\it PDB_REDO} server for macromolecular structure model optimization. IUCrJ, 1, 213. (13) Mansell, D. J.; Toogood, H. S.; Waller, J.; Hughes, J. M. X.; Levy, C. W.; Gardiner, J. M.; Scrutton, N. S. Adv. Synth. Catal. 2013, 3, 370. (14) Morokutti, A.; Lyskowski, A.; Sollner, S.; Pointner, E.; Fitzpatrick, T. B.; Kratky, C.; Gruber, K.; Macheroux, P. Biochemistry 2005, 44, 13724.
S38
Better than Nature: nicotinamide biomimetics that outperform natural coenzymes Tanja Knaus,a,‡ Caroline E. Paul,b,‡ Colin W. Levy a, Simon de Vries,b Francesco G. Mutti a, Frank Hollmann,*,b and Nigel S. Scrutton*,a a
BBSRC/EPSRC Centre for Synthetic Biology of Fine and Speciality Chemicals, Manchester Institute of Biotechnology, Faculty of Life Sciences, 131 Princess Street, Manchester M1 7DN, United Kingdom, [email protected]
b
Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628BL Delft (The Netherlands) [email protected]
Table of Contents 1.
General information
2
2.
Synthesis of the nicotinamide coenzyme analogues (mNADHs, mNAD+s and tetrahydro‐mNADHs)
2
2.1. Reduction of mNADH nicotinamide biomimetics to 1,4,5,6 tetrahydro‐mNADH derivatives 2 (mNADH4s) 3.
Expression and purification of the ERs
16
4.
Determination of the molar extinction coefficient for mNADHs (1‐5)
16
5.
Reductive half‐reaction
18
6. Steady‐state kinetics for the conversion of 2‐cyclohexen‐1‐one by ERs using varied concentration of coenzyme 23 6.1.
Determination of the saturation concentration of substrate 2‐cyclohexen‐1‐one for ERs
23
6.2.
Steady‐state kinetics
24
7.
Biocatalytic transformations
28
7.1.
Reaction conditions
28
7.2.
Analytical methods
28
7.3.
GC chromatograms
28
7.4.
Enantiomeric excess of 6 reduced to (R)‐6a by various ERs with different coenzymes
29
8.
mNAD+s recycling with the rhodium complex
31
9.
X‐ray crystallography
33
9.1. 10.
Data Collection and Refinement Beyond the ER family: Enzymes that accept the biomimetic analogues
34 35
10.1. non flavin‐dependent double bond reductase from Nicotiana tabacum
35
10.2. NADPH‐dependent oxidase
37
11.
38
References
S1
1. General information All commercial reagents and solvents were purchased from Sigma‐Aldrich, Fluka, Acros Organics and Alfa Aesar with the highest purity available and used as received. NMR spectra were recorded on a Bruker AV‐ 300 spectrometer at 300 (1H) and 75 (13C) MHz or on a Bruker Spectrospin 400 Ultrashield spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) relative to Me4Si (δ 0.00) using deuterated solvent (DMSO‐d6, D2O or CDCl3) as an internal standard.
2. Synthesis of the nicotinamide coenzyme analogues (mNADHs, mNAD+s and tetrahydro‐ mNADHs) The mNADHs (1‐5) and mNAD+s (1‐5)a were synthesised and characterised as previously described.1 NMR data obtained corresponded to the previously reported characterisation. Once synthesised, the compounds were kept at ‐20 C for storage. 2.1. Reduction of mNADH nicotinamide biomimetics to 1,4,5,6 tetrahydro‐mNADH derivatives (mNADH4s) The aim was to generate a further reduced form of the reduced mNADHs (1‐5). The 1,4,5,6 tetrahydro‐ mNADHs (mNADH4 (1‐5)b) were synthesised to bind in the active site of the ERs generating a “dead‐end” complex which should be suitable for crystallisation and structural biochemical characterisation. The general structure of the tetrahydro‐biomimetics is as follows: O X N R
Synthesis of 1‐benzyl‐1,4,5,6‐tetrahydropyridine‐3‐carboxamide (1b) O
O NH2
N
H2 , 1 atm ethanol
1
NH2
Pd 10% on activated carbon N
1b
Pd catalyst on carbon (25 mg; extent of labelling 10% w w‐1 loading matrix activated carbon support, Sigma Cat. 20, 569.9) was added into a Schlenk tube containing dried ethanol (6 mL). Then, biomimetic 1 (100 mg, 0.467 mmol) was added under inert atmosphere (no stirring). The solution became yellow.
