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The Story of LIPITOR® - A Peek into the World
of Pharmaceutical Process Chemistry
Aman Desai
7th Feb. 2007
Chemical Synthesis
Biocatalysis
LIPITOR® – $12 billion/year sales (2005)
Chiral side chain (circled) – 220 ton/year
Atorvastatin calciumLIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
The Problem – The “Bad” Cholesterol
• Cholesterol – a very important biological molecule.
• Most cholesterol is not dietary, it is synthesized internally.
• Cholesterol is bound to lipoproteins and transported through blood.
• 2 kinds of lipoproteins
www.wikipedia.orgwww.americanheart.org
High Density Lipoprotein (HDL) – “good”
Low Density Lipoprotein (LDL) – “bad”
atherosclerosis
coronary heart disease & other cardiovascular diseases
One of the leading causes of death in the world today!
HOH
H
H
Cholesterol
The Solution – Suppressing Cholesterol Biosynthesis
http://www.med.unibs.it/~marchesi/cholest.html
O
SCoA
OHMG-CoA-Synthetase
SCoA
O
HSCoA
OH
SCoA
O
OO
HMG-CoA
2 NADPH + 2H+
2 NADP+ + HSCoA
HMG-CoA-Reductase
OH OH
OO
Mevalonic Acid
+
RateLimiting
Step
HHO
H
H
Cholesterol
H
>25 steps
The Solution – Suppressing Cholesterol Biosynthesis
OH
SCoA
O
OO
HMG-CoA
2 NADPH + 2H+
2 NADP+ + HSCoA
HMG-CoA-Reductase
OH OH
OO
Mevalonic Acid
RateLimiting
Step
Inhibition
Endo, A. J. Lipid Res. 1992, 33, 1569-1582.Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
H
O
OHO
O
O
Lovastatin (MEVACOR®)MERCK
9'
2''
HO
O
HO
CO2Na
HO
HO
Pravastatin (PRAVACOL®)BRISTOL - MYERS SQUIBB
9'
H
O
OHO
O
O
Mevastatin
9'
H
O
OHO
O
O
Simvastatin (ZOCOR®)MERCK
2''
Mechanism of Action of Statin Drugs
O
HMG-CoA
2 NADPH + 2H+
2 NADP+ + HSCoA
HMG-CoA-Reductase
OH OH
OO
Mevalonic Acid
RateLimiting
Step
Inhibition
SCoA
HO O
O
H
O
OHO
O
O
Lovastatin (MEVACOR®)MERCK
HO
O
HO
Pravastatin (PRAVACOL®)BRISTOL - MYERS SQUIBB
ONaO
HO
OH
H
O
OHO
O
O
Mevastatin
H
O
OHO
O
O
Simvastatin (ZOCOR®)MERCK
http://www.med.unibs.it/~marchesi/cholest.html
Mechanism of Action of Statin Drugs
Atorvastatin calcium (LIPITOR®)PFIZER
NF
O
NH
2
Ca2+
OH
OO
HO
http://www.med.unibs.it/~marchesi/cholest.html
H
O
OHO
O
O
Lovastatin (MEVACOR®)MERCK
HO
O
HO
Pravastatin (PRAVACOL®)BRISTOL - MYERS SQUIBB
ONaO
HO
OH
H
O
OHO
O
O
Mevastatin
H
O
OHO
O
O
Simvastatin (ZOCOR®)MERCK
Human HMGR with Natural Substrates
Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.
O
HMG-CoA
2 NADPH + 2H+
2 NADP+ + HSCoA
HMG-CoA-Reductase
OH OH
OO
Mevalonic Acid
RateLimiting
Step
Inhibition
SCoA
HO O
O
Human HMGR with Natural Substrates
Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.
O
HMG-CoA
SCoA
HO O
LIPITOR®
NF
O
NH
2
Ca2+
OH
OO
HO
O
Human HMGR with Natural Substrates
Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.
O
HMG-CoAS
OHO
O
CH2
CH2
NC OCH2
CH2
NC OC
H
H
OHHCH3C CH3
CH2
OR
R = 3'-phosphoadenosinediphosphate
LIPITOR®
NF
O
NH
2
Ca2+
OH
OO
HO
pantothenicacid
Human HMGR with LIPITOR®
Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.
LIPITOR®
NF
O
NH
2
Ca2+
OH
OO
HO
Human HMGR with LIPITOR®
Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.
LIPITOR®
NF
O
NH
2
Ca2+
OH
OO
HO
Circa 1995 – The Statin Drugs Market
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.
• Merck = cholesterol control
• At 20 mg, ZOCOR® lowered LDL by -29%.H
O
OHO
O
O
Lovastatin (MEVACOR®)MERCK
H
O
OHO
O
O
Simvastatin (ZOCOR®)MERCK
Circa 1995 – The Statin Drugs Market
• Merck = cholesterol control.
• At 20 mg, ZOCOR® lowered LDL by -29%.H
O
OHO
O
O
Lovastatin (MEVACOR®)MERCK
H
O
OHO
O
O
Simvastatin (ZOCOR®)MERCK
Spring 1997 – Pfizer launches LIPITOR®!
• At 20 mg, LIPITOR® lowered LDL by -46%.
• 2005 – $12 billion sales, used by over 45 million people.
Atorvastatin calciumLIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry
Chemical Synthesis
Biocatalysis
Drug Discovery
Process Development
Atorvastatin calciumLIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry
Chemical Synthesis
Biocatalysis
Drug Discovery
Process Development
Atorvastatin calciumLIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca+2
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.
H
O
OHO
O
O
Mevastatin
H
O
OHO
O
O
Lovastatin (MEVACOR®)MERCK
HO
O
HO
CO2Na
HO
HO
Pravastatin (PRAVACOL®)BRISTOL - MYERS SQUIBB
?
?
