protein-engineered biocatalysts in industry

Protein-Engineered Biocatalysts in Industry

June 18th, 2015Group Meeting

Stefan McCarver

N

O O

NN

N

FF

F

CF3

N

O

NN

N

FF

F

CF3

NH2

Sanchez, S.; Demain, A. L. Org. Process Res. Dev. 2011, 15 , 224–230.

■ World enzyme market in 2003

Protein-Engineered Biocatalysts in IndustryDiverse applications of enzymes

Product USD (Millions)

Detergents

Foods

Agriculture and feed

Textile processing

Pulp, paper, leather, and chemicals

789

634

376

237

222

Koeller, K. M.; Wong, C.-H. Nature 2001, 409, 232–240.

■ Enzymes can provide many advantages for chemical synthesis

Protein-Engineered Biocatalysts in IndustryDiverse applications of enzymes

Higher enantioselectivity and regioselectivity

Can be effective in both aqueous and organic media

Typically do not require protecting groups

Operate under mild conditions with high efficiency

HN

OAc

OHN

OAc

O

MeCHO

OH

N3

HOO

OPO32–

MeOH

N3 OH

OH

OOPO32–

lipase

aldolase

■ Naturally occuring enzymes usually do not have desired catalytic reactivity

Protein-Engineered Biocatalysts in IndustryUtilizing natural enzymes for chemical reactions

Naturally occuring enzyme Ideal biocatalyst

Activity for specific substrate(s)

Unstable under process conditions

Activity for desired substrate(s)

Tolerates process conditions

How do chemists transform natural enzymes into useful biocatalysts?

Bloom, J. D.; Arnold, F. H. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 , 9995–10000.

■ Proteins are modified through iterative mutation and screening


Parent enzyme

Modification of enzyme catalysts

mutagenesis screeningand

selection

Evolved enzyme

■ Evolved proteins are used as parents for another iteration

■ The process is continued until desired reactivity is reached

Mutant proteins

Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.; Wiley-Blackwell, 2006; ch. 8

■ Methods for mutagenesis

Protein-Engineered Biocatalysts in IndustryGenerating protein diversity

Error-prone PCR

Gene shuffling

Site-directed mutagenesisA number of methods are known for specifically substituting individual amino acids in a protein

Requires a lot of structural information to be useful, often a crystal structure and computational modeling

The polymerase chain reaction reaction is naturally error prone

This can be amplified by increasing Mg2+, adding Mn2+ and by using unequal nucleotide concentrations

DNA sequences from several similar proteins are fragmented and randomly recombined

A larger extent of fragmentation results in a greater number of single site mutations




Error-prone PCR

Gene shuffling




This can be amplified by replacing some Mg2+ with Mn2+ and by using excess of certain nucleotides


A larger extent of fragmentation results in a greater number of single site fragmentations




Error-prone PCR

Gene shuffling




This can be amplified by increasing Mg2+, adding Mn2+ and by using unequal nucleotide concentrations


A larger extent of fragmentation results in a greater number of single site mutations




Error-prone PCR

Gene shuffling




This can be amplified by replacing some Mg2+ with Mn2+ and by using excess of certain nucleotides


A larger extent of fragmentation results in a greater number of single site fragmentations

Protein-Engineered Biocatalysts in IndustryCase studies

NCO2Ca0.5

OH OHiPr

Ph

O

NHPh

F

N

O

NN

N

FF

F

CF3

NH2

Atorvastatin (Lipitor)

Me

OMe

O

HO

O

MeMe Me

Simvastatin (Zocor)

Sitagliptin (Januvia)

O

Protein-Engineered Biocatalysts in IndustrySynthesis of atorvastatin

NCO2Ca0.5

OH OHiPr

Ph

O

NHPh

F


■ Lowers blood cholesterol by inhibiting the HMG-CoA reductase enzyme

■ Discovered at Parke-Davis, later acquired by Pfizer

■ Best-selling drug of all time, with over $125 billion in total sales

Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.