S2
Finally, the Schlenk tube was evacuated and filled with hydrogen at atmospheric pressure. The reaction was run for approximately 20 h. A small aliquot of the reaction mixture was analysed by GC‐MS as well as TLC. After removal of the Pd/C, the solution was colourless, indicating that all the starting material was reduced. The work‐up was performed by filtering over celite and extensive washing with methanol. The clear solution was evaporated to afford a colourless oil (80 mg of crude product). Judging from GC‐MS analysis, the conversion into the desired reduced biomimetic 1b was 50% whereas the rest of the material were side‐products generated by further reduction and/or cleavage of 1b. Therefore, the reaction was repeated as previously described using biomimetic 1 (100 mg) and Pd/C (25 mg) in dried ethanol (6 mL). The reaction time was shortened to 2h and 30 min to determine whether a better chemoselectivity could be obtained. Reducing the reaction time led to the formation of higher amounts of the desired product 1b. The composition of the product mixture (determined by GC‐MS) was: 82% of 1b, 13% of the fully reduced by‐product (i.e. 1‐benzylpiperidine‐3‐carboxamide) and 4% of cleaved derivatives. The aliquots of the crude mixture from the first and the second reaction were combined and purified together by column chromatography on silica. Conditions for column chromatography: Column diameter: 1 cm, Elution: CH2Cl2, 50 mL, fractions 1 – 7; CH2Cl2 / CH3OH, 98:2, v v‐1, 50 mL, fractions 8 – 13; CH2Cl2 / CH3OH, 96:4, v v‐1, 50 mL, fractions 14 – 19; CH2Cl2 / CH3OH, 94:6, v v‐1, 50 mL, fractions 20 – 25 1b was obtained as a yellow oil: m = 147 mg (0.680 mmol), total yield: 73%
GC‐MS characterisation
O NH2 N
S3
NMR characterisation 1H‐NMR (CDCl , 400 MHz, δ (ppm), J (Hz)): 1.77 (2H, CH , m), 2.15 (2H, CH , t, 3J= 6.4 Hz), 2.89 (2H, CH , t, 3 2 2 2 3J= 5.6 Hz), 4.20 (2H, CH , s), 5.37 (2H, NH , broad), 7.12 – 7.15 (2H, CH aromatic, m), 7.20 – 7.27 (3H, CH, 2 2
aromatic). 13C‐NMR (CDCl
3, 400 MHz, δ (ppm): 19.1, 20.0, 43.6, 58.4, 94.2, 126.2, 126.3, 127.3, 135.7, 143.4, 169.7.
1H‐NMR:
S4
13C‐NMR
DEPT‐135
S5
Synthesis of 1‐butyl‐1,4,5,6‐tetrahydropyridine‐3‐carboxamide (2b) O
O NH2
N
NH2
Pd 10% on activated carbon H2, 1 atm ethanol
N
2
2b
Pd catalyst on carbon (30 mg; extent of labelling 10% w w‐1 loading matrix activated carbon support, Sigma Cat. 20, 569.9) was added into a Schlenk tube containing dried ethanol (6 mL). Then, biomimetic 2 (150 mg, 0.832 mmol) was added under inert atmosphere. Finally, the Schlenk tube was evacuated and filled with hydrogen at atmospheric pressure. The reaction was run for approximately 16 h. A small aliquot of the reaction mixture was analysed by GC‐MS as well as TLC. GC‐MS analysis showed that the “mono”‐reduced desired product 2b was formed with 31% conversion. The remaining was the “double”‐reduced compound (i.e. 1‐butylpiperidine‐3‐carboxamide). The work‐up was performed by filtering over celite and extensive washing with methanol. The clear solution was evaporated to afford a colourless crude oil that was purified by column chromatography. Conditions for column chromatography: The crude mixture was purified by column chromatography on silica. Column diameter: 1 cm; Elution: CH2Cl2 / CH3OH, 98:2, v v‐1, 200 mL, fractions 1 – 24; CH2Cl2 / CH3OH, 95:5, v v‐1, 200 mL, fractions 25 – 48 A green oil was obtained. The amount of the product 2b was: m = 48 mg (0.2634 mmol), yield: 32%.