The Decision of the Core TemplateThe Drug Discovery
F
O
OHO
MERCK
The Decision of the Core Template
H
O
OHO
O
O
Mevastatin
The Drug Discovery
Hydrolysis – 100-fold
loss in potency
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
hydrophobic group
template
correct spatial relationship
A Potent HMGR Inhibitor
X
O
OHO
F
O
OHO
MERCK
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
The Pyrrole TemplateThe Drug Discovery
N
X
R2R1
O
OHO
The Pyrrole TemplateThe Drug Discovery
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
O+
O
FNEt3
S
N
HO
BnCl
58%
H2NCN
HOAc, reflux
73%N
CN
F
DIBAL-H92%
N
CHO
F
CH3COCH2CO2CH3
NaH, n-BuLiTHF, -78 °C
64%N
F
CO2CH3
O
HO
NF
CO2HHO
HO
1) Bu3B, NaBH4
THF, -78 °C2) NaOH, H2O2
toluene, reflux
52%tr ans:cis 97:3
NF
O
OHO
F O O
20 mol%
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
The Pyrrole TemplateThe Drug Discovery
• Over 40 analogs prepared.
• Optimization studies for X, R1 and R2.
O+
O
FNEt3
S
N
HO
BnCl
58%
H2NCN
HOAc, reflux
73%N
CN
F
DIBAL-H92%
N
CHO
F
CH3COCH2CO2CH3
NaH, n-BuLiTHF, -78 °C
64%N
F
CO2CH3
O
HO
NF
CO2HHO
HO
1) Bu3B, NaBH4
THF, -78 °C2) NaOH, H2O2
toluene, reflux
52%tr ans:cis 97:3
N
X
R2R1
O
OHO
F O O
20 mol%
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
The Need for Additional FunctionalityThe Drug Discovery
N
O
OHO
F
• IC50
(analog): 0.40 µM.
• Limit of current synthetic route.
H
O
OHO
O
O
MevastatinMERCK
IC50
– 0.030 µM.
The Need for Additional FunctionalityThe Drug Discovery
N
O
OHO
F
Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
F
O
OHO
MERCK
F
O
OHO
An overlay
N
O
OHO
F
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
Incorporation of Additional FunctionalityThe Drug Discovery
N
O
OHO
F
X X
X = ClX = Br
TBDMSClimidazole, DMF
25 ºC100%
N
O
OTBDMSO
F
1) 2 eq NCS or NBSDMF, 0 ºC100%
2) TBAF, HOAcTHF, 25 ºC35%
0.028Br
0.028Cl
0.23H
IC50
(µM)X
N
O
OHO
F
H
O
OHO
O
O
MevastatinMERCK
IC50
– 0.030 µM.
Incorporation of Additional FunctionalityThe Drug Discovery
N
O
OHO
F
X X
X = ClX = Br
TBDMSClimidazole, DMF
25 ºC100%
N
O
OTBDMSO
F
1) 2 eq NCS or NBSDMF, 0 ºC100%
2) TBAF, HOAcTHF, 25 ºC35%
N
O
OHO
F
Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
Toxicity in early preclinical development! 0.028Br
0.028Cl
0.23H
IC50
(µM)X
H
O
OHO
O
O
MevastatinMERCK
IC50
– 0.030 µM.
Penta-substituted Pyrroles via [3+2]The Drug Discovery
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
Br
CO2Et
F H2N O
O
NEt3, CH3CN, 25 ºC82%
NH
CO2Et
F
OO1) O
Cl
NEt3, CH2Cl2, 0 ºC
2) NaOH
N
HO2C
F
OO
O97%
O
NHPhPh
Ac2O, 90 ºC
NF
PhO
NHPh
OO1) HCl, EtOHreflux
2) p-TSA,acetone-waterreflux
N
CHO
F
PhO
NHPh
43%
87%
NF
PhO
NHPh
O
OHO
Penta-substituted Pyrroles via [3+2]The Drug Discovery
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
NF
X Y
O
OHO
20 analogstested
NF
O
OHO
O
NH
(racemic)
IC50
– 0.025 µM
H
O
OHO
O
O
MevastatinMERCK
IC50
– 0.030 µM.
Penta-substituted Pyrroles via [3+2]The Drug Discovery
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
NF
O
OHO
O
NH
(racemic)
resolution NF
O
OHO
O
NH
(R, R)
NF
X Y
O
OHO
20 analogstested
H
O
OHO
O
O
MevastatinMERCK
IC50
– 0.030 µM.