■ Process route


key hydroxynitrile

Synthesis of atorvastatin

NC CO2EtOH

LDA, THF

Me OtBu

O

NCOH O

CO2tBu NCOH OH

CO2tBu

NaBH4, MeOBEt2

THF/MeOH –90 °C

2,2-DMP

Acetone, MsOH NCO O

CO2tBu

MeMeH2, Raney-Ni

MeOH, NH3

O OCO2tBu

MeMe

H2N

OiPr O

Ph

PhHN

OF

heptane, tolueneTHF, pivalic acid

NCO2tBu

O OiPr

Ph

O

NHPh

F

MeMe

deprotection

salt formation

Atorvastatincalcium


■ Previous routes to hydroxynitrile starting material


key hydroxynitrile


NC CO2EtOH

■ Estimated demand 100 mT per year

■ Simplified route that reduces waste is highly desirable

HO ClOH

HO CO2EtOH

Br CO2EtOH

NC CO2EtOH

from kineticresolution

3) EtOH, H+

1) NaCN2) NaOH HBr NaCN

kinetic resolution - max 50% yield

two steps involving the use of cyanide

HBr required to form bromohydrin




key hydroxynitrile


NC CO2EtOH



NC CO2EtOH

lactoseO O

HO HBr

EtOH

1) HO–, H2O2

2) H+ Br CO2EtOH NaCN

uses chiral pool materials

HBr required to form bromohydrin




key hydroxynitrile


NC CO2EtOH



NC CO2EtOH

O

O

Cl CO2EtO

Cl CO2EtOHasymmetric

hydrogenation

NaCNCl2

EtOH

high pressure H2 for asymmetric reduction

harsh cyanation conditions




key hydroxynitrile


NC CO2EtOH



Issues to be addressed

■ Requirement of chiral pool materials or high pressure hydrogenation to access alcohol

■ Cyanation requires harsh conditions, challenging to separate product from byproducts


■ Enzymatic route to hydroxynitrile starting material


key hydroxynitrile


NC CO2EtOH



ClO

CO2Et ClOH

CO2Et NCOH

CO2Et

ketonereductase

glucosedehydrogenase

NADPH NADP

glucosegluconate

CO2EtO– HCl HCN

Halohydrin dehalogenase

Kratzer, R.; Wilson, D. K.; Nidetzky, B. Life 2006, 58, 499–507.

■ Ketone reductase mechanism


ClO

CO2Et ClOH

CO2Et

ketonereductase


NADPH NADP

glucosegluconate

O

RlargeRsmall

N N H

His OH

Tyr

NH HH Lys

AspO

O–

N

H H O

NH2

OO

OHNADPH

H

■ Hydrogen bond to histidine orients substrate

■ Proton transfer occurs from tyrosine residue

■ Lysine provides electrostatic stabilization

■ NADPH positioned by hydrogen bond to asparagine


■ Enzymatic route to hydroxynitrile starting material


key hydroxynitrile


NC CO2EtOH



ClO

CO2Et ClOH

CO2Et NCOH

CO2Et

ketonereductase


NADPH NADP

glucosegluconate

CO2EtO– HCl HCN


Janssen, D. B. et. al. J. Bacteriol. 2001, 183 , 5058–5066.

■ Halohydrin dehalogenase mechanism


■ Deprotonation by tyrosine residue

■ Hydrogen bonding catalysis from serine

■ Binding pocket for departing halide

■ NADPH positioned by hydrogen bond to asparagine

NCOH

CO2Et

CN–


ClOH

CO2Et

ClOH

CO2Et

H2N N

ArgH

HO–

Tyr

OSerH

halidebinding pocket

CO2Et

H2N N

ArgH

HO

Tyr

OSerH

halidebinding pocket

H

O

Cl–

ClO

CO2Et ClOH

CO2Et NCOH

CO2Et

ketonereductase


NADPH NADP

glucosegluconate

CO2EtO– HCl HCN



■ Directed evolution of KRED and GDH enzymes using DNA shuffling



■ Directed evolution of KRED and GDH enzymes using DNA shuffling


Initial process (wild-type enzymes) Final process (evolved enzymes)

Substrate (g/L)

Reaction time (h)

Enzyme loading (g/L)

Isolated yield (%)

Product ee (%)

