The Story of LIPITOR® - A Peek into the World

of Pharmaceutical Process Chemistry

Ca2+

HO
Chemical Synthesis
CO 2
HO

F
N LIPITOR® – $12 billion/year sales (2005)
Chiral side chain (circled) – 220 ton/year
NH
O

2
Atorvastatin calcium Biocatalysis
LIPITOR ®

Aman Desai
7th Feb. 2007
 The Problem – The “Bad” Cholesterol

• Cholesterol – a very important biological molecule.

• Most cholesterol is not dietary, it is

H
synthesized internally.
H H
HO
• Cholesterol is bound to lipoproteins and transported Cholesterol
through blood.
High Density Lipoprotein (HDL) – “good”
• 2 kinds of lipoproteins
Low Density Lipoprotein (LDL) – “bad”

atherosclerosis

coronary heart disease & other cardiovascular diseases

One of the leading causes of death in the world today!

www.wikipedia.org
www.americanheart.org
 The Solution – Suppressing Cholesterol Biosynthesis

OH O
O O O
+ HMG-CoA-Synthetase
SCoA
SCoA SCoA
O O
HSCoA HMG-CoA

2 NADPH + 2H +

HMG-CoA-Reductase Rate
Limiting
2 NADP + + HSCoA Step

OH OH
H H

H H O O
HO
Cholesterol >25 steps Mevalonic Acid

http://www.med.unibs.it/~marchesi/cholest.html
 The Solution – Suppressing Cholesterol Biosynthesis
HO O OH O
HO O
O SCoA
O O
O 2''
O O O
O H
HMG-CoA
H
9'
9' 2 NADPH + 2H+
Lovastatin (MEVACOR®) HMG-CoA-Reductase Rate
Mevastatin Limiting
MERCK
2 NADP + + HSCoA Step

Inhibition
HO HO O
CO 2Na
HO O
O O
O 2'' OH OH
H O
H
9'
HO O O
Pravastatin (PRAVACOL®) Simvastatin (ZOCOR®) Mevalonic Acid
BRISTOL - MYERS SQUIBB MERCK

Endo, A. J. Lipid Res. 1992, 33, 1569-1582.

Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
 Mechanism of Action of Statin Drugs
HO O HO O
HO O O
O
O O O
O
O SCoA
O H
H HMG-CoA
2 NADPH + 2H+

HMG-CoA-Reductase Rate
® Limiting
Mevastatin Lovastatin (MEVACOR )
2 NADP + + HSCoA Step
MERCK
Inhibition
O
HO ONa HO O
OH
O O
O OH OH
O
H O
H
O O
HO
Mevalonic Acid
Pravastatin (PRAVACOL®) Simvastatin (ZOCOR ) ®
BRISTOL - MYERS SQUIBB MERCK

http://www.med.unibs.it/~marchesi/cholest.html
 Mechanism of Action of Statin Drugs
HO O
HO O
O
O O
O
O
O H O Ca 2+
H HO O

OH 2

Mevastatin Lovastatin (MEVACOR®) F

MERCK N

O NH
HO ONa HO O
O
OH
O O
O
Atorvastatin calcium (LIPITOR®)
O PFIZER
H O
H

HO
Pravastatin (PRAVACOL®) Simvastatin (ZOCOR®)
BRISTOL - MYERS SQUIBB MERCK

http://www.med.unibs.it/~marchesi/cholest.html
 Human HMGR with Natural Substrates

HO O
O

SCoA
HMG-CoA
2 NADPH + 2H+

HMG-CoA-Reductase Rate
Limiting
2 NADP + + HSCoA Step

Inhibition

OH OH

O O

Mevalonic Acid

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

 Human HMGR with Natural Substrates

HO O
O

SCoA
HMG-CoA

O Ca 2+
HO O

OH 2

F
N

NH
O

LIPITOR ®

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

 HO O
Human HMGR with Natural Substrates O

O
S
HMG-CoA
CH2
CH2
H N
C O
CH2
CH2 pantothenic
H N acid
C O
H C OH
H3C C CH3
CH2
O
R
R = 3'-phosphoadenosine
diphosphate
Ca 2+
O
HO O
2
OH

F
N

NH
O

LIPITOR®
Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.
 Human HMGR with LIPITOR®

O Ca 2+
HO O

OH 2

F
N

NH
O

LIPITOR®

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

 Human HMGR with LIPITOR®

O Ca 2+
HO O

OH 2

F
N

NH
O

LIPITOR®

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

 Circa 1995 – The Statin Drugs Market
HO O HO O
O • Merck = cholesterol control
O O
O
• At 20 mg, ZOCOR® lowered LDL by -29%.
O O
H H

Lovastatin (MEVACOR®) Simvastatin (ZOCOR®)

MERCK MERCK

Thayer, A. M. Chem. Eng. News, 2006, 84, 33, 26-27.

Jones P.; Kafonek, S.; Laurora, I.; Hunninghake, D. Am. J. Cardiol. 1998, 81, 582-587.
 Circa 1995 – The Statin Drugs Market
HO O HO O
O • Merck = cholesterol control.
O O
O
• At 20 mg, ZOCOR® lowered LDL by -29%.
O O
H H

Lovastatin (MEVACOR®) Simvastatin (ZOCOR®)

MERCK MERCK

Spring 1997 – Pfizer launches LIPITOR®!

Ca 2+

HO • At 20 mg, LIPITOR® lowered LDL by -46%.

CO 2
HO • 2005 – $12 billion sales, used by over 45 million
people.
F
N

NH
O Atorvastatin calcium
LIPITOR®
2
 The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry

Ca2+ Drug
Discovery
HO
CO 2 Chemical Synthesis
HO Process
Development
F
N

NH
O
Biocatalysis
2
Atorvastatin calcium
LIPITOR ®
 The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry

Ca+2 Drug
Discovery
HO
CO 2 Chemical Synthesis
HO Process
Development
F
N

NH
O
Biocatalysis
2
Atorvastatin calcium
LIPITOR ®
 The Drug
Discovery The Decision of the Core Template
HO O
HO O
O

?
O O
O
O
O H
H

Mevastatin Lovastatin (MEVACOR®)

MERCK

HO HO O
CO 2Na

?
HO O
O

O F
H

HO
Pravastatin (PRAVACOL®)
BRISTOL - MYERS SQUIBB MERCK

Willard, A. K.; Novello, F. C.; Hoffmann, W. F.; Cragoe, E.; E. J. Jr. USP 4459422, 1984.
Fortune, 2003, January 20.
Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
 The Drug
Discovery The Decision of the Core Template
HO O correct spatial relationship
O
O
HO O
O
H O

hydrophobic X
group
Mevastatin
template
Hydrolysis – 100-fold
loss in potency HO O
A Potent HMGR
O
Inhibitor
F