S6
GC‐MS characterisation: O NH2 N
NMR characterisation 1H‐NMR (CDCl
3, 400 MHz, δ (ppm), J (Hz)): 0.84 (3H, CH3,
3J= 7.2 Hz), 1.22 (2H, CH , m), 1.44 (2H, CH , m), 2 2
1.81 (2H, CH2, m), 2.15 (2H, CH2, 3J= 6.4 Hz), 3.02 (4H, CH2, m), 5.32 (2H, NH2, broad), 7.31 (1H, CH, s). 13C‐NMR (CDCl
3, 400 MHz, δ (ppm): 12.8, 18.8, 19.6, 20.5, 29.6, 44.3, 54.6, 93.2, 143.7, 170.0.
S7
1H‐NMR:
13C‐NMR:
S8
13C‐DEPT135‐NMR:
Synthesis of 1‐(1‐benzyl‐1,4,5,6‐tetrahydropyridin‐3‐yl) ethanone (3b) O
O Pd 10% on activated carbon
N
H2 , 1 atm ethanol
N
3
3b
Pd catalyst on carbon (30 mg; extent of labelling 10% w w‐1 loading matrix activated carbon support, Sigma Cat. 20, 569.9) was added into a Schlenk tube containing dried ethanol (6 mL). Then, biomimetic 3 (150 mg, 0.704 mmol) was added under inert atmosphere. Finally, the Schlenk tube was evacuated and filled with hydrogen at atmospheric pressure. The reaction was run for approximately 16 h. A small aliquot of the reaction mixture was analysed by GC‐MS as well as TLC. GC‐MS showed that the desired product 3b was formed with quantitative conversion (> 99%). Therefore, in this case, the hydrogenation was completely chemo‐ and regioselective, even when the reaction time was prolonged to 16 h. S9
The work‐up was performed by filtering over celite and extensive washing with methanol. The clear solution was evaporated to afford a red oil (150 mg, 0.697 mmol). The total yield was 99%. GC‐MS characterisation O
N
NMR characterisation 1H‐NMR (CDCl , 400 MHz, δ (ppm), J (Hz)): 1.71 (2H, CH ,m), 2.09 (3H, CH , s), 2.25 (2H, CH , t, 3J= 6.4 Hz), 3 2 3 2
2.97 (2H, CH2, t, 3J= 5.6 Hz), 4.28 (2H, CH2, s), 7.14 – 7.16 (2H, CH aromatic), 7.21 – 7.32 (3H, CH aromatic), 7.39 (1H, CH, s). 13C‐NMR (CDCl
3, 400 MHz, δ (ppm): 19.1, 22.0, 43.9, 58.2, 125.4, 126.1, 127.0, 134.6, 146.2, 191.5.
S10
1H‐NMR:
13C‐NMR:
S11
13C‐DEP135‐NMR:
Synthesis of 1‐benzyl‐1,4,5,6‐tetrahydropyridine‐3‐carbonitrile (5b) N
N Pd(OH)2 10 - 20% on activated charcoal
N
H2 , 1 atm EtOAc
5
N
5b
Pd(OH)2 (60 mg on activated charcoal, moistened with water, 10‐20% Pd basis based on dry substance; Sigma Cat. 76063‐5G) was added into a Schlenk tube containing dried ethyl acetate (12 mL). Then, biomimetic 5 (150 mg, 0.764 mmol) was added under inert atmosphere. Finally, the Schlenk tube was evacuated and filled with hydrogen at atmospheric pressure. The reaction was monitored over time (3 h – 6h – 9 h) by GC‐MS. The reaction was complete after 9 h, yielding the desired product 5b. The work‐up was performed by filtering over celite and extensive washing with EtOAc. The clear solution was evaporated to afford a red oil: m = 151 mg (0.761 mmol). The total yield was >99%.
