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Biocatalytic Retrosynthesis Nicholas J. Turner School of Chemistry & Manchester Institute of Biotechnology, University of Manchester, UK ROBOX/Carbazymes Summer School Certosa di Pontignano, Siena, IT 3 rd – 6 th October 2017
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Page 1: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Biocatalytic Retrosynthesis

Nicholas J. Turner

School of Chemistry & Manchester Institute of Biotechnology, University of Manchester, UK

ROBOX/Carbazymes Summer School

Certosa di Pontignano, Siena, IT 3rd – 6th October 2017

Page 2: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

3 Approaches to (un)natural product synthesis

Biosynthesis Organic

Synthesis Biocatalysis

• Natural products • Biosynthetic pathways • Enzyme mechanism • Specialised enzymes

• New reagents • New catalysts • Retrosynthesis • Synthetic strategy

• Engineered biocatalysts • Broad specificity • Systems biocatalysis • Synthetic biology

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Biosynthetic retrosynthesis

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Biosynthetic retrosynthesis

Page 5: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Biosynthetic retrosynthesis

Page 6: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Biosynthetic retrosynthesis

Page 7: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Biosynthetic retrosynthesis

Page 8: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Diversity of natural products

1. All steps subjected to catalysis

2. No protecting groups

3. Relatively simple transformations (oxidation, reduction, hydrolysis)

4. Cascades used to build molecular complexity

Page 9: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Nature Chem. Biol., 2013, 9, 285-288; Angew. Chem. Int. Ed., 2017, 56, in press (with Erick Carreira)

Biocatalytic retrosynthesis

Page 10: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Design-Build-Test: Development of a cascade

S.P. France, L. Hepworth, N.J. Turner & S.L. Flitsch, ACS Catal., 2017, 7, 710-724.

Page 11: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

What makes a good cascade?

1. ‘One Pot’ – all transformations compatible

2. High selectivity/orthogonality of biocatalysts – no cross reactivity

3. Intrinsic pre-organisation of reaction sequences

4. Thermodynamics favourable

5. Comparable kinetics – no accumulation of reactive intermediates

6. Whole cells to avoid need for co-factors and enzyme isolation

A B C

Cat 1 Cat 2

S.P. France, L. Hepworth, N.J. Turner & S.L. Flitsch, ACS Catal., 2017, 7, 710-724.

Page 12: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Biocatalytic toolbox

Page 13: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

(Asymmetric) biocatalytic amine toolbox

Need new biocatalysts for (C-C) formation in addition to (C-N)

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Biocatalytic toolbox

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(S)-DIP-Cl @ -20oC

MLK-II MLK-III

D. Rozzell and J. Liang, Speciality Chemicals Magazine, 2008, 36.

Montelukast

Page 16: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Replaces (S)-DIP-Cl @ -20oC

D. Rozzell and J. Liang, Speciality Chemicals Magazine, 2008, 36.

MLK-II MLK-III

Montelukast

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Montelukast

Biocatalytic Process

100g/L Catalytic

45 oC 99.3%

Direct filtration >99.9%

6 IPA, H2O, toluene

Biodegradable enzyme, cofactor

D. Rozzell and J. Liang, Speciality Chemicals Magazine, 2008, 36.

(S)-DIP-Cl Process

100g/L 1.8 eq DIP-Cl

-25 oC Not provided

Extraction with high dilution 99.2% (after recryst.)

30-50 DCM, THF

Non-biodegradable borate salts Other inorganics, 3.6 eq. pinene

Parameter

Ketone Concentration Catalytic/Stoichiometric

Temperature Conversion

Product Isolation Enantiomeric Excess

Solvent/MLK-III (L/Kg) Solvents Used

Other Waste Generation

Comparison of biocatalytic and (S)-DIP-Cl process metrics for MLK-II to MLK-III

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DSM

tonne-scale production

• 7-step process • Cost reduction and IP issues became important

PAL mediated synthesis of perindopril

Page 19: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

PAL mediated synthesis of perindopril

Anti-hypertensive

DSM

96% yield; 99% e.e.

tonne-scale production

B. de Lange et al., ChemCatChem, 2011, 3, 289.

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K.B. Hansen et al., J. Am. Chem. Soc., 2009, 131, 8798.