MERCK

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

 The Drug
Discovery The Pyrrole Template

HO O

X
R1 N R2

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

 The Drug
Discovery The Pyrrole Template
CN
F H2 N CN
NEt3 F HOAc, reflux
O O F
20 mol% Bn N
O 73%
+ N Cl
O
S
HO DIBAL-H
58% 92%
O
CO 2CH 3
HO CHO
CH 3COCH 2CO 2CH3
NaH, n-BuLi F
1) Bu3B, NaBH4 F THF, -78 °C
N N
THF, -78 °C
2) NaOH, H2O 2 64%

HO HO O
CO 2H
HO O

F toluene, reflux F
N N
52%
tr ans:cis 97:3

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

 The Drug
Discovery The Pyrrole Template
CN
F H2 N CN
NEt3 F HOAc, reflux
O O F
20 mol% Bn N
O 73%
+ N Cl
O
S
HO DIBAL-H
58% 92%
O
CO 2CH 3
HO CHO
CH 3COCH 2CO 2CH3
NaH, n-BuLi F
1) Bu 3B, NaBH4 F THF, -78 °C
N N
THF, -78 °C
2) NaOH, H2O 2 64%

HO
CO 2H HO O • Over 40 analogs
HO O prepared.

F toluene, reflux X
• Optimization studies
N
R1 N R2
for X, R1 and R2.
52%
tr ans:cis 97:3

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

 The Drug
Discovery The Need for Additional Functionality

HO O
HO O O
O
O
O
H
F
N

Mevastatin
MERCK
• IC50 (analog): 0.40 µM.
IC50 – 0.030 µM.
• Limit of current synthetic route.

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

 The Drug
Discovery The Need for Additional Functionality

HO O
HO O
O
O
F

F
N

MERCK
An overlay

HO O
O

F
F
N

Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.

 The Drug
Discovery Incorporation of Additional Functionality

HO O
HO O TBDMSO O O
1) 2 eq NCS or NBS
O O
TBDMSCl DMF, 0 ºC
imidazole, DMF 100% F
N
F F 2) TBAF, HOAc
25 ºC N
N THF, 25 ºC
100% 35% X X
X = Cl
X = Br

HO O
X IC50
(µM) O
O
H 0.23 O
H
Cl 0.028
Br 0.028
Mevastatin
MERCK
IC50 – 0.030 µM.
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
 The Drug
Discovery Incorporation of Additional Functionality

HO O
HO O TBDMSO O O
1) 2 eq NCS or NBS
O O
TBDMSCl DMF, 0 ºC
imidazole, DMF 100% F
N
F F 2) TBAF, HOAc
25 ºC N
N THF, 25 ºC
100% 35% X X
X = Cl
X = Br

HO O
X IC50
(µM) O
O
H 0.23 O
H
Cl 0.028
Toxicity in early preclinical
development! Br 0.028
Mevastatin
MERCK
IC50 – 0.030 µM.
Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
 The Drug
Discovery Penta-substituted Pyrroles via [3+2]
1) O
O O O O O
Cl
F O F F
H2N
NEt3 , CH2Cl2, 0 ºC
Br NH N
NEt3, CH 3CN, 25 ºC
CO2 Et 82% 2) NaOH
CO2 Et HO2C O
97%

NHPh
Ph 43%
O
Ac2O, 90 ºC
HO O
CHO
O 1) HCl, EtOH O O
reflux
F 2) p-TSA,
N acetone-water
F F
reflux N
N
Ph NHPh 87%
NHPh O Ph NHPh
Ph
O O

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

 The Drug
Discovery Penta-substituted Pyrroles via [3+2]
HO O
HO O
O
O
20 analogs
tested F
F N
N

NH
X Y O

(racemic)
HO O
IC50 – 0.025 µM
O
O

O
H

Mevastatin
MERCK
IC50 – 0.030 µM.

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

 The Drug
Discovery Penta-substituted Pyrroles via [3+2]
HO O HO O
HO O
O O
O
20 analogs
tested F F
F N resolution N
N

NH NH
X Y O O

(racemic) (R, R)
HO O
IC50 – 0.025 µM IC50(µM)
O
O
(R, R) – 0.007
O
H (S, S) – 0.44

Mevastatin
MERCK
IC50 – 0.030 µM.

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

 The Drug
Discovery The Birth of LIPITOR®!
HO O HO O
HO O
O O
O
20 analogs
tested F F
F N resolution N
N

NH NH
X Y O O

(racemic) (R, R)
Ca2+

HO
CO 2
HO

F
N

NH
O

2
Atorvastatin calcium
LIPITOR ®
 The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry

Ca2+ Drug
Discovery
HO
CO 2 Chemical Synthesis
HO Process
Development
F
N

NH
O
Biocatalysis
2
Atorvastatin calcium
LIPITOR ®
 The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry

Ca2+ Drug
Discovery
HO
CO 2 Chemical Synthesis
HO Process
Development
F
N

NH
O
Biocatalysis
2
Atorvastatin calcium
LIPITOR ®
 The Process
Development Scale-up Issues and Potential Solutions
O O NHPh O O
[3+2] Cycloaddition 1.6 eq Ph
Route F O
F
N N
Ac 2O, 90 ºC
HO2C O 43%
Ph NHPh
O
HO
Paal-Knorr Route: CO 2R O O
HO
Penta-substituted H2N
CO2 R

Pyrroles +
F F O O
N

NH NH
O
Failed Model O
Study
OEt
EtO OEt
Paal-Knorr Route: H 2N OEt
+
Tetra-substituted F F O O
Pyrroles N

Roth, B. D. Prog. Med.