S12
GC‐MS characterisation
N
N
NMR characterisation 1H‐NMR (CDCl , 400 MHz, δ (ppm), J (Hz)): 1.73 (2H, CH ,m), 2.12 (2H, CH , t, 3J= 6.4 Hz), 2.93 (2H, CH , t, 3 2 2 2 3J= 5.6 Hz), 4.14 (2H, CH , s), 6.84 (1H, CH, s, broad), 7.11 – 7.13 (2H, CH aromatic), 7.21 – 7.31 (3H, CH 2
aromatic). 13C‐NMR (CDCl
3, 400 MHz, δ (ppm): 19.8, 20.8, 44.0, 58.5, 72.0, 122.5, 126.5, 127.0, 127.8, 135.4, 146.4.
S13
1H‐NMR:
13C‐NMR:
S14
13C‐DEPT135‐NMR:
S15
3. Expression and purification of the ERs All the enzymes used during this study were expressed and purified as described previously.2‐4 TsOYE was additionally purified by anion exchange chromatography using a HiPrepQ HP 16/10 column (GE Healthcare). The heat‐shock purified protein was dissolved in start buffer (20 mM Tris/HCl, pH 8.0 buffer) and loaded onto the column. The elution of the protein was performed with a gradient between start buffer and elution buffer (20 mM Tris/HCl, 1M NaCl, pH 8.0 buffer). All proteins were stored as concentrated stocks in 50 mM MOPS‐NaOH, pH 7.0 buffer, supplemented with 5 mM CaCl2 at ‐20 °C.
4. Determination of the molar extinction coefficient for mNADHs (1‐5) 24‐32 mg of mNADHs (1‐5) were dissolved in a certain amount of DMSO (600 µL for 1 and 2, 2.5 mL for 3 and 4 and 50 mL for 5) and filled up with buffer (50 mM MOPS‐NaOH, pH 7.0, supplemented with 5 mM CaCl2) to 500 mL using a volumetric flask. Then, 4 dilutions were prepared by pipetting 5, 10, 20 and 40 mL in 100 mL volumetric flasks. UV‐vis absorbance spectra were recorded and the absorbance of selected wavelengths was plotted against the concentration of the biomimetic‐solution. The slope of the linear fit represents the molar extinction coefficient.
A summary of the calculated molar extinction coefficients can be found in Table S1 Table S1. Calculated extinction coefficients for mNADHs (1‐5). mNADH
wavelength [nm]
ε [M‐1∙cm‐1]
1
360
7254
1
380
4784
1
395
1965
2
364
6450
2
390
3277
3
378
10831
3
409
5264
3
424
1876
4
283
8988
5
342
5925
Figure S1 shows the UV‐vis absorbance spectra of the mNADHs (1‐5).
S16
Figure S1. UV‐vis absorbance spectra of mNADHs (1‐5) at 4 different concentrations. The insets represent the absorbance at the peak‐maximum plotted against the biomimetic concentration for obtaining the molar extinction coefficient.
S17
5. Reductive half‐reaction The reductive half‐reactions between ERs, NAD(P)H and mNADHs were investigated using a High‐Tech stopped‐flow spectrophotometer (TgK Scientific Limited). The experiments were prepared and run under anaerobic conditions in N2‐environment as previously described. All measurements were acquired in MOPS‐NaOH buffer (50 mM, pH 7) supplemented with CaCl2 (5 mM) at 30 °C. After mixing of the two reactant solutions the enzyme concentration was between 8 and 10 µM. Measurements were taken with various coenzyme concentrations (25 µM‐20 mM). The reduction of FMN was monitored continuously by measuring the decrease in absorbance at the corresponding wavelength (464 nm for PETNR and TOYE, 458 nm for XenA and 457 nm for TsOYE). Initial rates were calculated by fitting the curves with Kinetic Studio (TgK Scientific Limited) using a single exponential equation. 5‐6 Shots were made for each experimental condition and reaction transients averaged. The reductive half‐reaction was modelled as shown in (1) (1)
ER(ox) + NADPH
k1 k‐1
[ER(ox)
NAD(P)H]
kred k‐red
ER(red) + NAD(P)+
where ER(ox) is the enzyme bound flavin in its oxidised state, [ER(ox)—NAD(P)H] is the enzyme‐coenzyme complex and ER(red) is the enzyme bound flavin in its reduced state upon immediate NAD(P)+ release after the reduction process. The dissociation constant KD is therefore determined according to equation (2): (2)
Figure S2 to Figure S5 show the hyperbolic fits for the reductive half‐reaction of ERs with NAD(P)H and mNADHs.