2nd generation process

Januvia (Sitagliptin)

oral anti-diabetic DPP-4 inhibitor

Sitagliptin

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Januvia (Sitagliptin)

oral anti-diabetic DPP-4 inhibitor

C.J. Savile et al., Science, 2010, 329, 305.

(R)-selective transaminase ‘substrate walking’ to evolve larger binding pocket

3rd generation process

• Increased yield (by 13%) • Higher e.e. • Higher productivity (by 53%) • Reduced waste (by 19%)

92% yield >99.95% e.e.

Sitagliptin

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Retrosynthesis of lipitor side-chain

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Sustainable low cost production of medicines for 3rd world

‘A process for the preparation of substituted prolyl peptides and similar peptidomimetics EP 2539320 A1’.

Synthesis of telaprevir (hepatitis C)

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Reagents and conditions: a) MAO-N, 100 mM KPO4, pH = 8.0, 37 °C, then: b)

1,2, CH2Cl2, 50%; c) K2CO3, MeOH; d) Dess-Martin, CH2Cl2, 50% over 2 steps.

telaprevir

Multi-component synthesis of telaprevir

A. Znabet, R. Orru, N.J. Turner et al., Chem. Commun., 2010, 7918.

83:17

Page 25: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Galactose oxidase (GOase)

J.B. Rannes et al., J. Am. Chem. Soc., 2011, 133, 8436-8439.

B. Yuan et al., Angew. Chem. Int Ed., 2010, 49, 7010; S. Staniland, et al. Chem. Eur. J. 2014, 20, 13084.

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Chiral auxilliary based on diarylether

Lipase catalysed desymmetrisation Asymmetric oxidation Asymmetric reduction

B. Yuan, J. Clayden, N.J. Turner et al., Angew. Chem. Int Ed., 2010, 49, 7010.

Page 27: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Desymmetrisation of diaryl ethers

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Origin of selectivity

B. Yuan, J. Clayden, N.J. Turner et al., Angew. Chem. Int Ed., 2010, 49, 7010.

(+)-P

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Entry KRED conv [%] ee [%] Config

1 102 21 7 R

2 105 4 >99 R

3 107 5 >99 R

4 108 100 96 S

5 110 59 99 S

6 111 5 >99 R

7 112 100 98 S

8 113 93 95 S

9 119 100 94 S

10 123 22 16 S

11 124 73 68 S

12 130 100 >99 R

DKR of racemic quinoline N-oxide

S. Staniland, N.J. Turner, J. Clayden et al., Angew. Chem. Int. Ed. 2016, 55, 10755.

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Tandem GOase/PaoABC oxidation

Susanne, Herter, Shane McKenna, Will Birmingham, Andrew Carnell

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Tandem GOase/XDH oxidation

B. Bechi, S. Herter, S. McKenna, C. Riley, S. Leimkühler, N. J. Turner and A.J. Carnell, Green

Chem. 2014, 16, 4524.

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Dibenz[c,e]azepines – biocatalytic retrosynthesis

• Possess carbon centred and axial chirality (atropisomers)

• Potentially interesting applications as chiral ligands

• Can possess biological activity e.g. Azapetine is a vasodilator.

Azapetine S. L. Pira, T. W. Wallace and J. P. Graham, Org. Lett., 2009, 11, 1663-1666.

Scott France

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Dibenz[c,e]azepines

Scott France

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Dibenz[c,e]azepines

Scott France

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Cascade reactions with IREDs

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ω-TA - IRED tandem reactions

S.P. France, S. Hussain, F. Leipold, A. Hill et al., ACS Catal., 2016, 6, 3753.

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S.P. France, S. Hussain, F. Leipold, A. Hill et al., ACS Catal., 2016, 6, 3753.

Cascade reactions with IREDs

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CAR - ω-TA - IRED tandem reactions

O’Reilly et al., Angew. Chem. Int. Ed. 2014, 53, 10714 - 10717 (VIP).

S.P. France, S. Hussain, F. Leipold, A. Hill et al., ACS Catal., 2016, 6, 3753.

D. Gahloth et al., Nature Chem. Biol, 2017, 13, in press: (CAR structure).

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In vitro generation of chiral piperidines

S.P. France, S. Hussain, F. Leipold, A. Hill et al., ACS Catal., 2016, 6, 3753.