80 160

24 8

9 0.9

85 95

> 99.5 > 99.9

ClO

CO2Et ClOH

CO2Et NCOH

CO2Et

ketonereductase


NADPH NADP

glucosegluconate

CO2EtO– HCl HCN



■ Directed evolution of HDDH enzyme using DNA shuffling


ClO

CO2Et ClOH

CO2Et NCOH

CO2Et

ketonereductase


NADPH NADP

glucosegluconate

CO2EtO– HCl HCN



■ Directed evolution of HDDH enzyme using DNA shuffling


Inital process (wild-type enzyme) Final process (evolved enzyme)

Substrate (g/L)

Reaction time (h)

Enzyme loading (g/L)

Isolated yield (%)

Product ee (%)

20 140

72 5

30 1.2

67 92

> 99.5 > 99.5

ClO

CO2Et ClOH

CO2Et NCOH

CO2Et

ketonereductase


NADPH NADP

glucosegluconate

CO2EtO– HCl HCN


Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin

■ Antidiabetic dipeptidyl peptidase-4 inhibitor

■ Developed and marketed by Merck

■ Often used as a combination therapy with other medicines

N

O

NN

N

FF

F

CF3

NH2


Desai, A. A. Angew. Chem. Int. Ed. 2011, 50, 1974–1976.

■ First generation process route


FF

F

OCO2Me

1) [(S)-binapRuCl2]HBr, H2

2) NaOH83%

FF

F

OH

1) BnONH2•HClEDC, LiOH

2) DIAD, PPh3

81%

CO2H

FF

F

N O

BnO

FF

F

NHBnO

N

O

NN

N

CF3

FF

F

NH3+

N

O

NN

N

CF3

H2PO4–1) H2, Pd/C2) H3PO4

78% (4 steps)

1) base2) EDC, NMM

HNN

NN

CF3

Desai, A. A. Angew. Chem. Int. Ed. 2011, 50, 1974–1976.

■ Second generation process route


FF

F

CO2H

1) [{Rh(cod)Cl}2], NH4Cl,tBu-josiphos, H2

2) heptane/iPrOH3) H3PO4

FF

F

O–

then NH4OAc

FF

F

NH2

N

O

NN

N

CF3

FF

F

NH3+

N

O

NN

N

CF3

H2PO4–

HNN

NN

CF3

tBuCOCl, iPr2NEt83%

O

O

O

O

MeMe

O

O

O

O

MeMe

iPr2HNEt+

CF3CO2H

one pot, 82% yield 81% yield> 99.9% ee

•HCl

Savile, C. K. et. al. Science 2010, 329, 305–309.

■ Third generation process route


transaminasepyridoxal phosphate

FF

F

O

N

O

NN

N

CF3

FF

F

NH2

N

O

NN

N

CF3

iPrNH2

■ Transition metal catalyzed hydrogenation required carbon treatment to remove Rh as well as ee upgrade through recrystallization

■ Biocatalysis presents an opportunity to attain excellent yield and near perfect selectivity under mild conditions

O

Me

NH2

MeATA-117

> 99.9% ee

Chem. Commun. 2009, 2127–2129.


Protein-Engineered Biocatalysts in IndustryTransaminase mechanism

NH

OH

Me

NH

H2O3PO

Lys

NH

OH

Me

NH

H2O3PO

MeMe

NH2

Lys

H

NH

OH

Me

NH

H2O3PO

MeMe

NH3

Lys

NH

OH

Me

NH

H2O3PO

MeMe

NH2

Lys

NH

OH

Me

NH2

H2O3PO

NH3

Lys

NH

OH

Me

NH

H2O3PO

NH2

Lys

PhMe

NH

OH

Me

NH

H2O3PO

PhMe

NH3

Lys

NH

OH

Me

NH

H2O3PO

PhMe

NH2

Lys

NH2

PhMe

O

PhMe

H2O

NH2

MeMe

NH2

MeMe

O

PhMe

NH2

PhMe

O

MeMe

H


■ Design of a transaminase catalyst - site saturation libraries in small and large pockets




Orange: Large pocket

Green: Catalytic residues

Teal: Small Pocket





FF

F

O

N

O

NN

N

CF3

FF

F

NH2

N

O

NN

N

CF3

iPrNH2

■ Site saturation and combinatorial libraries of binding pocket screened for activity

■ Most active mutant subjected to directed evolution for activity and stability under process conditions