S18
Figure S2. Hyperbolic fit for the reductive half‐reaction of PETNR with NAD(P)H and mNADHs.
S19
Figure S3. Hyperbolic fit for the reductive half‐reaction of TOYE with NAD(P)H and mNADHs.
S20
Figure S4. Hyperbolic and linear fits for the reductive half‐reaction of XenA with NAD(P)H and mNADHs.
S21
Figure S5. Hyperbolic and linear fits for the reductive half‐reaction of TsER with NAD(P)H and mNADHs.
S22
6. Steady‐state kinetics for the conversion of 2‐cyclohexen‐1‐one by ERs using varied concentration of coenzyme 6.1. Determination of the saturation concentration of substrate 2‐cyclohexen‐1‐one for ERs The saturation concentration of the substrate 2‐cyclohexen‐1‐one for the ERs PETNR, TOYE and XenA was determined by following the oxidation of NADPH (ε340 = 6220 M‐1∙cm‐1) under anaerobic conditions in N2‐ environment at 30 °C using a Varian Cary 50 Bio UV‐visible spectrophotometer. Measurements were carried out at 30 °C in 50 mM MOPS‐NaOH, pH 7.0 buffer, supplemented with 5 mM CaCl2. Variable concentrations of 2‐cyclohexen‐1‐one were added as a stock solution (500 mM, dissolved in DMSO) to a fixed concentration of enzyme (100 nM), and the reaction was started by the addition of NADPH. The path length of the cuvette was chosen so that the initial absorbance was always below 1. The initial velocity was calculated from the linear range of the fitted trend line of the progress curve and plotted against the substrate concentration to obtain the kinetic parameter. Table S2 summarises the reaction conditions for the assay. Table S2. Kinetic assay for the determination of the saturation concentration of 2‐cyclohexen‐1‐one for ERs. ER
enzyme [nM]
substrate [mM]
NADPH [µM]
kcat [sec‐1]
KM [mM]
KI [mM]
conc. substrate employed for steady state kinetics
PETNR
100
0‐10
250
6.9
1.8
18.9
5.5 mM
TOYE
100
0‐45
50
9.8
6.6
‐
35 mM
XenA
100
0‐2
150
24.6
0.145
23.1
1.5 mM
Figure S6. Michaelis‐Menten plots for the determination of the saturation concentration of 2‐cyclohexen‐1‐one for ERs. S23
6.2. Steady‐state kinetics Steady‐state kinetic data for the conversion of 2‐cyclohexen‐1‐one by ERs using different coenzymes were performed under anaerobic conditions in N2‐environment by continuously following the oxidation of the reduced coenzymes on a Cary 50 Bio UV‐visible spectrophotometer (Varian) or on a Hi‐Tech stopped flow device (TgK Scientific Limited). Measurements were carried out at 30 °C in 50 mM MOPS‐NaOH, pH 7.0 buffer, supplemented with 5 mM CaCl2. The reaction was started by the addition of varied concentrations of the coenzyme to a constant concentration of substrate which was determined in the previous experiment and enzyme. Initial rates were obtained from the slope of the fitted trend line from the linear range of the progress curve. For spectrophotometric detection the path length of the cuvette (1 cm, 0.4 cm or 0.2 cm) as well as the wavelength for monitoring coenzyme oxidation were chosen and varied in a way that the initial absorbance was always below 1. The extinction coefficients for the mNADHs (1‐5) used for calculating kobs and KM are summarised in paragraph 4. Due to an overlap between the UV‐vis absorbance of mNADH 4 and 2‐cyclohexen‐1‐one the kinetic parameters for this coenzyme have not been determined. The experimental conditions for the single enzyme‐coenzyme combinations are summarised below:
