protein-engineered biocatalysts in industry

Protein-Engineered Biocatalysts in Industry

June 18th, 2015Group Meeting

Stefan McCarver

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

Agriculture and feed

Textile processing

Pulp, paper, leather, and chemicals

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

OPO32–

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

Atorvastatin (Lipitor)

MeMe Me

Simvastatin (Zocor)

Sitagliptin (Januvia)

Protein-Engineered Biocatalysts in IndustrySynthesis of atorvastatin

NCO2Ca0.5

OH OHiPr

■ 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

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

OiPr O

heptane, tolueneTHF, pivalic acid

NCO2tBu

O OiPr

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

lactoseO O

HO HBr

1) HO–, H2O2

2) H+ Br CO2EtOH NaCN

uses chiral pool materials

HBr required to form bromohydrin

key hydroxynitrile

NC CO2EtOH

Cl CO2EtO

Cl CO2EtOHasymmetric

hydrogenation

NaCNCl2

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

CO2Et ClOH

CO2Et NCOH

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

CO2Et ClOH

ketonereductase

NADPH NADP

glucosegluconate

RlargeRsmall

His OH

NH HH Lys

OHNADPH

■ 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

CO2Et ClOH

CO2Et NCOH

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

halidebinding pocket

CO2Et ClOH

CO2Et NCOH

ketonereductase

NADPH NADP

glucosegluconate

CO2EtO– HCl HCN

■ 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

> 99.5 > 99.9

CO2Et ClOH

CO2Et NCOH

ketonereductase

NADPH NADP

glucosegluconate

CO2EtO– HCl HCN

■ Directed evolution of HDDH enzyme using DNA shuffling

CO2Et ClOH

CO2Et NCOH

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

30 1.2

> 99.5 > 99.5

CO2Et ClOH

CO2Et NCOH

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

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

■ First generation process route

OCO2Me

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

2) NaOH83%

1) BnONH2•HClEDC, LiOH

2) DIAD, PPh3

H2PO4–1) H2, Pd/C2) H3PO4

78% (4 steps)

1) base2) EDC, NMM

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

■ Second generation process route

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

2) heptane/iPrOH3) H3PO4

then NH4OAc

H2PO4–

tBuCOCl, iPr2NEt83%

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

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

MeATA-117

> 99.9% ee

Chem. Commun. 2009, 2127–2129.

Protein-Engineered Biocatalysts in IndustryTransaminase mechanism

H2O3PO

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

Orange: Large pocket

Green: Catalytic residues

Teal: Small Pocket

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

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

38% conversion

10 g/L enzyme

2 g/L substrate

0.5 M iPrNH2

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

38% conversion

10 g/L enzyme

2 g/L substrate

0.5 M iPrNH2

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

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

MeMe Me

Simvastatin (Zocor)

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

■ Produced by semisynthesis

xxxsteps

xxxxxx

MeMe Me

Lovastatin Simvastatin

Isolated from Aspergillus terreus

Hydroxymethylglutaryl CoA reductase inhibitor

Increased inhibitory properties

Lower undesirable side effects

■ Typical semisynthetic route

xxxxxx

MeMe Me

xxxhydrolysis

xxxxxx

TBSO O

xxxxxx

MeMe Me

xxxxxx

deprotect

■ Potential synthesis utilizing biocatalysis

MeMe Me

xxxhydrolysis

xxxxxx

Direct enzymatic route:

■ Higher yielding process, cost savings

■ Remove need for protecting groups

MeMe Me

US Patent WO/2011/041231

■ Selective biocatalytic acylation reaction

MeMe Me

Directed

evolution

xxxsaturation mutagenesis

error-prone PCRxxx

Improved thermal stability

Improved acyltransferase activity

Use of a small molecule acyl donor

MeMe Me

■ Directed evolution of acyltransferase

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

Directed evolution round

Improved Catalytic Activity

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

Improved Protein Solubility

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

Improved Protein Solubility

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

T m (°

Improved Protein Stability

MeMe Me

■ 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

MeMe Me

Simvastatin (Zocor)

protein-engineered biocatalysts in industry

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