IC50
– 0.025 µM
(S, S) – 0.44
(R, R) – 0.007
IC50
(µM)
The Birth of LIPITOR®!The Drug Discovery
NF
O
OHO
O
NH
(racemic)
resolution NF
O
OHO
O
NH
(R, R)
Atorvastatin calciumLIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
NF
X Y
O
OHO
20 analogstested
The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry
Chemical Synthesis
Biocatalysis
Drug Discovery
Process Development
Atorvastatin calciumLIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry
Chemical Synthesis
Biocatalysis
Drug Discovery
Process Development
Atorvastatin calciumLIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
[3+2] Cycloaddition Route
N
HO2C
F
OO
O
O
NHPhPh1.6 eq
Ac2O, 90 ºC
43%
NF
PhO
NHPh
OO
Paal-Knorr Route: Penta-substituted
Pyrroles
Scale-up Issues and Potential SolutionsThe Process Development
Failed Model Study
Paal-Knorr Route: Tetra-substituted
Pyrroles
Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
H2N
NF
OEt
OEtOEtEtO
F O O+
F
O
NH
O ONF
CO2RHO
HO
+
H2NCO2R
O O
NH
O
From Tetra- to Penta-substituted PyrrolesThe Process Development
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
NF
OEtEtO1) NBS, DMF 0 ºC, 100%2) n-BuLi, THF, PhNCO, 69%3) H3O+, 86%N
CHO
F
CONHPh
H2N
p-TSA, toluenereflux
OEt
OEt
71%
O PhCHO, p-TSAtoluene, reflux
80%
OO
F
NEt3
S
N
HO
BnCl
30%
2) NaOH, CH3OH, rt
O O O O
F O O
20 mol%
From Tetra- to Penta-substituted PyrrolesThe Process Development
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
38%
-78 ºC
-10 ºC
0 ºC
-78 ºC
-70 ºC
O
O
2 eq. LDA, MgBr2N
F
CONHPh
O
OHO1)
2) Recrystallization
N
CHO
F
CONHPh
1) NaOCH368%
2)
OtBu
OLi
78%
NF
CONHPh
OCO2
tBuHO
1) Et3B. NaBH42) H2O2, NaOH3) toluene, refluxN
F
CONHPh
O
OHO
>99% ee
dr 98:2
Ph
Ph
OHPh Ph
OH
Ph
Ph
From Tetra- to Penta-substituted PyrrolesThe Process Development
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
38%
-78 ºC
-10 ºC
0 ºC
-78 ºC
-70 ºC
O
O
2 eq. LDA, MgBr2N
F
CONHPh
O
OHO1)
2) Recrystallization
N
CHO
F
CONHPh
1) NaOCH368%
2)
OtBu
OLi
78%
NF
CONHPh
OCO2
tBuHO
1) Et3B. NaBH42) H2O2, NaOH3) toluene, refluxN
F
CONHPh
O
OHO
>99% ee
dr 98:2
Ph
Ph
OHPh Ph
OH
Ph
Ph
From Tetra- to Penta-substituted PyrrolesThe Process Development
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
38%
-78 ºC
-10 ºC
0 ºC
-78 ºC
-70 ºC
3 columns & 1 crystallization7% yield
O
O
2 eq. LDA, MgBr2N
F
CONHPh
O
OHO1)
2) Recrystallization
N
CHO
F
CONHPh
1) NaOCH368%
2)
OtBu
OLi
78%
NF
CONHPh
OCO2
tBuHO
1) Et3B. NaBH42) H2O2, NaOH3) toluene, refluxN
F
CONHPh
O
OHO
>99% ee
dr 98:2
Ph
Ph
OHPh Ph
OH
Ph
Ph
A Recap – The FailuresThe Process Development
N
HO2C
F
OO
O
O
NHPhPh1.6 eq
Ac2O, 90 ºC
43%
NF
PhO
NHPh
OO[3+2] Cycloaddition Route
Paal Knorr Route: Tetra-substituted
Pyrroles
Paal Knorr Route: Penta-substituted
Pyrroles
H2N
HOAc, reflux71%
NF
OEt
OEt OEtEtOF O O
F
O
NH
O ONF
CO2RHO
HO
+
H2NCO2R
O O
NH
O
Failed Model Study
Paal Knorr Route: Penta-substituted PyrrolesThe Process Development
Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
N
CO2RR2O
R1O
F
O
NH
NH2
CO2RR2O
R1O
F
O
NH
O O
+
B
A
F
O
NH
O O
+
H2N OEt
OEt
NF
THF, reflux
OEtEtO
43%
OH
O1 eq
O
NH
Pfizer’s Commercial Route: Fragment AThe Process Development
Dr. Bruce D. Roth (VP, Global Research & Development, Pfizer), personal communication.Baumann, K. L. et al. Tetrahedron Lett. 1992, 33, 2283-2284.
F
ONH
O O
A
O
NHPh
O ββββ -Alanine, AcOHhexane, heat
85%
O
NHPh
O
F
CHONEt3, EtOH, heat
80%
S
NBr
HO
Ph
PhCHO
20 mol%
Pfizer’s Commercial Route: Fragment BThe Process Development
Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282.Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.
NH2
CO2RR2O
R1O
B
OO
OHHO
HOOH
HH2O2, CaCO3
K2CO3HO CO2K
OH
OH
HBr, HOAcCH3OH
Br CO2CH3
OH
Br
H2, Pd/CCH3COONa/CH3COOH
60%(over 3 steps)
Br CO2CH3
OH1) TBDMS-Cl
imid., 4-DMAP
2) NaCN, DMSONC CO2CH3
OTBDMS
Pfizer’s Commercial Route: Fragment BThe Process Development
Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication.Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282.
1) NaOH2) CDI
Mg(O2CCH2CO2tBu)
3) TBAF, HOAc, THFNC CO2CH3
OTBDMSNC
OH O
CO2tBu
NH2
CO2RR2O
R1O
B
NC CO2CH3
OH3 eq LiCH2CO2tBu
THF
75%
Pfizer’s Commercial Route: Fragment BThe Process Development
Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication.Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282.
1) NaOH2) CDI
Mg(O2CCH2CO2tBu)
3) TBAF, HOAc, THFNC CO2CH3
OTBDMS
liquid N2
liquid N2
Cryogenic reactor employed
NC
OH O
CO2tBu NC CO2CH3
OH3 eq LiCH2CO2tBu
THF
75%
1) NaBH4, Et2BOMeCH3OH, -90 °C
2) (CH3)2C(OCH3)2CH3SO3H
NC
O O
CO2tBu
Raney-Ni, MeOH50 psi H2
95%
O O
CO2tBu
H2N
>99.5% ee
B
dr 350:1ee >99.5%
Pfizer’s Commercial RouteThe Process Development
Baumann, K. L. et al. Tetrahedron Lett. 1992, 33, 2283-2284.Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication.
NH2
CO2tBu
O
O
F
ONH
O O
+ 1 eq OH
O
1:4:1 toluene:heptane:THFreflux75%
N
CO2tBu
O
O
F
O
NH
a) HCl, MeOHb) NaOHc) Ca(OAc)2
84%
A B
Atorvastatin calciumLIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
>99.5% ee
• Highly convergent.
• Yields >75% at each step.
• One low temperature reaction.