■ Process optimization


Me

NH2

N

O

NN

N

CF3

NH2

N

O

NN

N

CF3

Activity on model substrate Activity on sitagliptin ketone

38% conversion

10 g/L enzyme

2 g/L substrate

0.5 M iPrNH2

5% DMSO / triethanolamine

10 g/L enzyme

2 g/L substrate

0.5 M iPrNH2


76% conversion

Optimized process

0.6 g/L enzyme

200 g/L substrate

1 M iPrNH2


92% yield, > 99.9% ee

F

F

F

NH2

N

O

NN

N

CF3

F

F

F





FF

F

O

N

O

NN

N

CF3

FF

F

NH2

N

O

NN

N

CF3

iPrNH2

■ Increase in overall yield relative to second generation process: 13%

■ Waste reduction relative to second generation process: 19%

Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin

■ Lipid lowering medication - HMG CoA reductase inhibitor

■ Developed and marketed by Merck

■ Produced by semisynthesis from lovastatin

Me

OMe

O

HO

O

MeMe Me

Simvastatin (Zocor)

O

Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C. C.; Tang, Y. Chem. Biol. 2006, 13 , 1161–1169.

■ Produced by semisynthesis


xxxsteps

xxxxxx

Me

OMe

O

HO

O

MeMe Me

O

Me

OMe

O

HO

O

Me

O

Me

Lovastatin Simvastatin

Isolated from Aspergillus terreus

Hydroxymethylglutaryl CoA reductase inhibitor

Increased inhibitory properties

Lower undesirable side effects


■ Typical semisynthetic route


xxx

xxxxxx

Me

OMe

O

HO

O

MeMe Me

O

Me

OMe

O

HO

O

Me

O

Me

xxxhydrolysis

xxxxxx

Me

OHMe

O

HO O

Me

OHMe

O

TBSO O

TBSCl

Me

OHMe

O

TBSO O

xxx

xxxxxx

Cl

O

MeMe Me

Me

OMe

O

TBSO

O

MeMe Me

O

xxx

xxxxxx

deprotect


■ Potential synthesis utilizing biocatalysis


Me

OMe

O

HO

O

MeMe Me

O

Me

OMe

O

HO

O

Me

O

Me

xxxhydrolysis

xxxxxx

Me

OHMe

O

HO O

xxxxxx

Direct enzymatic route:

■ Higher yielding process, cost savings

■ Remove need for protecting groups

S

O

MeMe Me

CoA

US Patent WO/2011/041231

■ Selective biocatalytic acylation reaction





Me

OMe

O

HO

O

MeMe Me

O

Me

OHMe

O

HO O

Directed

evolution

xxxsaturation mutagenesis

error-prone PCRxxx

Improved thermal stability

Improved acyltransferase activity

Use of a small molecule acyl donor

S

O

MeMe Me

CO2Me


■ Directed evolution of acyltransferase





0"

1"

2"

3"

4"

5"

6"

0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10"

k cat

(min

-1)

Directed evolution round

Improved Catalytic Activity




0"

1"

2"

3"

4"

5"

6"

0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10"

k cat

(min

-1)



0"

50"

100"

150"

200"

250"

0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10"

Solu

bilit

y (m

g/L)


Improved Protein Solubility




0"

1"

2"

3"

4"

5"

6"

0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10"

k cat

(min

-1)



0"

50"

100"

150"

200"

250"

0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10"

Solu

bilit

y (m

g/L)


Improved Protein Solubility

35#

37#

39#

41#

43#

45#

47#

49#

51#

0# 1# 2# 3# 4# 5# 6# 7# 8# 9# 10#

T m (°

C)


Improved Protein Stability




Me

OMe

O

HO

O

MeMe Me

O

xxx

Me

OHMe

O

HO O

S

O

MeMe Me

CO2Me

■ Reduced use of hazardous substances and solvents in synthesis

■ Catalyst from renewable feedstocks, byproducts all recycled

■ Produced in 97% yield compared to < 70% for previous processes

EPA Presidential Green Chemistry Award 2012



Protein-Engineered Biocatalysts in IndustryCase studies

NCO2Ca0.5

OH OHiPr

Ph

O

NHPh

F

N

O

NN

N

FF

F

CF3

NH2


Me

OMe

O

HO

O

MeMe Me

Simvastatin (Zocor)


O

protein-engineered biocatalysts in industry

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