Chem. 2002, 40, 1-22.
 The Process
Development From Tetra- to Penta-substituted Pyrroles
F

F O O
O PhCHO, p-TSA O
O NEt3
toluene, reflux

80% 20 mol% Bn
O O O O
N Cl

S OEt
HO
2) NaOH, CH3 OH, rt H2 N OEt
71%
30% p-TSA, toluene
reflux

CHO EtO OEt

1) NBS, DMF 0 ºC, 100%
F 2) n-BuLi, THF, PhNCO, 69%
N 3) H3O +, 86% F
N

CONHPh

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
 The Process
Development From Tetra- to Penta-substituted Pyrroles
O Ph Ph
1) OH Ph
CHO Ph HO O
O OH
Ph O Ph
F F
N N
2 eq. LDA, MgBr2 -78 ºC

CONHPh 2) Recrystallization -10 ºC

CONHPh
dr 98:2
38%

1) NaOCH 3
0 ºC
68%
2) OLi -70 ºC
Ot Bu
78%

HO O
HO
O CO 2t Bu
O
1) Et3 B. NaBH 4 -78 ºC F
F 2) H 2O2, NaOH N
N 3) toluene, reflux

CONHPh
CONHPh

>99% ee
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
 The Process
Development From Tetra- to Penta-substituted Pyrroles
O Ph Ph
1) OH Ph
CHO Ph HO O
O OH
Ph O Ph
F F
N N
2 eq. LDA, MgBr2 -78 ºC

CONHPh 2) Recrystallization -10 ºC

CONHPh
dr 98:2
38%

1) NaOCH 3
0 ºC
68%
2) OLi -70 ºC
Ot Bu
78%

HO O
HO
O CO 2t Bu
O
1) Et3 B. NaBH 4 -78 ºC F
F 2) H 2O2, NaOH N
N 3) toluene, reflux

CONHPh
CONHPh

>99% ee
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
 The Process
Development From Tetra- to Penta-substituted Pyrroles
O Ph Ph
1) OH Ph
CHO Ph HO O
O OH
Ph O Ph
F F
N N
2 eq. LDA, MgBr2 -78 ºC

CONHPh 2) Recrystallization -10 ºC

CONHPh
dr 98:2
38%

1) NaOCH 3
0 ºC
68%
2) OLi -70 ºC
Ot Bu
78%

HO O
HO
O CO 2t Bu
O
1) Et3 B. NaBH 4 -78 ºC F
F 2) H 2O2, NaOH N
N 3) toluene, reflux

3 columns & 1 crystallization CONHPh

CONHPh 7% yield
>99% ee
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
 The Process
Development A Recap – The Failures
[3+2] Cycloaddition O O NHPh O O
1.6 eq Ph
Route F
O
F
N N
Ac2O, 90 ºC
HO2C O
43% Ph NHPh
O

Paal Knorr Route: F

OEt EtO OEt
O O
Tetra-substituted H2N OEt
Pyrroles F
HOAc, reflux N
71%

HO
Paal Knorr Route: CO 2R O O
Penta-substituted HO
H2N
CO2 R
Pyrroles +
F F O O
N

Failed Model
NH NH
Study
O O
 The Process
Development Paal Knorr Route: Penta-substituted Pyrroles
OEt
1 eq O EtO OEt
H2 N OEt
+ OH
F
F O O N
THF, reflux

43%
NH
NH
O
O

R2O
CO 2R
R2O
CO 2R R1O
R1O B

NH 2
F
N +
F O O

NH
O NH
O

Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.

 The Process
Development Pfizer’s Commercial Route: Fragment A

PhCHO
β -Alanine, AcOH O O
O O
hexane, heat
NHPh
NHPh
85%
Ph

20 mol%
F Br
N
CHO S
NEt3 , EtOH, heat HO
80%

F O O

NH
O

Dr. Bruce D. Roth (VP, Global Research & Development, Pfizer), personal communication.
Baumann, K. L. et al. Tetrahedron Lett. 1992, 33, 2283-2284.
 The Process
Development Pfizer’s Commercial Route: Fragment B

OH H2 O2, CaCO 3 HBr, HOAc OH

HO H K2CO3 OH CH3 OH
O HO CO2 K Br CO2 CH3
O
OH Br
HO
OH

R2O 60%
CO2R H 2, Pd/C
CH 3COONa/CH3COOH (over 3 steps)
R 1O
B

NH2

1) TBDMS-Cl
OTBDMS imid., 4-DMAP OH
NC CO2 CH 3 Br CO2 CH 3
2) NaCN, DMSO

Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282.

Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
 The Process
Development Pfizer’s Commercial Route: Fragment B
1) NaOH
2) CDI 3 eq LiCH2 CO 2t Bu
Mg(O 2CCH2 CO2 t Bu) OH O OH
OTBDMS THF
NC CO 2t Bu NC CO2CH3
NC CO 2CH 3
3) TBAF, HOAc, THF 75%

R2O
CO2R
R 1O
B

NH2

Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication.
Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282.
 The Process
Development Pfizer’s Commercial Route: Fragment B
1) NaOH
2) CDI 3 eq LiCH 2CO 2t Bu
Mg(O 2CCH2 CO2 t Bu) OH O OH
OTBDMS THF
NC CO 2t Bu NC CO2CH3
NC CO 2CH 3
3) TBAF, HOAc, THF 75%

1) NaBH4, Et2 BOMe

CH3OH, -90 °C
Cryogenic reactor
2) (CH 3)2C(OCH 3)2 employed
CH3SO3 H

O O Raney-Ni, MeOH
CO 2t Bu 50 psi H2 O O
H 2N
NC CO 2t Bu
B 95%
>99.5% ee dr 350:1 liquid
ee >99.5% N2

liquid
N2

Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication.
Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282.
 The Process
Development Pfizer’s Commercial Route
F O
O O O CO2t Bu
CO2t Bu O
O
O
1 eq OH
+
NH
F
O N
1:4:1 toluene:heptane:THF
NH 2 reflux
75%
A B NH
O

• Highly convergent. a) HCl, MeOH

b) NaOH 84%
• Yields >75% at each step. c) Ca(OAc)2 Ca2+

• One low temperature reaction. HO

CO2 2
• One special equipment requirement. HO

• No chromatography.
F
N Atorvastatin calcium
• Scalable to ton quantities. LIPITOR ®
>99.5% ee
NH
O

Baumann, K. L. et al. Tetrahedron Lett. 1992, 33, 2283-2284.

Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication.
 LIPITOR®: Drug tackled, the struggles remained…

“The number of factors, internal and external, that had to come together for
the drug to be a success really boggles the mind" – Bruce D. Roth

The problems: And the facts now:

• By 1987, three statins in market. • #1 statin in the market.
• A decade since the first statin • Top selling drug in history.
introduced.
• 2005: $12 billion sales & used by >45
• Warner-Lambert was floundering. million people.
• Come on, how good could it be? • “LIPITOR® is on track to have greater
benefit for more people than any other
• On the verge of terminating
drug in the history of the industry in terms
LIPITOR®.
of lives improved and saved.”
- Nobel Laureate Michael Brown

Fortune, 2003, January 20.

