green chemistry: highly selective biocatalytic hydrolysis of nitrile compounds csir conference dr...

Green Chemistry: highly selective biocatalytic hydrolysis of nitrile compounds

CSIR Conference

Dr Dean Brady

Research Fellow (Biosciences)

28 February 2006

Slide 2 © CSIR 2006 www.csir.co.za

The need for Green Chemistry

• On average 300 kg of chemicals are required to produce 1 Kg of a fine chemical or active pharmaceutical intermediate.

• This is due to the lack of specificity of most catalysts, requiring extensive use of protecting groups and additional reaction steps.

• Reaction yields are often low.• The reactions often require extreme conditions of pressure

and temperature, with special pressure reactors.• Polluting waste levels are high, and solvent recycling

costs add to the final product price.

Slide 3 © CSIR 2006 www.csir.co.za

Biocatalysis

• Selectivity: • Enantiomeric• Regioselectivity• Chemoselectivity

• Safety and environment:• Low pressure and temperature reactions

(improved safety and CAPEX costs)• Less waste products (minimise or eliminate

organic solvents)• Improved atom economy (less protecting groups

and enantiomeric “ballast” leads to savings).

Slide 4 © CSIR 2006 www.csir.co.za

Nitrile Hydratase

Slide 5 © CSIR 2006 www.csir.co.za

• In 2000, sales in the chemical industry exceeded $1.7 trillion.

• $50 billion via biological routes (cellular biotransformations or biocatalysts)

• By 2015 this will have grown to approximately $250 billion.

• 15% of chiral technology is now achieved by biocatalysis, and is projected as 30% by 2009!

Slide 6 © CSIR 2006 www.csir.co.za

Biocatalysis in Application

• Lonza's Biotechnology microbial fermentation unit has 17 products (worth a $100 million) in its pharmaceutical and "nutraceutical" pipeline.

• Avecia: five of the firm's top 10 commercial pharmaceutical intermediates involves at least one biotransformation step.

• DSM Fine Chemicals: 30% of the more than 80 pharmaceuticals in development for third parties at DSM involve biocatalytic steps.

Slide 7 © CSIR 2006 www.csir.co.za

• Fluka’s products, 5% of are now made using biocatalysis,

• Dutch chemicals giant DSM uses 25 biocatalysis-based processes at large scale.

• Other companies involved in biotransformations include: Degussa and Direvo biotech (Germany)

• Daicel Chemical Industries (Japan)

• Dow Chemical, Diversa, and Du Pont (USA)

Slide 8 © CSIR 2006 www.csir.co.za

• Growing drug industry demand for enantiomerically pure compounds is the driver for pursuing biocatalytic technology.

• SA imports R14 billion p.a. speciality and fine chemicals.

Slide 9 © CSIR 2006 www.csir.co.za

Biocatalysis at the CSIR

• The biocatalytic resolution of naproxen (as part of a commercial synthetic process).

• The biocatalytic resolution of menthol (as part of a commercial synthetic process).

• The epoxide technology platform has been progressed to the product stage.

Slide 10 © CSIR 2006 www.csir.co.za

The versatility of the nitrile group

R1

N

R1

N

R1

NH2

R1

OH

O

R1

NH2

OR1 CN

2H2O

H2O

+H2

Slide 11 © CSIR 2006 www.csir.co.za

Chemical Nitrile Hydrolysis

R N 2H2OKOH

200 oC R

O

OH

R N 2H2OR

O

OH100 oC

conc. HCl

Slide 12 © CSIR 2006 www.csir.co.za

Nitrile Biocatalysis

R NH2O

Nitrile Hydratase R

O

NH2

H2O

Amidase R

O

OHNH3

2H2O

Nitrilase

Slide 13 © CSIR 2006 www.csir.co.za

Regioselectivity

N

N

N

NH2 O

NH2

NH2 O

H2H2O

Nitrile hydratase

Slide 14 © CSIR 2006 www.csir.co.za

Chemoselectivity

O

N

OO

OH

O

O

OH

OH

OHOOH NH3

OH

NH3

+ +

3H2O

2H2O +Nitrilase

Slide 15 © CSIR 2006 www.csir.co.za

Stereoselectivity

N

OH OH

OH

O

N

OH

2H2O

Slide 16 © CSIR 2006 www.csir.co.za

Problems with nitrile biocatalysts

• Availability of both nitrilases and nitrile hydratases

• Stability of nitrilases

• Enantioselectivity of nitrile hydratases

Slide 17 © CSIR 2006 www.csir.co.za

Alpha-subsituted Substrates

N

R2 N

OH

N

NH2

N

CH3

Hydroxy-phenyl-acetonitrile

Amino-phenyl-acetonitrile

2-Phenyl-propionitrile

-substituted phenyl propionitriles

(2-phenylglycinonitrile)