• One special equipment requirement.
• No chromatography.
• Scalable to ton quantities.
LIPITOR®: Drug tackled, the struggles remained…
Fortune, 2003, January 20.
The problems:
• By 1987, three statins in market.
• A decade since the first statin introduced.
• Warner-Lambert was floundering.
• Come on, how good could it be?
• On the verge of terminating LIPITOR®.
“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
And the facts now:
• #1 statin in the market.
• Top selling drug in history.
• 2005: $12 billion sales & used by >45 million people.
• “LIPITOR® is on track to have greater benefit for more people than any other drug in the history of the industry in terms
of lives improved and saved.”
- Nobel Laureate Michael Brown
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 plaindumb luck!”
Fortune, 2003, January 20.
The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry
Chemical Synthesis
Biocatalysis
Drug Discovery
Process Development
Atorvastatin calciumLIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry
Chemical Synthesis
Biocatalysis
Drug Discovery
Process Development
220 ton/year market ($500 million)
Atorvastatin calciumLIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
The Story of LIPITOR® - a Peek into the World
of Pharmaceutical Process Chemistry
Chemical Synthesis
Biocatalysis
Drug Discovery
Process Development
220 ton/year market ($500 million)
Atorvastatin calciumLIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
Existing Route & the Need for ImprovementNC
OHOH O
OR
NaCN, DMSO
HBr, HOAcCH3OH
1) NaBH4, Et2BOMeCH3OH, -90 °C
OO
OHHO
HOOH
H H2O2, CaCO3K2CO3 HO CO2K
OH
OH
NC CO2CH3
OH
NC
OH O
CO2tBu
3 eq LiCH2CO2tBu
THF
75%
NC
O O
CO2tBu
>99.5% ee
Br CO2CH3
OH
Br
60%(over 3 steps)
Br CO2CH3
OH
H2, Pd/CCH3COONa/CH3COOH
2) (CH3)2C(OCH3)2CH3SO3H
Existing Route & the Need for ImprovementNC
OHOH O
OR
• SHE issues.
• Cryogenic step – special requirements/waste.
• With purification – 11 steps.
• Month’s supply: $66 LIPITOR® vs $120 ZOCOR®.
• But patent expires ~ 2010.
OO
OHHO
HOOH
H H2O2, CaCO3K2CO3 HO CO2K
OH
OH
NC CO2CH3
OH
NC
OH O
CO2tBu
3 eq LiCH2CO2tBu
THF
75%
NC
O O
CO2tBu
>99.5% ee
Br CO2CH3
OH
Br
60%(over 3 steps)
Br CO2CH3
OH
H2, Pd/CCH3COONa/CH3COOH
2) (CH3)2C(OCH3)2CH3SO3H
1) NaBH4, Et2BOMeCH3OH, -90 °C
NaCN, DMSO
HBr, HOAcCH3OH
Biocatalytic Routes for the Chiral Side Chain
Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
NC
OHOH O
OR
OEt
OOH
Cl
HO OEt
OO OH
NCOH
OOH
NCOEt
OOH
O OHX
OH
OEt
OO
Cl
ADHGDH
Lipase
EtO OEt
OO OR1
NitrilaseCNNC
OH
1) ADH
2) Dehalo-genase
ClOEt
OO
AldolaseX
H
O+
H
O2
KANEKA
CIBA
DOWPHARMA(DIVERSA)
CODEXIS
DIVERSA
NC
OHOH O
OR
Biocatalytic Routes for the Chiral Side Chain
OEt
OOH
Cl
HO OEt
OO OH
NCOH
OOH
NCOEt
OOH
O OHX
OH
OEt
OO
Cl
ADHGDH
Lipase
EtO OEt
OO OR1
NitrilaseCNNC
OH
1) ADH
2) Dehalo-genase
ClOEt
OO
AldolaseX
H
O+
H
O2
NC
OHOH O
OR
NC
OHOH O
OR
KANEKA
CIBA
DOWPHARMA(DIVERSA)
CODEXIS
DIVERSA
Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
Biocatalytic Routes for the Chiral Side Chain
OEt
OOHCl
HO OEt
OO OH
NCOH
OOH
NCOEt
OOH
O OHX
OH
OEt
OO
Cl
ADHGDH
Lipase
EtO OEt
OO OR1
NitrilaseCNNC
OH
1) ADH
2) Dehalo-genase
ClOEt
OO
AldolaseX
H
O+
H
O2
NC
OHOH O
OR
NC
OHOH O
OR
KANEKA
CIBA
DOWPHARMA(DIVERSA)
CODEXIS
DIVERSA
Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
Biocatalytic Routes for the Chiral Side Chain
OEt
OOHCl
HO OEt
OO OH
NCOH
OOH
NCOEt
OOH
O OHX
OH
OEt
OO
Cl
ADHGDH
Lipase
EtO OEt
OO OR1
NitrilaseCNNC
OH
1) ADH
2) Dehalo-genase
ClOEt
OO
AldolaseX
H
O+
H
O2
NC
OHOH O
OR
NC
OHOH O
OR
KANEKA
CIBA
DOWPHARMA(DIVERSA)
CODEXIS
DIVERSA
Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
Biocatalytic Routes for the Chiral Side Chain
OEt
OOHCl
HO OEt
OO OH
NCOH
OOH
NCOEt
OOH
O OHX
OH
OEt
OO
Cl
ADHGDH
Lipase
EtO OEt
OO OR1
NitrilaseCNNC
OH
1) ADH
2) Dehalo-genase
ClOEt
OO
AldolaseX
H
O+
H
O2
NC
OHOH O
OR
NC
OHOH O
OR
KANEKA
CIBA
DOWPHARMA(DIVERSA)
CODEXIS
DIVERSA
Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
Biocatalytic Routes for the Chiral Side Chain
OEt
OOHCl
HO OEt
OO OH
NCOH
OOH
NCOEt
OOH
O OHX
OH
OEt
OO
Cl
ADHGDH
Lipase
EtO OEt
OO OR1
NitrilaseCNNC
OH
1) ADH
2) Dehalo-genase
ClOEt
OO
AldolaseX
H
O+
H
O2
NC
OHOH O
OR
NC
OHOH O
OR
KANEKA
CIBA
DOWPHARMA(DIVERSA)
CODEXIS
DIVERSA
Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
Kaneka’s Route
Kizaki, N. et al. Appl. Microbiol. Biotechnol. 2001, 55, 590-595.Yasohara, Y. et al. Tetrahedron: Asymmetry 2001, 12, 1713-1718.