 The Story of LIPITOR®

“The story of how Pfizer acquired the rights to an improved statin

and turned it into the all-time biggest blockbuster is a tale of
hyperaggresive marketing, deft timing, financial power and plain
dumb luck!”

Fortune, 2003, January 20.

 The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry

Ca2+ Drug
Discovery
HO
CO 2 Chemical Synthesis
HO Process
Development
F
N

NH
O
Biocatalysis
2
Atorvastatin calcium
LIPITOR ®
 The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry

Ca2+ Drug
Discovery
HO
CO 2 Chemical Synthesis
HO Process
Development
F 220 ton/year market
N
($500 million)
NH
O
Biocatalysis
2
Atorvastatin calcium
LIPITOR ®
 The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry

Ca2+ Drug
Discovery
HO
CO 2 Chemical Synthesis
HO Process
Development
F 220 ton/year market
N
($500 million)
NH
O
Biocatalysis
2
Atorvastatin calcium
LIPITOR ®
 OH OH O
NC
OR
Existing Route & the Need for Improvement
OH HBr, HOAc OH
H H2O 2, CaCO3 OH CH3 OH
HO
O K 2CO 3 HO CO2K Br CO 2CH3
O
OH Br
HO
OH
H 2, Pd/C 60%
CH3COONa/CH 3COOH (over 3 steps)

3 eq LiCH 2CO 2t Bu OH OH
OH O THF
NaCN, DMSO
NC CO2 CH 3 Br CO2 CH 3
NC CO2 t Bu
75%

1) NaBH4, Et2BOMe
CH3OH, -90 °C
2) (CH 3)2C(OCH 3)2
CH3SO3 H

O O
NC CO 2t Bu
>99.5% ee
 OH OH O
NC
OR
Existing Route & the Need for Improvement
OH HBr, HOAc OH
H H2O 2, CaCO3 OH CH3 OH
HO
O K 2CO 3 HO CO2K Br CO 2CH3
O
OH Br
HO
OH
H 2, Pd/C 60%
CH3COONa/CH 3COOH (over 3 steps)

3 eq LiCH 2CO 2t Bu OH OH
OH O THF
NaCN, DMSO
NC CO2 CH 3 Br CO2 CH 3
NC CO2 t Bu
75%

1) NaBH4, Et2BOMe
CH3OH, -90 °C
2) (CH 3)2C(OCH 3)2 • SHE issues.
CH3SO3 H
• Cryogenic step – special requirements/waste.
• With purification – 11 steps.
O O
NC CO 2t Bu • Month’s supply: $66 LIPITOR® vs $120 ZOCOR®.
>99.5% ee
• But patent expires ~ 2010.
 OH OH O
NC
OR
Biocatalytic Routes for the Chiral Side Chain

ADH O O
OH O GDH
KANEKA
Cl Cl
OEt OEt

O OH O Lipase O OR1 O
CIBA
HO OEt EtO OEt

DOWPHARMA
OH OH O (DIVERSA) OH O Nitrilase OH
NC NC NC CN
OR OH

CODEXIS OH O 1) ADH O O
NC Cl
OEt OEt
2) Dehalo-
genase
DIVERSA O OH Aldolase O O
X + 2
X
H H

OH

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.

Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
 OH OH O
NC
OR
Biocatalytic Routes for the Chiral Side Chain

ADH O O
OH O GDH
KANEKA
Cl Cl
OEt OEt

O OH O Lipase O OR1 O
CIBA
HO OEt EtO OEt

DOWPHARMA
(DIVERSA) OH O Nitrilase OH
OH OH O
NC NC NC CN
OR
OH

CODEXIS OH O 1) ADH O O
NC Cl
OEt OEt
2) Dehalo-
genase
DIVERSA O OH Aldolase O O
X + 2
X
H H

OH

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.

Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
 OH OH O
NC
OR
Biocatalytic Routes for the Chiral Side Chain

ADH O O
OH O GDH
KANEKA
Cl Cl
OEt OEt

O OH O Lipase O OR1 O
CIBA
HO OEt EtO OEt

DOWPHARMA
(DIVERSA) OH O Nitrilase OH
OH OH O
NC NC NC CN
OR
OH

CODEXIS OH O 1) ADH O O
NC Cl
OEt OEt
2) Dehalo-
genase
DIVERSA O OH Aldolase O O
X + 2
X
H H

OH

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.

Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
 OH OH O
NC
OR
Biocatalytic Routes for the Chiral Side Chain

ADH O O
OH O GDH
KANEKA
Cl Cl
OEt OEt

O OH O Lipase O OR1 O
CIBA
HO OEt EtO OEt

DOWPHARMA
(DIVERSA) OH O Nitrilase OH
OH OH O
NC NC NC CN
OR
OH

CODEXIS OH O 1) ADH O O
NC Cl
OEt OEt
2) Dehalo-
genase
DIVERSA O OH Aldolase O O
X + 2
X
H H

OH

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.

Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
 OH OH O
NC
OR
Biocatalytic Routes for the Chiral Side Chain

ADH O O
OH O GDH
KANEKA
Cl Cl
OEt OEt

O OH O Lipase O OR1 O
CIBA
HO OEt EtO OEt

DOWPHARMA
(DIVERSA) OH O Nitrilase OH
OH OH O
NC NC NC CN
OR
OH

CODEXIS OH O 1) ADH O O
NC Cl
OEt OEt
2) Dehalo-
genase
DIVERSA O OH Aldolase O O
X + 2
X
H H

OH

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.

Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
 OH OH O
NC
OR
Biocatalytic Routes for the Chiral Side Chain

ADH O O
OH O GDH
KANEKA
Cl Cl
OEt OEt

O OH O Lipase O OR1 O
CIBA
HO OEt EtO OEt

DOWPHARMA
(DIVERSA) OH O Nitrilase OH
OH OH O
NC NC NC CN
OR
OH

CODEXIS OH O 1) ADH O O
NC Cl
OEt OEt
2) Dehalo-
genase
DIVERSA O OH Aldolase O O
X + 2
X
H H

OH

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.

Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
 OH OH O
NC
OR
Kaneka’s Route

OH O
D-glucose NADP+ Cl Et
O

glucose carbonyl
dehydrogenase reductase
recombinant E.
coli

O O
D-gluconolactone NADPH Cl Et
O
spontaneous
Aqueous phase Organic phase
D-glutonic acid (n-butyl acetate)

• >99.9% ee, 89% yield.