(Mandelonitrile)

(-methyl-benzylcyanide)

Slide 18 © CSIR 2006 www.csir.co.za

Strecker Synthesis

O

N

NH2

MeOH/H2O

R

KCN, NH3

R

R = H, Cl, F, OH, NO2, CH3

Slide 19 © CSIR 2006 www.csir.co.za

Beta-substituted substrates

R2

N

OH

N

NH2

N

3-Hydroxy-3-phenyl-propionitrile

3-Amino-3-phenyl-propionitrile

-substituted phenyl propionitriles

Slide 20 © CSIR 2006 www.csir.co.za

Beta-hydroxy substrates

OKCN

OH

NMeOH/H2O

ba

Styrene oxide 3-hydroxy-3-phenyl-propionitrile

Slide 21 © CSIR 2006 www.csir.co.za

N

R R

N

NH

N

R1

N

R2

N

N

N+

O O

N

N NH

NOH

N

O

N

O

NN

N R

NN

NN N

N

N

R

N

O

N+O

O

OH

O

N

N+O

O

OH

O

R

O

O

O

S OO

NH2 O O

O

O

1 R = H2 R = CH3

3 R = NH2

4 R = OH5 R = CH2CH3

6 R = H7 R = OH

8 9

10 R1 = CH3, R2 = H11 R1 = OCH3, R2 = OCH3

12 13 14 15

16

17 R = Br18 R = C(O)OCH2CH3

19 R = OCH(O)Ph

20 21

22 R = H23 R = NO2

24 2526 R = OCH3

27 R = H

31

28

29 30 32 33

Slide 22 © CSIR 2006 www.csir.co.za

Commercial vs Isolated Organisms

Compound

Biocatalyst CN

P. fluorescens nitrilase 100% 21% 89% 74% 1% 1% 0% 0% 11% 0% 0% BioCatalytics nitrilase-1001 100% 0% 0% 0% 0% 319% 0% 639% 12% 23% 0% BioCatalytics nitrilase-1004 100% 0% 0% 1% 0% 18% 0% 11% 3% 22% 2% BioCatalytics nitrilase-1005 100% 0% 0% 0% 0% 64% 0% 128% 62% 128% 128% BioCatalytics nitrilase-1006 100% 0% 14% 41% 0% 0% 0% 7% 3% 0% 0% Arabidopsis thaliana nitrilase 100% 0% 0% 0% 0% 4011% 0% 332% 278% 0% 0%

Rhodococcus BCT-ABIs nitrile hydratase 100% 8% 5% 4% 10% 351% 167% 305% 118% 49% 37% Rhodococcus BCT-ABFGs nitrile hydratase 100% 0% 0% 3% 0% ND 9% 10% 0% 29% 11% Rhodococcus DSMZ 44519 nitrile hydratase 100% 46% 12% 9% 2% ND 76% 83% 29% 100% 43% Rhodococcus NOVO SP361 nitrile hydratase 100% 15% 4% 4% 10% ND 30% 7% 9% 27% 13% ND Not determined

C N C N O H C N C H 3

C N N H 2

C N C H

3 O H C N N H C N

C N C N

C H 3

C N O

O C H 3 C H 3

C N

Brady D., Beeton A, Kgaje C, Zeevaart J, van Rantwijk F, Sheldon RA (2004)

Characterisation of Nitrilase and Nitrile Hydratase Biocatalytic Systems.

Applied Microbiology and Biotechnology. 64: 76-85.

