engineering microorganisms for synthesis of medicinally important molecules
TRANSCRIPT
Engineering microorganisms for synthesis of medicinally important molecules
Innovación Para el Desarrollo Sostenible, Mérida. Mayo 2014
N
N
O H C
HO H
OO
O A cHNH
N
H
O H
M e OO
M e O
H O
O
H O
NM e N
O
O
C O 2 M e
NO
O
N
H
H
H
OH
O
H
H
HO
O
O
H
H
H
HO
O H
OO H O H
O H
O H
O H
O
O
OHH
H
HOH
N
H
H
OH
H3C
NO
O
HMe
O
H
H
O
O
O
OH
O HH
M e O
O
N HO
O
M e O
H C l
O
OO H
H O
O H
O H
O HO
O
OH OH
O
O
NO2
MeO
O
O
O
OO
O O
M e
M e
H H
O
NH2
MeO
OMe
MeO
NH
NO
P
O
O
HO
H
O
HO
MeO
OH
OH
OMe
Tarahumaras
Biocatalysis in Organic Chemistry
• Why microorganisms to do organic transformations?
• Epibatidine – an alkaloid from Ecuadorian frogs
• Selective hydroxylation of a piperidine?
• Modafinil – a unique CNS stimulant
• Other uses of lipases --Oxidations
The 12 Principles of Green Chemistry
1. It is better to prevent waste than to treat or clean up waste after it is formed.2. Synthetic methods should be designed to maximize the incorporation of all materials
used in the process into the final product.3. Wherever practicable, synthetic methodologies should be designed to use and
generate substances that possess little or no toxicity to human health and the environment.
4. Chemical products should be designed to preserve efficacy of function while reducing toxicity.
5. The use of auxiliary substances (e.g. solvents, separation agents, etc.) should be made unnecessary wherever possible and innocuous when used.
6. Energy requirements should be recognized for their environmental and economic impacts and should be minimized. Synthetic methods should be conducted at ambient temperature and pressure.
7. A raw material or feedstock should be renewable rather than depleting wherever technically and economically practicable.
8. Reduce derivatives - Unnecessary derivatization (blocking group, protection/ deprotection, temporary modification) should be avoided whenever possible.
9. Catalytic reagents (as selective as possible) are superior to stoichiometric reagents.10.Chemical products should be designed so that at the end of their function they do not
persist in the environment and break down into innocuous degradation products.11.Analytical methodologies need to be further developed to allow for real-time, in-
process monitoring and control prior to the formation of hazardous substances.12.Substances and the form of a substance used in a chemical process should be chosen to
minimize potential for chemical accidents, including releases, explosions, and fires.
Paul Anastas & John Warner
Hormone Steroids
O
O
H
H
HHO
O
H
H
H
O
OH
H
H
H
O
O
H
H
H
OOH
OH
O
OHC
H
H
H
HO
OH
O
progesterone estrone testosterone
cortisone aldosterone
Natural sources of steroidsDioscorea mexicana, “cabeza de negro”
Dioscorea composita, “barbasco”
HO
O
O
H
H
H
H
diosgenine
Marker’s Degradation of diosgenine
H O
O
O
H
H
H
HA c 2 O
2 0 0 C
A c O
O
H
H
H
H
O A c
d io s g e n in e
AcO
O
H
H
H
H
OAc
CrO3
AcOH
AcO
O
O OAc
O
Russell E. Marker
AcO
O
O OAc
O
NaOH
EtOH
HO
O
H O
O
O
O
O
O
O H
O
O
O
O H
O
O H
O
O HM e
p r o g e s te r o n e a n d r o s te n e d io n e e th is te r o n e
te s to s te r o n e m e th y lte s to s te r o n eh a lo te s t in
2 1 -h y d r o x y p r o g e s te ro n e
Laboratorios Syntex SA (1944)
O
O
O
O?