• Product concentration: 450 g/L.
• NADP+ TON: 16,200 mol/mol.
• Problematic product separation.
Kizaki, N. et al. Appl. Microbiol. Biotechnol. 2001, 55, 590-595.
Yasohara, Y. et al. Tetrahedron: Asymmetry 2001, 12, 1713-1718.
 OH OH O
NC
OR
Daicel’s Route
OH O
formic acid NADP+ Cl Et
O

formate carbonyl
dehydrogenase reductase
recombinant E.
coli

O O
NADPH Cl Et
CO2 O

• >99% ee.
• Product concentration: 50 g/L.
• Easy product separation.
• Commercially used: >100 ton/year.

Rouhi, A. M. Chem. Eng. News 2004, 82, (24), 47-62.

 OH OH O
NC
OR
Ciba’s Route
O O
O O
O
O O O O α-chymotrypsin
O OH O Cl O O O
0.008 mol%
EtO OEt pyridine EtO OEt 24-30 h HO OEt
0 °C to rt 94%
~ 12 h 98.2% ee
98% 6 steps
44% overall
yield

O O O

N3 OEt

The good things The bad things

• 94% yield, 98.2% ee. • Follow up chemistry – long.
• Substrate concentration: 285 g/L. • Low temperature reactions.
• Kilogram scale. • Column chromatography.
• Cheap & robust biocatalyst.

Öhrlein, R.; Baischf, G. Adv. Synth. Catal. 2003, 345, 713-715.

 OH OH O
NC
OR
Diversa/Dowpharma’s Route

OH nitrilase OH
NC CN NC COOH
pH 7.5 NaH2PO4
27 °C, 16 h
81%
98.8% ee

DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025.

DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.
 OH OH O
NC
OR
Diversa/Dowpharma’s Route
HCN, base NaCN (aq.)
OH pH=10, 4 h, 50 °C OH
(n-Bu)4N+Br-
O NC CN
Cl NC Cl
12 h, 45 °C 45%
92%
pH 7.5 NaH 2PO4
nitrilase 27 °C, 16 h
81%
98.8% ee
EtOH, H+
OH 3 h, 80 °C OH
NC CO 2Et NC COOH
62%

The good things The bad things

• Cheap starting material. • HCN under heated alkaline
conditions.
• Efficient enzymatic step: 3 M [substrate]
& 619 g L-1 d-1. • Special equipment for purification.
• Low cost of catalyst by expression in • Some low yield steps.
Pseudomonas fluorescens developed by
Dow.
Scale-up economics good!
Bergeron, S. et al. Org. Process Res. Dev. 2006, 10, 661-665.
 OH OH O
NC
OR
Diversa’s Route
O
O O DERA OH O OH OH O
+ Cl Aldol
Cl Cl reaction
25 °C DERA

DERA = deoxyribose-
5-phosphate aldolase
Wild Type O OH
DERA Cl

Catalyst Load 20% w/w

OH
Product Isolation Difficult
Reaction Time 6 days
[chloroacetaldehyde] 100 mM

Volumetric 2 g L-1 d-1 OH OH O

Productivity Cl
OR
ee (de) Unknown
Practical? NO

Gijsen, H. J. M.; Wong, C.-H. J. Am. Chem. Soc. 1994, 116, 8422-8423.
Wong, C.-H. et al. J. Am. Chem. Soc. 1995, 117, 3333-3339.
 OH OH O
NC
OR
Diversa’s Route
O
O O DERA OH O OH OH O
+ Cl Aldol
Cl Cl reaction
25 °C DERA

DERA = deoxyribose-
5-phosphate aldolase
Wild Type Improved O OH
DERA DERA Cl

Catalyst Load 20% w/w 2% w/w

OH
Product Isolation Difficult Simple
Reaction Time 6 days 3h
[chloroacetaldehyde] 100 mM Fed-batch
process
Volumetric 2 g L-1 d-1 735 g L-1 d-1
O O O
Productivity
NC
OMe
ee & de Unknown ≥ 99.9%
23% yield
Practical? NO YES 3 overall steps

Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.
 OH OH O
NC
OR
Codexis Route

O O OH O OH O
KRED/GDH HHDH
Cl Cl NC
OEt OEt OEt
not purified oil, not purified
- HCl
O
+ HCl O + HCN
OEt
KRED = Ketoreductase
GDH = Glucose dehydrogenase pK a ~9 HCNaq HClaq pKa <0 chemical
HHDH = Halohydrin dehalogenase chain
NaClaq NaCNaq elongation
neutral pH
two phase, no solvent

chemical
O O protection OH OH KRED/GDH OH O
NC CO 2t Bu NC CO2t Bu NC CO 2t Bu

first crystalline intermediate oil, not purified oil, not purified

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
 OH OH O
NC
OR
Codexis Route

O O OH O OH O
KRED/GDH HHDH
Cl Cl NC
OEt OEt OEt
not purified oil, not purified
- HCl
O
+ HCl O + HCN
OEt
KRED = Ketoreductase
GDH = Glucose dehydrogenase pK a ~9 HCNaq HClaq pKa <0 chemical
HHDH = Halohydrin dehalogenase chain
NaClaq NaCNaq elongation
neutral pH
two phase, no solvent

chemical
O O protection OH OH KRED/GDH OH O
NC CO2 t Bu NC CO 2t Bu NC CO2 t Bu

first crystalline intermediate oil, not purified oil, not purified

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
 OH OH O
NC
OR
Codexis Route

1) KRED/GDH
O O 2) HHDH O O
Cl NC CO2t Bu
OEt 3) Chemical chain
elongation "TBIN"
4) KRED/GDH First crystalline intermediate
5) Protection

• Excellent selectivities and yields.

• Purification only at the last step – reduced requirements.
• Green and safe.
• Reduced by-products.
• Mild reaction conditions.
• Efficient, scalable and cost effective.
• Commercialized: (1) Arch Pharmalabs, India – Codexis
production partner.
(2) Lonza – a Pfizer supplier.

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
 OH OH O
NC
OR
Biocatalytic Routes for the Chiral Side Chain

ADH O O
OH O GDH
KANEKA
Cl Cl
OEt OEt

O OH O Lipase O OR1 O
CIBA
HO OEt EtO OEt

DOWPHARMA
OH OH O (DIVERSA) OH O Nitrilase OH
NC NC NC CN
OR OH

CODEXIS OH O 1) ADH O O
NC Cl
OEt OEt
2) Dehalo-
genase
DIVERSA O OH Aldolase O O
X + 2
X
H H

OH

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.

Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
 OH OH O
NC
OR
Biocatalytic Routes for the Chiral Side Chain

ADH O O
OH O GDH
KANEKA
Cl Cl
OEt OEt

O OH O Lipase O OR1 O
CIBA
HO OEt EtO OEt

DOWPHARMA
(DIVERSA) OH O Nitrilase OH
OH OH O
NC NC NC CN
OR
OH

CODEXIS OH O 1) ADH O O
NC Cl
OEt OEt
2) Dehalo-
genase
DIVERSA O OH Aldolase O O
X + 2
X
H H

OH

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.

Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
 An Extremely Attractive Chemo-enzymatic Approach

BIOCATALYSIS O O
at CODEXIS
Cl O

Ca2+
O
CO 2t Bu
HO
CO2 2 O
HO

F NH 2
N
+
F O O
NH CHEMICAL
SYNTHESIS
O at PFIZER
NH
LIPITOR® O
O O

NHPh
CHEMICAL
SYNTHESIS
at PFIZER
 An Extremely Attractive Chemo-enzymatic Approach

BIOCATALYSIS O O
at CODEXIS
Cl O

Ca2+
O
CO 2t Bu
HO
CO2 2 O
HO
Why not other pharmaceuticals and natural
F products also?
NH 2
N
+
F O O
NH CHEMICAL
SYNTHESIS
O at PFIZER
NH
LIPITOR® O
O O

NHPh
CHEMICAL
SYNTHESIS
at PFIZER
 The Take-Home Message

The Story of LIPITOR®: Drug Discovery & Chemical Development

• The world of process chemistry works on the same basic principles as us

here in this building.
• But in many respects, it is a vastly different world, driven by an entirely
different set of considerations.

The Story of LIPITOR®: Biocatalytic Routes for the Sidechain

• State-of-art enzymatic transformations have reached an extraordinary level,

making them valuable and competitive methods for use in the
pharmaceutical industry.
• Biocatalysis has been emerging
 The Take-Home Message

The Story of LIPITOR®: Drug Discovery & Chemical Development

• The world of process chemistry works on the same basic principles as us

here in this building.
• But in many respects, it is a vastly different world, driven by an entirely
different set of considerations.

The Story of LIPITOR®: Biocatalytic Routes for the Sidechain

• State-of-art enzymatic transformations have reached an extraordinary level,

making them valuable and competitive methods for use in the
pharmaceutical industry.
• Biocatalysis has been emerging clearly emerged as a very attractive tool in
a synthetic chemist’s toolkit.
 Acknowledgements

Dr. Wulff
Dr. Walker Dr. Borhan Dr. Maleczka

Dr. Bruce Roth (VP, Global R&D, Pfizer)

Dr. Donald Butler (Former Process Development Leader, Pfizer
Chief Science Advisor, Austin Chemical Company)
Dr. Peter Seufer-Wasserthal (VP, Head of Pharma Services, Codexis)
Dr. Stephen Ritter (Senior Editor, C&EN)

Dr. Christopher Schmid (Lilly) Professor Samir Zard

Dr. Jos Brands (Merck) Dr. Michael Lipton (SPCorp)

Keith, Ding, Cory, Zhenjie, Gang, Chunrui, Alex, Kostas, Dima, Nilanjana, Anil,
Munmun, Li, Yong

Janelle

Aman K., Toyin, Luis, Brian and Alli & Dan

 Other Beneficial Effects of Statin Drugs

• Promotion of new blood vessel growth.

• Stimulation of bone formation.

• Protection against modification of low density lipoproteins.

• Reduction of clot-forming process so important in plaque formation.

• Improvement in endothelial function.

• Reduction of inflammation in blood vessel tissue.

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

Rosanoff, A.; Seelig, M. S. Journal of the American College of Nutrition 2004, 23, 501S-505.
 Some Rare Adverse Effects of Statin Drugs

• The most common side effects are gas, constipation, stomach pain and
heartburn.

• Rare Myalgia (muscle pain). possibly life threatening!

• Rare Rhabdomyolosis (skeletal muscle failure – loss of kidney function).

Uncommon side reactions occur mainly when statins are co-prescribed with other
interacting drugs.

www.lipitor.com
Rosanoff, A.; Seelig, M. S. Journal of the American College of Nutrition 2004, 23, 501S-505.
 Statin Drugs
O
HO O
HO O HO O HO O
C OH
O O HO O
O O O
F
O O O
H H H N

HO

Lovastatin (MEVACOR ®) Simvastatin (ZOCOR®) Pravastatin (PRAVACOL ®) Fluvastatin (LESCOL®)

MERCK MERCK BRISTOL - MYERS SQUIBB NOVARTIS
O
O
HO O O
CO 2 HO O HO O
HO
HO OH OH
HO
F F
F
N

O N F N
NH N N
O O
N
O S
Cerivastatin (LIPOBAY®)
Atorvastatin (LIPITOR®) BAYCOL Rosuvastatin (CRESTOR ®) Pitavastatin (LIVALO®)
PFIZER withdr awn in 2001 ASTRAZENECA KOWA
 Chemo-catalytic vs. Bio-catalytic: Wacker Specialty Chemicals

Asymmetric Hydrogenation Biocatalytic Reduction

(Ruthenium Catalyst + (Alcohol Dehydrogenase
O O Diphosphine Ligand) OH O Enzyme) O O
Cl Cl Cl
OR OR OR

Chemo-catalysis Bio-catalysis
%ee (yield) 98 (95) >99.9 (97)
Scale Multi-ton Multi-ton
Cost/kg (product) < $85 < $100
SHE issues 100 °C, MeOH solvent, None – ambient temp. &
handling H2 pressure
Equipment Standard Standard
Waste/kg (product) < 100 g 2L
Relative Throughput 3 1 (dilute conditions)

Requirement of price & purity will dictate the route.

Rouhi, A. M. Chem. Eng. News 2004, 82, (24), 47-62.

 The Drug
Discovery Other Templates Attempted

R F OH
N N

F O O
H
H R1
O
N
HO O

R = Ph (IC 50 = 0.035 µM) Compound R1 IC50 (µM)

1 Cl 0.032
mevastatin: IC 50 = 0.030 µM 2 OCH3 0.013
HO O 3 (N-oxide) F 0.018
O
O

O
H

Sliskovic, D. R. et al. J. Med. Chem. 1991, 34, 367-373.