Slide 23 © CSIR 2006 www.csir.co.za

HO-

[Fe]3+

R N

O

H

H HB

HOH

[Fe]3+

R NH

OH

HO-

[Fe]3+

R NH2

O HBB

NITRILE HYDRATASE MECHANISM

Huang et al (1997), Structure 5:691-699.

Slide 24 © CSIR 2006 www.csir.co.za

S

S N

N

O

O F e

S

N O

OC y s 1 1 2

C y s 1 1 4 S e r 1 1 3

C y s 1 0 9

I n a c t i v e A c t i v e

S

S N

N

O

O F e

S

O

OC y s 1 1 2

C y s 1 1 4 S e r 1 1 3

C y s 1 0 9

N O

h v

H

N I T R I L E H Y D R A T A S E M E C H A N I S M

Endo et al (1999) TIBTECH 17:244-249.

Slide 25 © CSIR 2006 www.csir.co.za

Ribbon model of the Rhodococcus sp R312 nitrilse hydratase with 3-hydroxy-3-phenylpropionitrile in the active site

Slide 26 © CSIR 2006 www.csir.co.za

Superimposition of 2-phenylbutyronitrile and 3-hydroxy-3-phenylpropionitrile in the active site of the nitrile hydratase

Slide 27 © CSIR 2006 www.csir.co.za

Two-step reaction

On

OH

CNn

OH

COOHn

alkaline pHstereospecific biocatalyst

aldehyde (R,S)-a-hydroxynitrile (S)-a-hydroxycarboxylic acid

H2OHCN

Slide 28 © CSIR 2006 www.csir.co.za

Synthesis of carboxylic acids from aldehydesAldehyde compound incubated with cyanide

and biocatalystConversion to homologous -

hydroxy acid (%)

benzaldehyde 95

4-methylbenzaldehyde 51

4-hydroxybenzaldehyde 55

2-nitrobenzaldehyde 20

2-fluorobenzaldehyde 100

3-chlorobenzaldehyde 100

4-chlorobenzaldehyde 100

4-nitrobenzaldehyde 100

2-chlorobenzaldehyde 90

Vanillin 0

4- methoxy-benzaldehyde (p-anisaldehyde) 0

4-cyanobenzaldehyde 100

4-methylsulpnonyl-benzaldehyde 0

Trimethoxybenzaldehyde 0

Slide 29 © CSIR 2006 www.csir.co.za

Multi-step transformation of phenyl-acetaldehyde to 2-hydroxy-3-phenylpropionic acid.