(I) PETNR, 2‐cyclohexen‐1‐one (5.5 mM, added as a 1 M DMSO stock solution): spectrophotometric: NADPH: PETNR (100 nM), NADPH (0‐1 mM), 340 nm. 1: PETNR (100 nM), 1 (0‐700 µM, added as 500 mM stock solution in DMSO), 360 nm. 2: PETNR (100 nM), 2 (0‐700 µM, added as 500 mM stock solution in DMSO), 364 nm. 3: PETNR (100 nM), 3 (0‐2.3 mM, added as 150 mM stock solution in DMSO), 409 or 424 nm. NADH: PETNR (450 nM), NADH (0‐2.6 mM), 340 nm. (II) TOYE, 2‐cyclohexen‐1‐one (35 mM, added as a DMSO stock solution of 3.5 M): spectrophotometric: 2: TOYE (100 nM), 2 (0‐1.2 mM, added as DMSO stock solution 500 mM), 390 nm. 1: TOYE (100 nM), 1 (0‐2.2 mM, added as DMSO stock solution 500 mM), 380 or 395 nm. 3: TOYE (100 nM), 3 (0‐2.3 mM, added as DMSO stock solution 150 mM), 409 or 424 nm. Stopped flow (concentrations after mixing of the two syringes): NADPH: TOYE (100 nM), NADPH (5‐60 µM), 340 nm. NADH: TOYE (500 nM), NADH (5‐70 µM), 340 nm. (III) XenA, 2‐cyclohexen‐1‐one (1.5 mM, added as a DMSO stock solution of 750 mM): spectrophotometric: NADH: XenA (250 nM), NADH (0‐2.6 mM), 340 nm. Stopped flow (concentrations after mixing of the two syringes): NADPH: XenA (100 nM), NADPH (7‐150 µM), 340 nm. 3: XenA (100 nM), 3 (5‐70 µM, added as a 150 mM DMSO stock solution), 378 nm. 1: XenA (100 nM), 1 (5‐120 µM, added as a 150 mM DMSO stock solution), 360 nm. 2: XenA (100 nM), 2 (7‐110 µM, added as a 150 mM DMSO stock solution), 364 nm.
S24
Figure S7. Steady‐state kinetics for PETNR with a fixed concentration of 2‐cyclohexen‐1‐one (5.5 mM) and varied concentrations of NAD(P)H and mNADHs.
S25
Figure S8. Steady‐state kinetics for TOYE with a fixed concentration of 2‐cyclohexen‐1‐one (35 mM) and varied concentrations of NAD(P)H and mNADHs.
S26
Figure S9. Steady‐state kinetics for XenA with a fixed concentration of 2‐cyclohexen‐1‐one (1.5 mM) and varied concentrations of NAD(P)H and mNADHs.
S27
7. Biocatalytic transformations 7.1. Reaction conditions Standard reactions for screening of the ERs with ketoisophorone 6 were performed using the following procedure: in a 2 mL microcentrifuge plastic tube, MOPS buffer (50 mM, pH 7) supplemented with CaCl2 (5 mM) was deoxygenized and ketoisophorone (10 mM), coenzyme (11 mM), enzyme (3‐5 µM) were consecutively added, to a final volume of 1 mL. The reactions were run at 30 °C, 800 rpm, for 4 h unless otherwise stated. Then, the product was extracted with EtOAc (2 × 500 µL), dried over MgSO4 and analysed by GC using a calibration curve, with 2 mM dodecane as an internal standard. Reactions with a NAD(P)H recycling system were performed using GDH with the following concentrations: [NAD(P)H] = 20 μM, [glucose] = 30 mM, [GDH] = 20 U.