• >99.9% ee, 89% yield.
• Product concentration: 450 g/L.
• NADP+ TON: 16,200 mol/mol.
• Problematic product separation.
ClO
O
OEt
Cl
OH
O
O
EtNADP+
NADPH
D-glucose
D-gluconolactone
spontaneous
D-glutonic acidAqueous phase Organic phase
(n-butyl acetate)
NC
OHOH O
OR
glucose dehydrogenase
carbonyl reductase
recombinant E. coli
Daicel’s Route
• >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.
Cl
O
O
O
Et
Cl
OH
O
O
EtNADP+
NADPH
formic acid
CO2
NC
OHOH O
OR
formate dehydrogenase
carbonyl reductase
recombinant E. coli
Ciba’s Route
Öhrlein, R.; Baischf, G. Adv. Synth. Catal. 2003, 345, 713-715.
The good things
• 94% yield, 98.2% ee.
• Substrate concentration: 285 g/L.
• Kilogram scale.
• Cheap & robust biocatalyst.
EtO OEt
OO OH Cl
O
O
pyridine0 °C to rt
~ 12 h98%
EtO OEt
OO O
O
αααα-chymotrypsin0.008 mol%
HO OEt
OO O
O
24-30 h
O O
6 steps44% overall
yield
OEt
OO O
N3
94%98.2% ee
NC
OHOH O
OR
The bad things
• Follow up chemistry – long.
• Low temperature reactions.
• Column chromatography.
Diversa/Dowpharma’s Route
DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025.DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.
NC
OHOH O
OR
NC CNOH nitrilase
pH 7.5 NaH2PO427 °C, 16 h
81%98.8% ee
NC COOHOH
Diversa/Dowpharma’s RouteNC
OHOH O
OR
Bergeron, S. et al. Org. Process Res. Dev. 2006, 10, 661-665.
The good things
• Cheap starting material.
• Efficient enzymatic step: 3 M [substrate]& 619 g L-1 d-1.
• Low cost of catalyst by expression in Pseudomonas fluorescens developed by Dow.
Scale-up economics good!
The bad things
• HCN under heated alkaline conditions.
• Special equipment for purification.
• Some low yield steps.
NC CN
OH
nitrilase
ClO
HCN, base(n-Bu)4N+Br-
NaCN (aq.)pH=10, 4 h, 50 °C
pH 7.5 NaH2PO427 °C, 16 h
81%98.8% ee
EtOH, H+
3 h, 80 °CNC CO2Et
OH
62%
92%
NC Cl
OH
45%
NC COOH
OH
12 h, 45 °C
Diversa’s Route
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.
OCl
OH
OH
Cl
OH OH O
OR
DERA = deoxyribose-5-phosphate aldolase
ClO DERA
+O
ClOH O
O
DERACl
OH OH OAldol
reaction25 °C
NC
OHOH O
OR
Unknownee (de)
NOPractical?
2 g L-1 d-1Volumetric Productivity
100 mM[chloroacetaldehyde]
6 daysReaction Time
DifficultProduct Isolation
20% w/wCatalyst Load
Wild Type DERA
Diversa’s Route
OCl
OH
OH
DERA = deoxyribose-5-phosphate aldolase
NC
OHOH O
OR
≥ 99.9%Unknownee & de
YESNOPractical?
735 g L-1 d-12 g L-1 d-1Volumetric Productivity
Fed-batch process
100 mM[chloroacetaldehyde]
3 h6 daysReaction Time
SimpleDifficultProduct Isolation
2% w/w20% w/wCatalyst Load
Improved DERA
Wild Type DERA
Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.
NC
O O O
OMe
23% yield3 overall steps
ClO DERA
+O
ClOH O
O
DERACl
OH OH OAldol
reaction25 °C
Codexis Route
Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
NC
OHOH O
OR
NCOH O
CO2tBu
oil, not purified
KRED/GDHNC
O O
CO2tBu
first crystalline intermediate
NC
OH OH
CO2tBu
oil, not purified
chemicalchain
elongation
chemicalprotection
Cl
O
OEt
OKRED/GDH
ClOH
OEt
O HHDHNC
OH
OEt
O
not purif ied oil, not purified
KRED = KetoreductaseGDH = Glucose dehydrogenaseHHDH = Halohydrin dehalogenase
- HCl
+ HClOEt
OO
pKa ~9 HCNaq HClaq pKa <0
NaCNaqNaClaqneutral pH
two phase, no solvent
+ HCN
Codexis Route
Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
NC
OHOH O
OR
NC
OH O
CO2tBu
oil, not purified
KRED/GDHNC
O O
CO2tBu
first crystalline intermediate
NC
OH OH
CO2tBu
oil, not purif ied
chemicalchain
elongation
chemicalprotection
Cl
O
OEt
OKRED/GDH
Cl
OH
OEt
O HHDHNC
OH
OEt
O
not purified oil, not purified
KRED = KetoreductaseGDH = Glucose dehydrogenaseHHDH = Halohydrin dehalogenase
- HCl
+ HClOEt
OO
pKa ~9 HCNaq HClaq pKa <0
NaCNaqNaClaqneutral pH
two phase, no solvent
+ HCN
Codexis Route
Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
• 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.