O

OHN

OH

NH2

OOH

OH

O

KCN

Phenyl-acetaldehyde

2-Hydroxy-3-phenyl-propionitrile

2-Hydroxy-3-phenyl-propionamide

2-Hydroxy-3-phenyl-propionic acid

Nitrile hydratase Amidase

DEPA

Slide 30 © CSIR 2006 www.csir.co.za

Biocatalysis tool boxes

Biocatalyst tool boxes

N

N

CN

CN

CN

OH OH

CN

CH3

CN

Slide 31 © CSIR 2006 www.csir.co.za

CN

OH

CN

NH2

CN

O O

CN CN

CN CN

CN

CN

CN

CN

CN

CN

CN

NCCN

Cl

CN

N

CN

OH

CN

CNNC

NCCN

NH2

NH2

1 2 3 4

5 6 7

8 9 10

11 1213 14

15 16 17

18

Slide 32 © CSIR 2006 www.csir.co.za

Beta-amino compounds

N NH2

CN

Ts

Steps

NH2

N

Slide 33 © CSIR 2006 www.csir.co.za

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

12.5

81

16.8

81

17.2

44

LC-MS

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

50.60

51.10

77.31

105.18

121.30

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

m/z

100.00 200.00 300.00 400.00 500.00 600.00

51.18

52.1363.22

65.22

77.36

89.26

90.97

92.30

103.27

115.30

121.30

161.40

0.00

0.50

1.00

1.50

226.3

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

228.6

273.4

0.00

0.50

1.00

1.50

2.00

2.50

nm

250.00 300.00 350.00 400.00 450.00 500.00 550.00

211.1

258.1

Slide 34 © CSIR 2006 www.csir.co.za

Substrate

Strain

Acr

ylon

itrile

Adi

pam

ide

Adi

poni

trile

Ace

toxy

-phe

nytl

prop

initr

ile

Ace

toxy

phe

nyl a

ceta

mid

e

-m

ethy

l ben

zyl c

yani

de

Ben

zylid

ene

mal

onon

itrile

Ben

zoni

trile

Ben

zam

ide

4-cy

anop

yrid

ine

Dia

min

o m

alen

itrile

Fum

aron

itrile

Isob

utyr

onitr

ile

Mal

onon

itrile

Phe

nylg

lyci

noni

trile

Pro

pion

itrile

Pro

pion

amid

e

Aureobacterium ND ND 3 ND ND 0 2 0 ND 0 ND ND ND ND 4 3 ND

Alcaligenes faecalis 1 ND ND 4 ND ND 0 0 0 ND ND ND ND ND ND 4 4 ND

A. faecalis ATCC 875011 1 2 ND ND ND ND ND ND 4 ND 1 1 1 0 ND ND 3

Bacillus licheniformis 1 3 1 3 1 3 1 0 1 1 1 1 1 2 1 2 0 1

Bacillus licheniformis 2 2 1 3 1 1 0 0 0 1 0 1 0 1 1 3 3 1

Bacillus subtilis 1 4 3 3 0 4 3 0 0 4 2 0 0 3 0 4 4 4

Bacillus subtilis 2 3 2 3 2 4 0 0 0 4 0 1 0 2 1 4 4 3

Bacillus subtilis 3 1 1 3 1 1 0 0 3 1 3 1 1 1 1 2 4 1

Bacillus subtilis 4 3 3 3 0 0 0 0 1 1 2 0 0 2 0 1 3 0

Bacillus subtilis 5 2 1 4 0 0 0 0 1 2 1 0 0 2 0 4 5 0

Chryseomonas luteola 4 3 4 4 5 0 0 0 5 0 1 0 4 3 4 4 5

Microbacterium 1 4 4 4 3 1 0 2 0 3 0 1 0 4 1 4 4 5

Microbacterium 2 3 3 2 4 1 0 0 0 3 0 1 1 3 1 2 2 3

Pseudomonas diminuata 2 0 1 0 1 2 0 2 0 1 0 0 0 0 2 0

Pseudomonas alcaligenes 1 ND ND 3 ND ND 1 0 3 ND 1 ND ND ND ND 1 3 ND

Pseudomonas alcaligenes 2 ND ND 1 ND ND 0 0 0 ND 0 ND ND ND ND 3 3 ND

Pseudomonas alcaligenes 3 1 2 0 1 1 0 0 0 1 0 1 1 1 1 0 1 1

Pseudomonas alcaligenes 4 3 3 3 1 4 0 0 0 2 0 1 1 2 1 4 3 3

Slide 35 © CSIR 2006 www.csir.co.za

Pseudomonas alcaligenes 2 ND ND 1 ND ND 0 0 0 ND 0 ND ND ND ND 3 3 ND

Pseudomonas alcaligenes 3 1 2 0 1 1 0 0 0 1 0 1 1 1 1 0 1 1

Pseudomonas alcaligenes 4 3 3 3 1 4 0 0 0 2 0 1 1 2 1 4 3 3