7.2. Analytical methods Data were analysed by GC with a flame ionization detector (FID) using a Chirasil Dex CB column (Agilent, 25 m × 0.32 mm × 0.25 µm); 250 °C injection, split ratio: 50, linear velocity: 25.4 cm/s. Temperature program: 80 °C hold 2 min; 5 °C/min to 90 °C hold 2 min; 5 °C/min to 100 °C hold 2 min; 5 °C/min to 110 °C hold 2 min; 5 °C/min to 120 °C hold 2 min; 40 °C/min to 200 °C hold 2 min. Retention times:
ketoisophorone 6 15.6 min
levodione 6a
16.9 min (R), 17.3 min (S)
7.3. GC chromatograms rac‐Levodione 0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 15.00
15.25
15.50
15.75
16.00
16.25
16.50
16.75
17.00
17.25
16.00
16.25
16.50
16.75
17.00
17.25
17.50
17.75
18.00
18.25
18.50
18.75
min
(R)‐Levodione 0.30 0.25 0.20 0.15 0.10 0.05 0.00 -0.05 15.00
15.25
15.50
15.75
17.50
17.75
18.00
18.25
18.50
18.75
min
S28
7.4. Enantiomeric excess of 6 reduced to (R)‐6a by various ERs with different coenzymes The following table shows the enantiomeric excess for the conversion of ketoisophorone 6 to levodione (R)‐6a using 12 different ERs. The table with conversions can be found in the main paper (Table 3). Discussion of the results from biotransformations
The applicability of the mNAD biomimetics in organic synthesis as inexpensive alternatives to natural nicotinamide coenzymes was evaluated for the asymmetric reduction of an activated alkene as the test substrate. An extended panel of 12 ERs from the OYE family (PETNR, TOYE, OYE2, OYE3, XenA, XenB, LeOPR1, NerA, MR, TsOYE, DrOYE and RmOYE) was employed for the conversion of ketoisophorone 6 to levodione (R)‐6a (see Table 3, main paper). The results of the screening showed that the biomimetics were overall well accepted, the only exception being biomimetic 5. This finding is in line with the kinetic parameters previously described in Table 1Error! Reference source not found. and Table 2 (see main paper). The mNADH 5 stands out because it lacks the oxygen atom of the carbonyl moiety that may form a hydrogen bond with other residues within the enzyme pocket. Nevertheless, a conversion of 80% was still obtained with XenA suggesting that this ER might use a different binding mode for the coenzyme biomimetic. The biomimetics 1‐4 gave conversions up to >99%. Among the others, TsOYE, DrOYE and RmOYE afforded >99% conversion for the alkene reduction employing at least 2 biomimetics out of 5. We note that the conversions for alkene reduction catalysed by PETNR with biomimetic 3 (98%), TOYE with biomimetic 1 (90%), TsOYE with biomimetics 1‐4 (>99%), DrOYE with biomimetic 1‐4 (98 ‐ >99%) and RmOYE with biomimetic 1,4 (>99%) were equal or superseded the conversions for the same reactions using stoichiometric amounts of the natural coenzyme (i.e. 93% for PETNR, 56% for TOYE, >99% for TsOYE, 64% for DrOYE and 63% for RmOYE). In general, a minimum of 59% conversion was obtained with most of the ERs tested. The exceptions were the reductions catalysed by OYE2, OYE3 and MR, that generally afforded poor conversions (99
n.d.
NADH
79
n.d.
1
21
2
2
5
0.5
3
18
1
4
n.d.
n.d.
5
5
2
n.d. not detectable
Kinetic parameter for the conversion of cinnamaldehyde by NtDBR using NAD(P)H and mimics 1 and 3 Steady‐state kinetic data for the conversion of cinnamaldehyde by NtDBR using NADPH as coenzyme were performed spectrophotometrically at 30 °C in KPi buffer (50 mM, pH 7.5) by following the NADPH oxidation at 365 nm (ε = 3256 M‐1 cm‐1). The reaction was started by the addition of varied concentrations of the substrate (50 µM to 1 mM, dissolved in DMSO as a 50 mM stock solution) to a constant concentration of NADPH (150 µM) and enzyme (3 µM); the initial rates were plotted against the substrate concentration. After fitting of the curve, a kapp value of 40 min‐1 and a KM value of 91 µM could be obtained (Figure S11).
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Figure S11. Steady‐state kinetics for NtDBR with a fixed concentration of NADPH (150 µM) and varied concentrations of cinnamaldehyde.