Cl
O
OEt
O
NC
O O
CO2tBu
1) KRED/GDH2) HHDH
3) Chemical chainelongation
4) KRED/GDH5) Protection
"TBIN"First crystalline intermediate
NC
OHOH O
OR
Biocatalytic Routes for the Chiral Side Chain
Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.
NC
OHOH O
OR
OEt
OOH
Cl
HO OEt
OO OH
NCOH
OOH
NCOEt
OOH
O OHX
OH
OEt
OOCl
ADHGDH
Lipase
EtO OEt
OO OR1
NitrilaseCNNC
OH
1) ADH
2) Dehalo-genase
ClOEt
OO
AldolaseX
H
O+
H
O2
KANEKA
CIBA
DOWPHARMA(DIVERSA)
CODEXIS
DIVERSA
NC
OHOH O
OR
Biocatalytic Routes for the Chiral Side Chain
OEt
OOHCl
HO OEt
OO OH
NCOH
OOH
NCOEt
OOH
O OHX
OH
OEt
OO
Cl
ADHGDH
Lipase
EtO OEt
OO OR1
NitrilaseCNNC
OH
1) ADH
2) Dehalo-genase
ClOEt
OO
AldolaseX
H
O+
H
O2
NC
OHOH O
OR
NC
OHOH O
OR
KANEKA
CIBA
DOWPHARMA(DIVERSA)
CODEXIS
DIVERSA
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
NH2
CO2tBu
O
O
F
ONH
O O
LIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
+
CHEMICALSYNTHESISat PFIZER
BIOCATALYSISat CODEXIS
Cl
O
O
O
CHEMICALSYNTHESISat PFIZER
O
NHPh
O
An Extremely Attractive Chemo-enzymatic Approach
NH2
CO2tBu
O
O
F
O
NH
O O
LIPITOR®
N
CO2
HO
HO
F
O
NH
2
Ca2+
+
CHEMICALSYNTHESISat PFIZER
BIOCATALYSISat CODEXIS
Cl
O
O
O
CHEMICALSYNTHESISat PFIZER
O
NHPh
O
Why not other pharmaceuticals and natural products also?
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. WulffDr. 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 ZardDr. 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
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.
• 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.
Some Rare Adverse Effects of Statin Drugs
www.lipitor.comRosanoff, A.; Seelig, M. S. Journal of the American College of Nutrition 2004, 23, 501S-505.
• The most common side effects are gas, constipation, stomach pain and heartburn.
• Rare Myalgia (muscle pain).
• Rare Rhabdomyolosis (skeletal muscle failure – loss of kidney function).
possibly life threatening!
Uncommon side reactions occur mainly when statins are co-prescribed with other
interacting drugs.
Statin Drugs
N
OO
HO
HO
H
O
OHO
O
O
Lovastatin (MEVACOR®)MERCK
H
O
OHO
O
O
Simvastatin (ZOCOR®)MERCK
HO
O
HO
C
HO
HO
Pravastatin (PRAVACOL®)BRISTOL - MYERS SQUIBB
Atorvastatin (LIPITOR®)PFIZER
N
CO2
HO
HO
F
O
NH
N
OO
HO
OH
F
Fluvastatin (LESCOL®)NOVARTIS
N
F
OO
HO
OH
O
Cerivastatin (LIPOBAY®)BAYCOL
withdrawn in 2001
N N
OO
HO
OH
N
F
SO
O
Rosuvastatin (CRESTOR®)ASTRAZENECA
F
Pitavastatin (LIVALO®)KOWA
O
O
Chemo-catalytic vs. Bio-catalytic: Wacker Specialty Chemicals
OR
OOH
Cl
Asymmetric Hydrogenation(Ruthenium Catalyst +Diphosphine Ligand)
OR
OO
Cl
Biocatalytic Reduction(Alcohol Dehydrogenase
Enzyme)
OR
OO
Cl
Standard StandardEquipment
2 L< 100 gWaste/kg (product)
1 (dilute conditions)3Relative Throughput
None – ambient temp. & pressure
100 °C, MeOH solvent, handling H2
SHE issues
< $100< $85Cost/kg (product)
Multi-tonMulti-tonScale
>99.9 (97)98 (95)%ee (yield)
Bio-catalysisChemo-catalysis
Requirement of price & purity will dictate the route.
Rouhi, A. M. Chem. Eng. News 2004, 82, (24), 47-62.
Sliskovic, D. R. et al. J. Med. Chem. 1991, 34, 367-373.Sliskovic, D. R. et al. J. Med. Chem. 1990, 33, 31-38.
Other Templates AttemptedThe Drug Discovery
0.018F3 (N-oxide)
0.013OCH32
0.032Cl1
IC50 (µM)R1Compound
NNR
F
O
OHO
H
R = Ph (IC50 = 0.035 µM)
N
R1
F
O O
OH
H
mevastatin: IC50 = 0.030 µM
H
O
OHO
O
O
Human HMGR with LIPITOR
Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.
The specificity & tight binding achieved by:
• hydrogen bonds
• ionic interactions
• polar interactions
LIPITOR®
NF
O
NH
2
Ca2+
OH
OO
HO
Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.
Human HMGR with Statin Drugs
Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.
The solution – suppressing cholesterol biosynthesis
www.cellml.org
Cholesterol Biosynthesis
Acetyl-CoA
Acetoacetyl-CoA
Hydroxymethylglutaryl-CoA(HMG-CoA)
Mevalonate
5-phosphomevalonate
5-pyrophosphomevalonate
Dimethylallylpyrophosphate
Geranylpyrophosphate
Farnesylpyrophosphate
Squalene
Squalene-2,3-epoxideLanosterol18 reactions
DesmosterolCholesterol
The solution – suppressing cholesterol biosynthesis
Steinberg, D.; Avigan, J. J. Biol. Chem. 1960, 235, 3127-3129.