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Biosynthesis of chiral piperidines via PKS

H. Peng, et al., ACS Chem. Biol., 2016, 11, 3278-3283.

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• Can we develop guidelines for route design for synthetic

chemists (biocatalytic retrosynthesis).

• How many different biocatalyst classes do we have/need to be able to do real organic synthesis? 50 /250.

• How many distinct retrosynthetic disconnections? (ca. 250).

• Where are the gaps in biocatalysis – which reactions are under-represented in the biocatalysis toolbox (C-C bond formation)?

• Need biocatalysts with broad substrate scope that are active and stable under the conditions of a chemical process (fit

• biocatalyst to process rather than vice-versa).

• Need to increase application of biocatalysis in medicinal chemistry.

Challenges for biocatalysis

Page 42: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,
Page 43: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Engineered Biocatalysts for Chiral Amine Synthesis

Nicholas J. Turner

School of Chemistry & Manchester Institute of Biotechnology, University of Manchester, UK

ROBOX/Carbazymes Summer School

Certosa di Pontignano, Siena, IT 3rd – 6th October 2017

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Protein Evolution

Biocatalysts Synthesis Mechanism-based Discovery

Design – Evolution - Synthesis

Design features: • Activity • Selectivity • Specificity • Stability

Sequence-based Discovery

(Bioinformatics)

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Amines in organic chemistry

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Amine biocatalysis

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ACS Catal, 2016, 6, 3753; ACIE, 2014, 53, 2447.

Transaminase

Page 48: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Januvia (Sitagliptin)

oral anti-diabetic DPP-4 inhibitor

C.J. Savile et al., Science, 2010, 329, 305.

(R)-selective transaminase ‘substrate walking’ to evolve larger binding pocket

3rd generation process

• Increased yield (by 13%) • Higher e.e. • Higher productivity (by 53%) • Reduced waste (by 19%)

92% yield >99.95% e.e.

Sitagliptin

Page 49: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

DKR with transaminases

J. Limanto et al., Org. Lett., 2014, 16, 2716.

C.K. Cheung et al., OPRD, 2014, 18, 215.

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DKR with transaminases

Z. Peng et al., Org. Lett., 2014, 16, 860 (Pfizer).

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A

The PAL (ammonia lyase) and PAM (ammonia

mutase) toolbox

A = Wild-type PcPAL / RgPAL

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B

A

Wu et al., Angew Chem Int Ed 2012, 51, 482.

B = Directed evolution of TcPAM

The PAL (ammonia lyase) and PAM (ammonia

mutase) toolbox

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B

A

C

,

C = Combining AvPAL, deaminase and chemical reduction steps

The PAL (ammonia lyase) and PAM (ammonia

mutase) toolbox

Parmeggiani et al., Angew Chem Int Ed, 2015, 54, 4608.

Page 54: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

B

D

A

C

The PAL (ammonia lyase) and PAM (ammonia

mutase) toolbox

A = Wild-type PcPAL / RgPAL

B = Directed evolution of TcPAM

C = Combining AvPAL, deaminase and chemical reduction steps

D = Rational Design of EncP

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(S)-selective MIO-dependent amination of

cinnamate derivatives by EncP

N.J. Weise et al., J. Am. Chem. Soc., 2015, 137, 12977-12983.

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(a)

(b)

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EncP whole cells

4M (NH4)2SO4, 55oC

Cinnamate β-Phenylalanine α-Phenylalanine

EncPwt 56 : 44 EncPR299K 88 : 12 EncPE293Q 43 : 57 EncPE293M 18 : 82

Rationally designed point mutations: regioselectivity amenable to shifting in both directions

EncP variants with altered regioselectivity

N.J. Weise et al., J. Am. Chem. Soc., 2015, 137, 12977-12983.

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N.J. Weise et al., J. Am. Chem. Soc., 2015, 137, 12977-12983.

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0

20

40

60

80

100

1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 1m 1n 1o 1p 1q 1r 1s 1t 1u 1v

0

20

40

60

80

100

1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 1m 1n 1o 1p 1q 1r 1s 1t 1u 1v

0

20

40

60

80

100

1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k 1l 1m 1n 1o 1p 1q 1r 1s 1t 1u 1v

>95

>95

75

83

>95

>95

>95

>95

n.d.