The reaction rate for NtDBR with cinnamaldehyde and NADH, mNADH 1 and mNADH 3 were determined using biocatalytic reactions in KPi buffer (50 mM, pH 7.5). The reactions consisted of a constant amount of enzyme (10 µM) and coenzyme (6 mM) and various concentrations of substrate (1 to 5 mM) in 500 µL of total volume. The reactions were run at 30 °C, 800 rpm and, for each substrate concentration, the initial rate was determined by quenching the reactions at different time points. Then, the product was extracted with EtOAc (1 × 500 µL), dried over MgSO4 and analysed by GC as described before. For a comparison of the coenzymes the observed rates at 5 mM substrate concentrations are summarized in Table S7
Figure S12. Reaction rates for NtDBR with a fixed concentration of coenzyme (6 mM) and varied concentrations of cinnamaldehyde. Table S7. Reaction rates for NADH and mNADHs 1 and 3 at 5 mM substrate concentration. coenzyme
kapp [min‐1]
NADH
4
1
0.15
3
0.08
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10.2. NADPH‐dependent oxidase YcnD was expressed and purified as described previously. 14 The activity for YcnD with NAD(P)H and biomimetics (1‐5) was tested spectrophotometrically. First, the UV‐vis absorbance spectrum of the coenzyme was recorded. Then, enzyme (final concentration 400 nM) was added and after an incubation time of 5 min the spectrum was recorded again. Immediately after the addition of the coenzyme, the solution containing the flavin‐dependent enzyme became colourless, indicating the reduction of the flavin. Then, the flavin underwent slowly reoxidation by molecular oxygen. A bleach of the UV‐vis absorbance spectra of the coenzyme showed activity for YcnD towards NAD(P)H and biomimetics 1, 2, 3 and 5 which can be seen in Figure S13.
Figure S13. UV‐vis absorbance spectra of YcnD (dotted line), reduced coenzymes (solid line) and coenzyme after incubation with YcnD for 5 min (dashed line). S37
11. References (1) Paul, C. E.; Gargiulo, S.; Opperman, D. J.; Lavandera, I.; Gotor‐Fernandez, V.; Gotor, V.; Taglieber, A.; Arends, I. W. C. E.; Hollmann, F. Org. Lett. 2013, 15, 180. (2) Opperman, D. J.; Sewell, B. T.; Litthauer, D.; Isupov, M. N.; Littlechild, J. A.; van Heerden, E. Biochem. Biophys. Res. Commun. 2010, 393, 426. (3) Knaus, T.; Mutti, F. G.; Humphreys, L. D.; Turner, N. J.; Scrutton, N. S. Org. Biomol. Chem. 2015, 13, 223. (4) Litthauer, S.; Gargiulo, S.; van Heerden, E.; Hollmann, F.; Opperman, D. J. J. Mol. Catal. B: Enzym. 2014, 99, 89. (5) Fryszkowska, A.; Toogood, H.; Sakuma, M.; Gardiner, J. M.; Stephens, G. M.; Scrutton, N. S. Adv. Synth. Catal. 2009, 351, 2976. (6) Poizat, M.; Arends, I. W. C. E.; Hollmann, F. J. Mol. Catal. B: Enzym. 2010, 63, 149. (7) Bernard, J.; van Heerden, E.; Arends, I. W. C. E.; Opperman, D. J.; Hollmann, F. ChemCatChem 2012, 4, 196. (8) Winter, G. J. Appl. Crystallogr. 2010, 43, 186. (9) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Acta Crystallogr. Sect. D Biol. Crystallography 2010, 66, 486. (10) Adams, P. D.; Afonine, P. V.; Bunkóczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse‐Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. Acta Crystallogr. Sect. D Biol. Crystallography 2010, 66, 213. (11) Chen, V. B.; Arendall, W. B.; Headd, J. J.; Keedy, D. A.; Immormino, R. M.; Kapral, G. J.; Murray, L. W.; Richardson, J. S.; Richardson, D. C. Acta Crystallogr. Sect. D Biol. Crystallography 2010, 66, 12. (12) The {\it PDB_REDO} server for macromolecular structure model optimization. IUCrJ, 1, 213. (13) Mansell, D. J.; Toogood, H. S.; Waller, J.; Hughes, J. M. X.; Levy, C. W.; Gardiner, J. M.; Scrutton, N. S. Adv. Synth. Catal. 2013, 3, 370. (14) Morokutti, A.; Lyskowski, A.; Sollner, S.; Pointner, E.; Fitzpatrick, T. B.; Kratky, C.; Gruber, K.; Macheroux, P. Biochemistry 2005, 44, 13724.
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