Pseudomonas alcaligenes 5 2 1 1 1 3 1 0 1 1 0 1 1 2 1 1 2 2

Pseudomonas alcaligenes 6 1 1 3 1 1 1 0 1 0 1 1 1 1 1 1 3 1

Pseudomonas alcaligenes 7 2 ND 3 1 ND 1 0 0 ND 0 1 1 2 1 1 3 ND

Pseudomonas alcaligenes 8 ND ND 2 ND ND 0 0 0 ND 0 0 ND ND ND 2 2 ND

Pseudomonas alcaligenes 9 5 5 3 2 3 0 0 0 4 0 0 1 5 0 3 4 4

Pseudomonas alcaligenes 10 4 2 1 0 4 0 0 0 4 0 0 0 3 0 2 3 4

Pseudomonas alcaligenes 11 4 3 3 0 4 0 0 0 3 0 0 0 2 0 3 4 3

Pseudomonas alcaligenes 12 1 3 2 1 1 0 0 0 1 0 1 0 1 1 4 3 2

Pseudomonas alcaligenes 13 2 3 2 5 2 0 0 2 2 1 1 0 3 2 4 3 5

Pseudomonas alcaligenes 14 1 1 5 0 0 2 0 2 0 2 0 1 1 0 4 5 0

Pseudomonas alcaligenes 15 2 2 3 3 5 0 1 0 1 0 0 0 1 4 4 3 5

Pseudomonas alcaligenes 16 3 3 3 0 5 2 0 2 2 2 0 0 2 4 2 2 5

Pseudomonas maltophilia 1 3 3 3 0 3 2 0 2 2 2 0 0 3 0 1 3 0

Pseudomonas maltophilia 2 2 2 3 3 5 0 0 0 2 0 1 0 1 5 2 3 5

Rhodococcus 1 4 4 4 1 3 1 0 1 5 1 1 1 4 2 2 4 5

Rhodococcus 2 4 4 3 4 2 0 0 0 4 0 1 0 4 2 1 3 5

Rhodococcus 3 4 4 3 5 0 3 0 2 4 2 0 0 5 3 5 3 5

Rhodococcus 4 (Is) 5 5 4 3 4 0 0 1 5 1 0 0 5 4 4 5 5

Rhodococcus 5 0 0 4 0 0 0 0 0 0 0 0 0 0 0 4 4 0

Unidentified 1 1 1 2 2 1 0 0 0 1 0 2 ND 1 1 4 2 1

Unidentified 2 1 1 2 0 0 0 0 0 1 0 0 0 1 0 1 2 1

Unidentified 3 1 1 3 4 2 0 0 0 1 1 1 0 1 3 2 2 5

Key: 5 - Exceptional; 4 - Strong; 3 – Good; 2 – Reasonable; 1 – Poor; 0 - no growth; ND – not determined

Slide 36 © CSIR 2006 www.csir.co.za

Substrates Species A

cryl

onitr

ile

Adi

pam

ide

Adi

poni

trile

Ace

toxy

-phe

nytl

prop

initr

ile

Ace

toxy

phe

nyl

acet

amid

e

AD

B

enzo

nitr

ile

Ben

zam

ide

Dia

min

o m

alon

onitr

ile

Fum

aron

itrile

Isob

utyr

onitr

ile

Mal

onon

itrile

Phe

nylg

lyci

noni

trile

Pro

pion

itrile

Pro

pion

amid

e

Candida famata 4 5 3 1 5 0 5 1 1 4 1 3 3 5 Candida guillermondii 1 5 5 3 1 5 0 5 1 1 4 1 3 3 5 Candida guillermondii 2 5 5 3 5 5 0 5 1 1 5 2 4 3 5 Candida haemulonii 5 5 3 1 5 0 5 2 2 4 5 3 3 5 Candida magnoliae 1 1 3 1 1 3 0 1 1 1 2 1 0 1 2 Candida magnoliae 2 2 2 1 1 4 0 3 0 1 4 1 1 0 4 Candida parapsilosis 5 4 3 1 5 0 5 2 2 3 3 3 3 5 Candida rugosa 2 4 3 2 4 0 4 0 1 4 2 3 3 5 Candida tenius 2 5 3 3 4 0 4 0 1 4 2 2 3 5 Candida tropicalis 1 3 2 0 1 2 0 1 1 1 3 1 0 0 3 Candida tropicalis 2 5 5 3 3 5 0 5 5 3 5 5 4 3 5 Cryptococcus humicola 5 5 3 1 5 0 5 1 1 5 5 3 3 5 Debaryomyces hansenii 1 3 3 3 1 3 0 2 1 1 1 2 3 3 2 Debaryomyces hansenii 2 5 5 3 3 5 0 4 1 1 5 2 3 3 4 Debaryomyces hansenii 3 5 4 3 2 5 0 5 1 1 5 1 3 3 5 Debaryomyces hansenii 4 5 5 3 2 5 0 5 3 3 4 3 3 3 5 Debaryomyces hansenii 5 5 5 4 4 5 0 2 1 1 5 3 3 3 5 Debaryomyces hansenii 6 3 2 2 3 5 0 5 1 1 2 3 2 3 3 Debaryomyces hansenii 7 3 4 2 1 5 0 4 1 1 3 1 2 1 4 Debaryomyces hansenii 8 4 5 3 1 5 0 5 1 1 4 1 3 2 5 Pichia guillermondii 5 4 4 3 5 0 5 1 1 5 2 3 3 5 Rhodotorula sp. 3 3 2 1 2 0 2 1 1 2 1 2 3 2 Trichosporon beigelii 1 5 5 2 1 5 0 5 1 1 5 1 3 3 5 Trichosporon beigelii 2 2 3 3 2 4 0 4 0 1 4 1 3 3 4 Trichosporon mucoides 2 5 2 4 4 0 4 0 1 5 5 3 3 5