Cholesterol Biosynthesis
OH
Cl
CH3
OH2N
Triparanol (MER-29)
DesmosterolCholesterol
Severe side effects – failed!
Acetyl-CoA
Acetoacetyl-CoA
Hydroxymethylglutaryl-CoA(HMG-CoA)
Mevalonate
5-phosphomevalonate
5-pyrophosphomevalonate
Dimethylallylpyrophosphate
Geranylpyrophosphate
Farnesylpyrophosphate
Squalene
Squalene-2,3-epoxideLanosterol18 reactions
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
Structure-Activity Relationship StudiesThe Drug Discovery
F
O
OHO
1MERCK
N
X
R2R1
O
OHO
Structure-Activity Relationship StudiesThe Drug Discovery
N
X
O
OHO
F
X =
-CH2CH2CH2
-CH2CH2
N
O
OHO
F
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
Structure-Activity Relationship StudiesThe Drug Discovery
N
X
O
OHO
FNR1
O
OHO
R1 =
Ph
4-PhC6H4
4-MeOC6H4
4-ClC6H4
4-HOC6H4
4-FC6H4
3-F3CC6H4
3-MeOC6H4
3-HOC6H4
2-MeOC6H4
2-HOC6H4
2-naphthyl
1-naphthyl
cyclohexyl
N
O
OHO
F
N R2
O
OHO
R1
< 5.9 Å
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
Structure-Activity Relationship StudiesThe Drug Discovery
N
X
O
OHO
FNR1
O
OHO
N R2
O
OHO
F
R2 =
-CH3
N
O
OHO
F
N R2
O
OHO
R1
< 5.9 Å
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
Structure-Activity Relationship StudiesThe Drug Discovery
N
X
O
OHO
FNR1
O
OHO
N R2
O
OHO
F
R2 =
-CH3
N
O
OHO
F
N R2
O
OHO
R1
< 5.9 Å < 3.3 Å
< 10.6 Å
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
Structure-Activity Relationship StudiesThe Drug Discovery
N
X
O
OHO
FNR1
O
OHO
N R2
O
OHO
F
N R2
O
OHO
R1
< 5.9 Å < 3.3 Å
< 10.6 Å
3-FC6H4
2-FC6H4
2,4-F2C6H3
2-MeOC6H4
2-ClC6H4
2,6-(MeO)2C6H3
2,5-Me2C6H3
2-iPrOC6H4
O
N
O
OHO
FNR1
O
OHO
Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.
Pentasubstituted Pyrroles via [3+2]The Drug Discovery
Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.
“Munchnone”
N
OHOOC
F
OO
CO2Et
Ac2O, reflux
N
CO2Et
OO
F
N
OHOOC
OO
CO2Et
Ac2O, reflux
N
EtO2C
OO
F
N
R2
O
R3R1
HOOC
N
R2
R1
R4 R5
R3
Ac2Oreflux N
OO
R2
R3
R1 R4 R5
Regiocontrol:
Ciba’s Route
EtO OEt
OO OH Cl
OO
pyridine0 °C to rt
~ 12 h98%
EtO OEt
OO O
O
α-chymotrypsin0.008 mol%
HO OEt
OO O
O
24-30 h
(COCl)2, 0 °C100%
Cl OEt
OO O
Oethylene, AlCl3
DCE, 0 °C
89%OEt
OO O
O
Cl
pig-leveresterase
pH=776%OEt
OO OH
Cl
1) BEt3, NaBH4-78 °C, 91%
2) dimethoxypropaneH+, 98%
3) NaN3, DMF94% y, 98% ee, 97% de
OEt
OO O
N3
+
F O O
Ph CONHPh
O OH
PBu370%
N
CO2EtO
O
F
Ph CONHPh
O O
OO
Öhrlein, R.; Baischf, G. Adv. Synth. Catal. 2003, 345, 713-715.
NC
OHOH O
OR
Diversa/Dowpharma’s Route
DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025.DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.
Screen genomic libraries
Most effective WT nitrilase
0.24 M [substrate] – 98% y, 95% ee
3 M [substrate] – 88% ee
NC
OHOH O
OR
NC CN
OH nitrilaseNC COOH
OH
pH 7.5 NaH2PO427 °C, 16h
81% y, 98.8% ee
Diversa/Dowpharma’s Route
DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025.DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.
Screen genomic libraries
Most effective WT nitrilase
0.24 M [substrate] – 98% y, 95% ee
3 M [substrate] – 88% ee
NC
OHOH O
OR
NC CN
OH nitrilaseNC COOH
OH
pH 7.5 NaH2PO427 °C, 16h
81% y, 98.8% ee
Genetical Engineering gave another library
Best mutant
2.25 M [substrate] – 98% ee in 15 h
Diversa’s GSSM™: Gene Site Saturation Mutagenesis
1. Target Protein forImprovement
2. Change Amino Acid(s)
A new gene variant library isborn containing genes with everysingle site variation in sequence.
Identify protein for optimization through extensive screening of gene libraries.
Evolve the gene encoding the protein by systematically changing each amino acid in the sequence to every other possible amino acid.
3. Complete Variant Library
4. Select Variants
Through screening, identify the variants that demonstrate improved characteristics.
5. Combine Mutations
Tests all potential combinations of the single amino acid changes that demonstrated improved characteristics and select the optimal combination.
6. Generate Optimized Protein
www.diversa.com
Diversa’s High Throughput Screen for Improved Selectivity
DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.
NC C15N
OH
15N-(R)-12
R-selectivenitrilase
S-selectivenitrilase
NC COOHOH
(R)-13m/z = 129
HOOC C15NOH
15N-(S)-13
m/z = 130
Diversa’s Route
Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.