72

-

n.d.

n.d.

81

>95

>95

>90

>95

>90

>95

>95

>95

n.d.

>95

-

6

>95

92

n.d.

64

n.d.

>95

>95

>95

0

-

85

>95

>95

>95

84

-

n.d.

86

ee 2 (%)

ee 3 (%)

Co

nv.

(%)

(a)

>95

>95

65

>95

n.d.

>95

>95

-

n.d.

>95

-

n.d.

n.d.

>95

>95

>95

>90

-

>90

>95

>95

n.d.

n.d.

-

-

68

n.d.

>95

-

>95

n.d.

-

>95

-

0

-

84

-

>95

-

86

-

-

-

ee 2 (%)

ee 3 (%)

Co

nv.

(%)

>95

>95

-

56

n.d.

87

n.d.

>95

-

48

-

n.d.

-

60

-

74

>80

>95

-

72

-

62

n.d.

>95

-

20

-

78

-

70

-

>95

>95

>95

0

>95

>95

>95

>95

>95

82

>95

-

>95

ee 2 (%)

ee 3 (%)

(b)

(c)

Co

nv.

(%)

WT

E293M

R299K

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Phenylalanine ammonia lyase

PAL

(H359Y)

ACIE, 2015, 54, 4608.

R = o-NO2, m-NO2, p-NO2, p-CF3, p-CN yields 64-83%, ee ≥98%

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Directed evolution of MAO-N

NH

NH2

NH2NH2

NH2

D1 D5 D6/D7

simple achiral

1o amines

Wild-TypeMAO-N

chiral

1o amines

chiral

1o/2o/3o amines

broad spectrum(S)-selective

amine oxidase

1st random libraryca. 150,000 clones

2nd random libraryfollowed by

'hot-spot' librariesca. 20,000 clones

'active-site' librariesca. 10,000 clones

N

H

>103 improvement in kcat

e.e. >98%

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(S)-MAO-N biocatalyst toolbox

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Monoamine oxidase

solifenacin

(urinary tract)

levocetirizine

(anti-histamine)

telaprevir

(hepatitis c) argatroban

(blood cloting)

JACS, 2013, 135, 10863.

Nature Chem, 2013,5, 93.

ACIEE, 2015, 54, 4608.

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Engineering MAO-N for diarylaminomethanes

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Rational engineering MAO-N

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MAO-N D11 crystal structure

Annika Frank (University of York).

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Deracemisation of API building blocks

4-chlorobenzhydrylamine:

1-phenyltetrahydroisoquinoline:

(R)-selectivity

D. Ghislieri A.P. Green, M. Pontini, et al., J. Am. Chem. Soc., 2013, 135, 10863-10869.

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4 reactions: 1 x C-C; 2 x Ox; 1 x Red

Harmicine:

Conv.: 80%

ee: 99%

D. Ghislieri A.P. Green, M. Pontini, et al., J. Am. Chem. Soc., 2013, 135, 10863-10869.

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Piperidines

Marta Pontini and Bas Groenendaal

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Regio- and stereoselective

ω-transaminase/MAO-N cascades

E. O’Reilly et al., Angew. Chem. Int. Ed., 2014, 53, 2447-2450.

Page 71: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

ω-TA - MAO-N tandem reaction

E. O’Reilly et al., Angew. Chem. Int. Ed., 2014, 53, 2447.

Page 72: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

MAO-N/Berberine bridge enzyme

J. Schrittwieser, D. Ghisleri, W. Kroutil, N.J. Turner et al., Angew. Chem. Int. Ed., 2014, 53, 3731-3734.

Page 73: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

(R)-Selective amine oxidase

A) 20 mM (R)-nicotine B) 20 mM (S)-nicotine

G. Schulz et al., J. Mol. Biol., 2005, 352, 418-428.

Page 74: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Glu352

Glu350

Ala374 Leu375

CASTing libraries

Ala374/Leu375: no hits Glu350/Glu352: several hits including Leu350/Asp352

(R)-Selective amine oxidase

R. Heath et al., ChemCatChem, 2014, 6, 996-1002.

Page 75: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Substrate specificity of Leu350/Asp352 HDNO

all >95% e.e. and (S)-enantiomers

R. Heath et al., ChemCatChem, 2014, 6, 996-1002.

Page 76: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Imine reductases from Streptomyces sp.