Sliskovic, D. R. et al. J. Med. Chem. 1990, 33, 31-38.
 Ca2+
Human HMGR with LIPITOR HO
O
O
2
OH

F
N

NH
O

LIPITOR®

The specificity & tight

binding achieved by:
• hydrogen bonds
• ionic interactions
• polar interactions

Istvan,
Istvan,
E.E.
S.;S.;
Deisenhofer,
Deisenhofer,
J. J.
Science 2001,
Science 2001,
292, 1160-1164.
292, 1160-1164.
 Human HMGR with Statin Drugs

Istvan,
Istvan, E.E.
S.;S.; Deisenhofer,
Deisenhofer, J. J. Science
Science 2001,
2001, 292,
292, 1160-1164.
1160-1164.
 The solution – suppressing cholesterol biosynthesis
Acetyl-CoA
Acetoacetyl-CoA
Hydroxymethylglutaryl-CoA
(HMG-CoA)

Mevalonate

5-phosphomevalonate

5-pyrophosphomevalonate

Dimethylallyl
pyrophosphate Cholesterol Biosynthesis

Geranyl
pyrophosphate
Farnesyl
pyrophosphate
Squalene

Cholesterol Desmosterol Lanosterol Squalene-2,3-epoxide

18 reactions

www.cellml.org
 The solution – suppressing cholesterol biosynthesis
CH3
Acetyl-CoA
Acetoacetyl-CoA
Cl
Hydroxymethylglutaryl-CoA
(HMG-CoA)
OH
Mevalonate H 2N
O
Triparanol (MER-29)
5-phosphomevalonate

5-pyrophosphomevalonate Severe side

effects – failed!
Dimethylallyl
pyrophosphate Cholesterol Biosynthesis
Geranyl
pyrophosphate
Farnesyl
pyrophosphate
Squalene

Cholesterol Desmosterol Lanosterol Squalene-2,3-epoxide

18 reactions

Steinberg, D.; Avigan, J. J. Biol. Chem. 1960, 235, 3127-3129.

 The Drug
Discovery Structure-Activity Relationship Studies

HO O

X
R1 N R2

HO O

1
MERCK

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

 The Drug
Discovery Structure-Activity Relationship Studies
HO O
HO O
O
O

F X F
N N
X=

-CH 2CH 2CH 2

-CH 2CH 2

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

 The Drug
Discovery Structure-Activity Relationship Studies

HO O HO O HO O
O O O

F X
F
N N R1 = N
R1

Ph 2-naphthyl

4-PhC 6H 4 1-naphthyl

4-MeOC6H 4 cyclohexyl

HO O 4-ClC6H 4

O 4-HOC 6H4

4-FC6 H4

N 3-F3CC6H 4
R1 R2
3-MeOC6H 4
< 5.9 Å
3-HOC 6H4

2-MeOC6H 4

2-HOC 6H4

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

 The Drug
Discovery Structure-Activity Relationship Studies

HO O HO O HO O
O O O

X -CH 3
F
N F
R1 N N R2

R2 =

HO O

N HO O
R1 R2
O
< 5.9 Å

F
N

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

 The Drug
Discovery Structure-Activity Relationship Studies

HO O HO O HO O
O O O

X -CH 3
F
N F
R1 N N R2

R2 =

HO O

N HO O
R1 R2
O
< 5.9 Å < 3.3 Å
F
N
< 10.6 Å

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

 The Drug
Discovery Structure-Activity Relationship Studies

HO O HO O HO O
O O O

F X
N F
R1 N N R2

3-FC6 H4 2,6-(MeO)2C6 H3
HO O
2-FC6 H4 2,5-Me 2C6 H3
O
2,4-F2C 6H 3 2-iPrOC 6H4

2-MeOC6H 4 O
N HO O HO O
R1 R2
2-ClC6H 4
O O
< 5.9 Å < 3.3 Å
F
N R1 N
< 10.6 Å

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

 The Drug
Discovery Pentasubstituted Pyrroles via [3+2]
R2 Ac2O R1 R2 R2
R1 N R3 reflux N R4 R5
R1 N R3
HOOC O O O R3

“Munchnone” R4 R5

Regiocontrol: O O

O O
CO2Et F
F N

N
Ac 2O, reflux CO2 Et
HOOC O

O O

O O
CO2Et F
N

N
Ac 2O, reflux EtO 2C
HOOC O

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

 OH OH O
NC
OR
Ciba’s Route
O O
O O
O
O OH O O O O O α-chymotrypsin
Cl 0.008 mol% O O O
EtO OEt pyridine EtO OEt 24-30 h HO OEt
0 °C to rt
~ 12 h
98% (COCl)2, 0 °C
100%

O O
pig-lever O ethylene, AlCl3 O
esterase DCE, 0 °C O O O
O OH O O O O
pH=7 89% Cl OEt
Cl OEt Cl OEt
76%
1) BEt3 , NaBH4
-78 °C, 91%
2) dimethoxypropane O OH
H+, 98% O
CO 2Et
3) NaN3, DMF
O
94% y, 98% ee, 97% de

F O O F
O O O N
+
N3 OEt PBu3
70%
Ph CONHPh Ph CONHPh

Öhrlein, R.; Baischf, G. Adv. Synth. Catal. 2003, 345, 713-715.

 OH OH O
NC
OR
Diversa/Dowpharma’s Route

OH OH
nitrilase
NC CN NC COOH
pH 7.5 NaH2PO4
27 °C, 16h
81% y, 98.8% ee

Screen genomic libraries

Most effective WT nitrilase

0.24 M [substrate] – 98% y, 95% ee

3 M [substrate] – 88% ee

DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025.

DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.
 OH OH O
NC
OR
Diversa/Dowpharma’s Route

OH OH
nitrilase
NC CN NC COOH
pH 7.5 NaH2PO4
27 °C, 16h
81% y, 98.8% ee

Screen genomic libraries

Most effective WT nitrilase Genetical Engineering gave another library

0.24 M [substrate] – 98% y, 95% ee Best mutant

3 M [substrate] – 88% ee 2.25 M [substrate] – 98% ee in 15 h

DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025.

DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.
 Diversa’s GSSM™: Gene Site Saturation Mutagenesis

1. Target Protein for 2. Change Amino 3. Complete Variant Library

Improvement Acid(s)

Identify protein for Evolve the gene encoding the A new gene variant library is
optimization through extensive protein by systematically born containing genes with every
screening of gene libraries. changing each amino acid in the single site variation in sequence.
sequence to every other possible
amino acid.