Biotransformations
O
O
O
OHO
Murray and Patterson, J. Am. Chem. Soc. 1952, 74, 1871
Murray and Patterson, J. Am. Chem. Soc. 1952, 74, 5933
Biotransformations
O
O
O
OHO
O
O
O
OH O
O
O
O H
O HO
c o r t i s o n e
R h i z o p u s a r r h i z u s
Murray and Patterson, J. Am. Chem. Soc. 1952, 74, 1871
Murray and Patterson, J. Am. Chem. Soc. 1952, 74, 5933
C lP . p u t i d a
m u t a n t s t r a i n
C l
O H
O H
David T. Gibson
Tomáš Hudlický
Jack Rosazza
NH
O
O
OH O
OH
OH
OH
HOOH
OH
X
X
X
O
O
N
OH OH
OH
OH
NH
O
OH
N
HO
HO
OHH
O
HO
HO
NMe
microorganism
kifunensin
pancratistatin morphine
trihydroxyheliotridane
OH
O
O
O
HO
O
OH
O
OMe
OH
OO
NHO
erythromycin
N
HN
OO
H
CO2H
penicillin
Biotechnology
insulin
Isolated from Streptomyces erythreus
Isolated from Penicillium fungi
Recombinant DNA
Pros and cons of using whole cell microorganisms
Form Pros Cons
Any No cofactor recycling necessary
Expensive equipment, tedious workup due to large volumes, low productivity due to lower concentration tolerance, low tolerance of organic solvents, side reactions likely due to uncontrolled metabolism
Growing culture Higher activities Large biomass, more byproducts, process control difficult
Resting cells Workup easier, fewer byproducts
lower activities
Immobilized cells Cell re-use possible lower activities
Pros and cons of using isolated enzymes
Form Pros Cons
Any Simple apparatus,Simple workup, better productivity due to higher concentration tolerance
Cofactor recycling necessary
Dissolved in water High enzyme activity Side reactions possible, lipophilic substrates insoluble, workup requires extraction
Suspended in organic solvents Easy to perform, easy workup, lipophilic substrates soluble, enzyme recovery easy
Reduced activities
Immobilized Enzyme recovery easy Loss of activity during immobilization
epibatidine
o Isolation: from the skin of poison frogs Epipedobates tricolor in Ecuador
(< 1 mg from 700 frogs)
o Daly JW et al J. Am. Chem. Soc. 1992, 114, 3475.
o Biological Activity: Pain killer hundreds of times more potent than morphine
o Mode of Action: Found to act on nicotinic receptors, not opioid receptors!
o Synthesis was needed!
HN
N Cl
epibatidine
John W. Daly (March 5, 2008)Epipedobates tricolor
Can microorganisms help us shorten the route to epibatidine?
NR
NR
OH
microorganism
HN
N C l
NR
O H
NR
O+
N C l
I
N C l
Johnson RA, Herr ME, Murray HC, Reineke LM, Fonken GS J. Org. Chem. 1968, 33, 3195
N
C O P h
N
C O P h
H O
N
C O P h
H O
+
B e a u v e r i a b a s s i a n a
4 5 - 7 0 %
NCOPh NCOPh
HOBeauveria bassiana
45 - 70%
Hydroxylation of unfunctionalized carbons
Olivo HF, Hemenway MS, Gezginci MH Tetrahedron Lett. 1998, 39, 1309
Olivo HF, Hemenway MS J. Org. Chem. 1999, 64, 8968
O H
N H 2
i . B e n z o y l c h l o r i d e
E t 3 N , C H 2 C l 2 , 1 0 0 %
i i . C H 3 S O 2 C l
E t 3 N , C H 2 C l 2 , 7 8 %
i i i . K O t - B u , D M F
8 8 %
N
Oi v . B . b a s s i a n a
5 6 % , 2 2 % e e
N
O
O H
HN
N C l
v . T P A P , N M O
C H 2 C l 2 , 8 9 %
N
O
O
v i . 2 - c h l o r o5 - i o d o p y r i d i n e
n - B u L i , T H F - 7 8 C , 7 8 %
N
O
O H
N C l
v i i . C H 3 O ( C O ) 2 C l
2 , 6 - l u t i d i n e , D M A P , 1 0 0 %
v i i i . B u 3 S n H
A I B N , 9 8 %
N
O
H
N C l i x . t - B u O K , t - B u O H1 0 0 C , 3 3 %
x . 6 N H C l1 0 0 C , 9 4 %
r a c - e p i b a t i d i n e
Biotransformation of N-acetylphenyl-Piperidine
N
O B e a u v e r i a s u l f u r e s c e n s
A T C C - 7 1 5 9N
O
H O
3 d a y s , 2 0 % ( J o h n s o n , 1 9 9 2 )5 d a y s , 6 6 % ( R o b e r t s , 1 9 9 8 )3 d a y s , 2 0 - 4 0 % ( H o l l a n d , 1 9 9 9 )
Not a clean reaction…
O
O
N
H O
N
N
N
O
O
H
H
H
N
O
O H
H O
N
O H
O
N
O
O H
O M e
H O
H O
N H O
O
O H
N
O
O H
Osorio V, Tovar R, Olivo HF J. Molecular Catalysis: Enzymatic 1998, 55, 30-36.
modafinilS
N H 2
OO
• Novel CNS stimulant used clinically to treat narcolepsy [Provigil, by Cephalon]
• Unlike other CNS stimulants, it has a low abuse potential
– Gold LH, Balster RL Psychopharmacology 1996, 126, 286-292.