K. Mitsukura et al., Org. Biomol. Chem., 2010, 8, 4533.

K. Mitsukura et al., Biosci. Biotechnol. Biochem. 2011, 75, 1778–1782.

Page 77: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Imine reductases (IREDs)

Whole cell and isolated enzyme imine reduction

ACS Catal, 2016, 6, 3753.

ACS Catal, 2016, 6, 3880. ChemCatChem, 2013, 5, 3505.

IRED from Paenibacillus

lactis: Li et al., Adv. Synth.

Catal., 2015, 357, 1692.

Page 78: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Y. Asano et al, J. Bacteriol., 1989, 171, 4466.

Merck-Codexis, Biocatalysis GRC, July 2014; US WO 2013170050 A1 (2013).

Asymmetric reductive amination

H. Mihara et al., FEBS J. 2005, 272, 1117–1123.

N-methyl-L-amino acid dehydrogenase

Opine dehydrogenase

L-amino acid dehydrogenase

M. Abrahamson et al., ACIEE. 2012, 51, 3969–3972.

Page 79: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Relationship of RedAms to IREDs

Page 80: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Reductive amination with AspRedAm

Page 81: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

AspRedAm preparative-scale reactions

Turnover number (TON) of >30,000

Turnover frequency (TOF) of up to 300 min-1

[S] up to 0.4M amine & 0.2M ketone with new homologues

G. Aleku, S.P. France et al., Nature Chem., 2017, 9, doi:10.1038/nchem.2782.

Page 82: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Biocatalytic

Hydrogen Borrowing Cascades

Page 83: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Biocatalytic asymmetric hydrogen borrowing

Page 84: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Biocatalytic asymmetric hydrogen borrowing

F.G. Mutti. T. Knaus, N.S. Scrutton, M. Breuer and N.J. Turner, Science, 2015, 349, 1525-1529.

Page 85: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Biocatalytic asymmetric hydrogen borrowing

F.G. Mutti. T. Knaus, N.S. Scrutton, M. Breuer and N.J. Turner, Science, 2015, 349, 1525-1529.

Page 86: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

2o Amine synthesis via hydrogen borrowing?

S.L. Montgomery, J. Mangas-Sanchez et al., Angew. Chem. Int. Ed., 2017, 56, in press.

Page 87: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

ADH/AspRedAm hydrogen borrowing

S.L. Montgomery, J. Mangas-Sanchez et al., Angew. Chem. Int. Ed., 2017, 56, in press.

Page 88: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

ADH/AspRedAm hydrogen borrowing

0

10

20

30

40

50

60

70

80

0 20 40 60 80

Pro

du

ct

(%)

Time (hours)

pH 7.0, 20°C

pH 7.0, 30°C

pH 9.0, 20°C

pH 9.0, 30°C

For racemic 2o

alcohols need

Non-selective ADH

(ADH-306 from JM)

S.L. Montgomery, J. Mangas-Sanchez et al., Angew. Chem. Int. Ed., 2017, 56, in press.

Page 89: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

The Landscape of Amine Biocatalysis

Tall mountains dominate the view…

AMINE OXIDASES

DEHYDROGENASES

AMMONIA LYASES ω-TRANSAMINASES IMINE

REDUCTASES REDUCTIVE

AMINASES

…but there are also tiny peaks far away!

Page 90: No Slide Title...5. Comparable kinetics – no accumulation of reactive intermediates 6. Whole cells to avoid need for co-factors and enzyme isolation A CB Cat 1 Cat 2 S.P. France,

Acknowledgements

amine biocatalysis: Rachel Heath, James Galman, Juan Mangas-Sanchez, Godwin Aleku, Sarah Montgomery, Scott France, Iustina Slabu, Bruna

Costa, Cesar Iglesias, Jeremy Ramsden, Wojciech Zawodny, Agata Brzezniak, Frank Xu

Gideon Grogan, Henry Man, Mahima Sharma (York)

and everyone else in the group … Fabio Parmeggiani, Syed Ahmed, Nick Weise, Sasha Derrington,

Chantel Jensen, Will Birmingham, Ian Rowles, Mark Corbett, Jane Kwok, Navya Menon, Michele Tavanti, Matthew Thompson


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