Slide 37 © CSIR 2006 www.csir.co.za

Table 4: NITRILE BIOCATALYSIS: Formation of carboxylic acid products

Substrate

Biocatalyst

O-acetoxy-

phenyl-

acetonitrile

Benzo-

nitrile

Chloro-

benzo-

nitrile

Naproxen

nitrile

Phenyl-

glycino-

nitrile

Nit 101 (commercial

enzyme)

0% 55% 75% 0% 0%

Nit 105 (commercial

enzyme)

0% 100% 100% 0% 0%

Nit 106 (commercial

enzyme)

100% 100% 85% 0% 100%

P. alcaligenes 0% 0% 0% 0% 7.41%

R. rhodochrous Is 9.0% 100% 100% 100% 18.3%

Microbacterium 0% 0% 0% 39.8% 9.44%

A. faecalis 0% 0% 0% 2.22% 12.1%

C. tropicalis 0% 0% 6% 0% 11.3%

D. hansenii 0% 0% 0% 0% 5.0%

Slide 38 © CSIR 2006 www.csir.co.za

Comparison with other studies:

• Bacterial genera: Rhodococcus, Pseudomonas, Bacillus and Alcaligenes

• Yeast genera Candida, Debaryomyces, Pichia, Rhodotorula, Trichosporon

• Most nitrile biocatalysts have previously been found in these species.

Slide 39 © CSIR 2006 www.csir.co.za

Conclusion

• Brandão and co-workers (Appl. Environ. Microbiol. 69: 5754-5766, 2003) found that the nitrile hydratases produced from Rhodococcus erythropolis strains isolated from around the world had infra-species amino acid sequence differences, and this provided an explanation for the variability of nitrile substrate usage within the species.

• Our data supports this result.

• South African microbial diversity will include unique biocatalysts.

Slide 40 © CSIR 2006 www.csir.co.za

The Future

• Nitrile hydratase isolation and characterisation (Joni Fredericks)

• Nitrilase kinetics – new assay method (Dr Neeresh Rohitlall).

• Nitrile hdrolysing activity in yeasts (Dr Mapitso Molefe)

• Nitrilase structure and function (Prof. Trevor Sewell, UCT)

• Nitrile Biocatalysts application – biocatalytic reactions and immobilisation (COST group)

Slide 41 © CSIR 2006 www.csir.co.za

ACKNOWLEDGEMENTS

Department of Science and Technology (DST) for financial support for the research, and in particular Mr Dan du Toit for support for this initiative. Dr Martínková, Dr Fred van Rantwijk and the COST Action D25 working group 2 on nitrile biocatalysts for valuable inputs.

A Beeton, Dr Gert Marais, Dr R Mitra and Dr A Botes for cultures.

Dr M Henton and V Chhiba for assistance with microbial taxonomy.U Horn, Dr C Kenyon and Dr N Rohitlall for protein modelling

Slide 42 © CSIR 2006 www.csir.co.za

CSIR• Nosisa Dube• Alison Beeton• Dr Mapitso Molefe• Dr Neeresh Rohitlall• Joni Fredericks

• Dr Riaan Petersen • Dr Jaco Zeevaart• Dr Chris van der Westhuizen

• Kgama Mathiba • Godfrey Kupi• Clement Stander• Neil Wilde• Dr Paul Steenkamp

TU Delft• Dr Fred van Rantwijk• Prof Roger Sheldon • Dr Luuk van Laagen• Dr Moira Bode

• UCT• Prof T Sewell• N Thuku

• WITS• Prof H Dirr

The End