4. Select Variants 5. Combine Mutations 6. Generate Optimized Protein

Through screening, identify the Tests all potential combinations

variants that demonstrate of the single amino acid
improved characteristics. changes that demonstrated
improved characteristics and
select the optimal combination.

www.diversa.com
 Diversa’s High Throughput Screen for Improved Selectivity

S-selective R-selective
OH nitrilase OH nitrilase OH
HOOC C15N NC C15N NC COOH

15 N-(S)-13 15N-(R)-12 (R)-13

m/ z = 130 m/ z = 129

DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.

 OH OH O
NC
OR
Diversa’s Route
O
O O DERA OH O OH OH O
+ Cl Aldol
Cl Cl reaction
25 °C DERA

DERA = deoxyribose-
O O NaOCl, HOAc 5-phosphate aldolase
a) NaCN, DMF, 5% H2O Cl H 2O, 25 °C, 3 h O OH
40 °C, 16 h Cl
b) H 2SO 4
c) (MeO)2CMe2 45% (over
OH 2 steps)
d) TMSCHN2 OH
white crystals
oil, not purified
>99.9% ee
99.8% de
OH O OH OH O
OH OH O
O NC
Cl ONa
ONa ONa

O O O O O O
NC
H2N OtBu OMe
Coupling partner oil, column chromatography
for the Paal-Knorr 48% (over 4 steps)

Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.
 Diversa’s Fluorogenic Activity Based Screen

1) 4-methylumbelliferone

O O OH

TosO O K 2CO 3, DMF, 75 °C, 16 h

O O
2) H 2O / 20% CH3CN O O O OH
HO
DOWEX 50WX8-100
5-toluenesulfonyl- 2 days HO
2-deoxyribose 62%

O O
+ DERA OH O
O O O H
OH O O O H
OH
spontaneous

O
+
O O OH O
fluorescent

Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.
 OH OH O
NC
OR
Codexis Biocatalyst Improvement
O O OH O
KRED
Cl Cl
OEt OEt

NADPH NADP +

Na-gluconate glucose
GDH

Parameter Initial Final

Substrate 80 g/L 180 g/L
loading
Reaction time 24 h 8h
Enzyme loading 10 g/L 0.7 g/L
Isolated yield ~80% 97%
Phase >1 h ~ 1 min
separation time
Volumetric 80 g/L.day 540 g/L.day
productivity

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
 OH OH O
NC
OR
Codexis Biocatalyst Improvement

OH O halohydrin
dehalogenase OH O
Cl NC
OEt OEt
- HCl
O
O + HCN
+ HCl
OEt

pKa ~9 HCNaq HClaq pKa <0

NaClaq NaCNaq
neutral pH
two phase, no solvent

Parameter Initial Final

Substrate 20 g/L 140 g/L
loading
Reaction time 72 h 5h
Enzyme loading 130 g/L 1.2 g/L
Isolated yield ~60% 92%

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
 OH OH O
NC
OR
Codexis Biocatalyst Improvement

OH O OH OH
KRED
NC CO2 tBu NC CO2 tBu

NADPH NADP +

Na-gluconate glucose
GDH

Parameter Initial Final

Substrate 120 g/L 300 g/L
loading
Reaction time 65 h 22 h
Conversion 87% 99.3%
Diastereomeric 100% 100%
excess
Work-up difficult facile

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
 Stetter Reaction Mechanism
O Bn
OH N O OH
R
R S R R
HO

O
Reversible benzoin formation
p.t.
R H
Cl HNEt3
Bn Bn Bn
N Cl N N OH
H + NEt3
S S p.t. S R
HO HO HO
H R
R1
O
p.t. O
Irreversible addition to the Michael Acceptor

Bn
O O N O
R1 R1
R OH
R
O S R
OH HO
R1

Hermann, S. Angew. Chem., Int. Ed. Engl. 1976, 15, 639-647.

 Syn-selective reduction of β-hydroxy ketones
attack from
less hindered
side

Bu H
R
Bu O B O 1
Bu R2 Bu
B
OH O O O
R1 R2 R1 R2 cis - diol
+ favored

Bu3B, THF
H
Bu
R1
O B Bu
R2
O

top H bottom

H
Bu O Bu
R1
B
R1 O B Bu Bu
O O
R2 R2
H

favored unfavored

Narasaka, K.; Pai, H. C. Chem. Lett. 1980, 1415-1418.

 Mechanism for H2O2 Oxidation of Ascorbic Acid
OH OH OH OH
HO HO HO
O H-O-O-H O O
O O O
HO HO O
OH O O
H2 O
OH
+
H2 O H-O-O-H

OH OH
HO OH OH
O HO HO
O O
HO O
HO O O
O OH
O O OH O OH
O OOH
OH OH
H2 O
(CO 2H)2

OH
HO
CO2 H

HO

Isbell, H. S.; Frush, H. L. Carbohyd. Res. 1979, 72, 301-304.

 O O O
Asp 102 His 57 Asp 102 Asp 102
His 57 His57
O O O
H H H
N N N
N N N
H2 O
HO Ser195 H Ser195
O Ser195 H
O
O R O R
O R
H
O O
O
H H OH H H
H H
N N N N
N N
Gly193 Ser195 Gly193 Ser195
Gly 193 Ser195
O
Asp 102 O
His57 Asp 102
O His57
O
H
N H
N
N
N
HO Ser195
O Ser195
H
Mechanism for Hydrolysis by HO R
HO
R

Chymotrypsin/PLE O O

H H H H
N N N N
Gly193 Ser195 Gly193 Ser195
 Mechanism for Hydrolysis by Nitrilase

NH
R C N + Enz SH R
H2O
S Enz

H
O
R NH2
S
Enz

O O
R + Enz SH R
OH NH3
S Enz
H2O

Faber, K. Biotransformations in Organic Chemistry, Springer-Verlag, 2004.

 Lys 201 Lys 201
Lys 201

NH3 H H
O H NH3 H NH2 O H
Asp102
O Asp102 O H Asp102
O O
O H
H
O HO
CH 3 OH OH
HN CH 3 N CH 3
HNH

Lys 167 Lys 167

Lys 167

Lys 201

Lys 201
NH3
Asp102 Ser238, 239
O Gly205
H OH
NH3 H
Asp102 O H
O N OPO 3 O
H OH H O
H 2O hydrophilic
site O
HN CH 2 OPO3
Lys 167
OH
Thr170, Lys172
hydrophobic Lys 167
site

O O
DERA O OH
DERA + H
Mechanism H OPO3 H OPO 3
OH OH
Bioorg. Med. Chem. 11, 2003, 43-52.