OCl
OH
OH
DERA = deoxyribose-5-phosphate aldolase
Cl
O DERA+
OCl
OH O
O
DERACl
OH OH OAldol
reaction25 °C
NaOCl, HOAcH2O, 25 °C, 3 h
45% (over2 steps)
OCl
OH
O
white crystals>99.9% ee99.8% de
a) NaCN, DMF, 5% H2O40 °C, 16 h
b) H2SO4c) (MeO)2CMe2d) TMSCHN2
oil, not purified
Cl
OH OH O
ONa
OH O
ONa
O NC
OH OH O
ONa
NC
O O O
OMe
oil, column chromatography48% (over 4 steps)
O O O
OtBuH2NCoupling partner
for the Paal-Knorr
NC
OHOH O
OR
Diversa’s Fluorogenic Activity Based Screen
Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.
OTosO
HO
O
OO OH
K2CO3, DMF, 75 °C, 16 h
OO O O
1)
2) H2O / 20% CH3CNDOWEX 50WX8-1002 days
62%
OH
HO
OO O H
O
OH
OHDERA
spontaneous
OO OH
+
O
O
fluorescent
OO O
OH
O+
H
O
5-toluenesulfonyl-2-deoxyribose
4-methylumbelliferone
Codexis Biocatalyst Improvement
Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
540 g/L.day80 g/L.dayVolumetric productivity
~ 1 min>1 hPhase separation time
97%~80%Isolated yield
0.7 g/L10 g/LEnzyme loading
8 h24 hReaction time
180 g/L80 g/LSubstrate loading
FinalInitialParameter
Cl
O
OEt
OKRED
Cl
OH
OEt
O
NADPH NADP+
GDHglucoseNa-gluconate
NC
OHOH O
OR
Codexis Biocatalyst Improvement
Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
92%~60%Isolated yield
1.2 g/L130 g/LEnzyme loading
5 h72 hReaction time
140 g/L20 g/LSubstrate loading
FinalInitialParameter
Cl
OH
OEt
O
NC
OH
OEt
O
- HCl
+ HClOEt
OO
pKa ~9 HCNaq HClaq pKa <0
NaCNaqNaClaqneutral pH
two phase, no solvent
+ HCN
halohydrindehalogenase
NC
OHOH O
OR
Codexis Biocatalyst Improvement
Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.
faciledifficultWork-up
100%100%Diastereomeric excess
99.3%87%Conversion
22 h65 hReaction time
300 g/L120 g/LSubstrate loading
FinalInitialParameter
KRED
NADPH NADP+
GDHglucoseNa-gluconate
NC
OH O
CO2tBu NC
OH OH
CO2tBu
NC
OHOH O
OR
Stetter Reaction Mechanism
Hermann, S. Angew. Chem., Int. Ed. Engl. 1976, 15, 639-647.
S
N
HO
BnCl
+ NEt3S
N
HO
BnCl HNEt3
O
H
Hp.t. S
N
HO
Bn
R
OH
R
R1
Op.t.
S
N
HO
BnO
RR1
OHR
OR1
OH
R
O
R1
O
Irreversible addition to the Michael Acceptor
R
O
Hp.t.
S
N
HO
BnO
R
OH
R
R
O
R
OH
Reversible benzoin formation
Syn-selective reduction of β-hydroxy ketones
Narasaka, K.; Pai, H. C. Chem. Lett. 1980, 1415-1418.
R1 R2
OH O
Bu3B, THF
OR2
BO
R1
HBu
Bu
favored
OR2
OB
Bu H
R1
Bu
attack fromless hindered
side
cis - diol+
O
BO
Bu
Bu
R2
R1
favored
R1 R2
O OB
BuBu
OB
O
Bu
Bu
H
H
R1
R2
unfavored
Htop bottom
Mechanism for H2O2 Oxidation of Ascorbic Acid
Isbell, H. S.; Frush, H. L. Carbohyd. Res. 1979, 72, 301-304.
OO
OHHO
HOOH
H-O-O-H OO
OHO
HOOH
OH+
H2O
H2O
OH
OO
OO
HOOH
H-O-O-H
OH
OO
O
HOOH
OOHOH
OO
HOOH
OHHO
OO
OH
OO
HOOH
OHHO
OO
OH
H2O(CO2H)2
HOOH
CO2H
HO
Mechanism for Hydrolysis by Chymotrypsin/PLE
Asp102
O
O
HN
N
His57
HO Ser195
R
O
O
H
N
Ser195
HN
Gly193
Asp102
O
O
HN
N
His57
O Ser195
R
O
O
H
N
Ser195
HN
Gly193
HH2O
OH
Asp102
O
O
HN
N
His57
O Ser195
R
O
H
N
Ser195
HN
Gly193
H
O
H
O
O
HN
N
His57
O Ser195
R
O
H
N
Ser195
HN
Gly193
Asp102
HO
H
O
O
HN
N
His57
HO Ser195
R
O
HO
H
N
Ser195
HN
Gly193
Asp102
Mechanism for Hydrolysis by Nitrilase
Faber, K. Biotransformations in Organic Chemistry, Springer-Verlag, 2004.
R C N + Enz SH RNH
S EnzH2O
R
OH
NH2
SEnz
NH3
RO
S EnzH2O
ROH
O
+ Enz SH
DERA Mechanism
Bioorg. Med. Chem. 11, 2003, 43-52.
H
O
H
O
OPO3
OH
+H
O
OPO3
OH
OHDERA
NH3
Lys 201
O
CH3
HO H
Lys 167
HNH
O
O
Asp102 NH3
Lys 201
H
CH3
HO H
Lys 167
O
OH
Asp102
HN
HO
NH2
Lys 201
H
CH3
HO H
Lys 167
O
OH
Asp102
N
NH3
Lys 201
H
CH2
HO H
Lys 167
O
O
Asp102
HN
O
OPO3
OH
NH3
Lys 201
H
Lys 167
O
O
Asp102
N OPO3OHH
OH
H2O hydrophilicsite
Ser238, 239Gly205
Thr170, Lys172
hydrophobicsite