• Currently being evaluated as a new treatment for ADHD, anticonvulsant, and treatment of cocaine and methamphetamine addiction
• Mechanism of action to promote wakefulness is currently unknown
Olivo HF, Osorio-Lozada A, Prisinzano T Tetrahedron: Asymmetry 2004, 15, 3811
Olivo HF, Osorio-Lozada A, Peeples TL Tetrahedron: Asymmetry 2005, 16, 3507
Olivo HF and Osorio-Lozada A. US Patent US Serial No. 11/460,532
modafinil synthesis
O H + H SO H
Oi . t r i f l u o r o a c e t i c a c i d
9 9 %
SO H
O
SOH
O
ii. Amycolaptosis orientalis
65%
SNH2
OO
(R,S)-modafinil
Olivo HF, Osorio-Lozada A, Prisinzano T Tetrahedron: Asymmetry 2004, 15, 3811
Olivo HF, Osorio-Lozada A, Peeples TL Tetrahedron: Asymmetry 2005, 16, 3507
Olivo HF and Osorio-Lozada A. US Patent US Serial No. 11/460,532
modafinil synthesis
O H + H SO H
Oi . t r i f l u o r o a c e t i c a c i d
9 9 %
SO H
O
SOH
OOii. Bacillus subtilis
99% ee, 68%
SNH2
OO
(S)-modafinil
SOH
O
ii. Beauveria bassiana
99% ee, 89%
SOH
OO
(S)-modafinic acid
LipasesCandida antarctica lipase-B
•Enzyme isolated originally from Antarctica
•317 amino acid residues, formula wt 33 273 Da
•3-Dimensional structure determined
•Ser105-His224-Asp187 cat. triad
•Enzyme expressed in Aspergillus oryzae
•Immobilized on acrylic resin
•Potential Applications:
• Detergents
• Pulp and paper industry
• Fine chemicals (broad substrate specificity
LipasesHydrolysis / Acylation
O
O
O
O
O
O
H 2 O
O
O
O
O H
O
H O
O
l i p a s e+
O
O H
O
O H
E t O H
E t O H
O
O E t
O
O E tH 2 O
H 2 Ol i p a s e
k 1
l i p a s e
k 2
+
+
+
+
OH
OH
AcOH
AcOH
OAc
OAcH2O
H2O+
+
lipase
k2
lipase
k1
+
+
Lipases-resolution-acetylation of alcohols
OH
OH
O
O
O
O
OAc
OAc
OH
OH
acetaldehyde
+
+lipase
k2
lipase
k1
+
+
O
Lipases-resolution-ester hydrolysis
O
O E t
O
O E t
H 2 O
H 2 O
O
O H
O
O H
E t O H
E t O H
+
+l i p a s e
k 2
l i p a s e
k 1
+
+
Reaction mechanism
Asp187
O
ON
N
His224
H
Ser105
O
H
N
Trp
H
Thr
NH R
O
O
R'
R
*RO
O
R*OHAsp
O
ON
N
His
H
Ser
O
H
N
Trp
H
Thr
NHR
O
O
Asp
O
ON
N
His
H
Asp
O
ON
N
His
H
Ser
O
H
N
Trp
H
Thr
NHR
O
O
R'
Ser
O
N
Trp
H
Thr
NHR
O
R'OH
free enzyme
acyl enzyme
Td1
Td2
R'
R 1 O H
O
R 1 O
O
R 3
H 2O
O R 1
O
ser
H 2O 2
R 1 NH
O
R 2
R 1 O
O
O H
acy l-enzym e in term ed ia teR 3-O H
R 2 -N H 2
Reaction mechanismNucleophile promiscuity
Lipases-perhydrolysis
H2O2
O
OH
O
OO
H
H2Operhydrolase
++
Bjorkling, F.; Godtfredsen, S. E.; Kirk, M. L. J. Chem. Soc., Chem. Commun. 1990, 1301
Lipases-perhydrolysis-epoxidation
H2O2O
O
O
O
OH
EtOH+
perhydrolase+
O
O H
O
O
O
OO
H
O
E tO H H 2O
H 2O 2
C an did a an tarctica
lip ase B
Ankudey EG, Peeples TL, Olivo HF Green Chemistry 2006, 8, 923-926
Ankudey EG, Peeples TL, Olivo HF Green Chemistry 2006, 8, 923-926
Alkene Epoxide Time Yield
O
40 hr 83%
O
2 hr 100%
Ph
Ph
O
28 hr 100%
O
11 hr 100%
O
H
60 hr 90%
O
5.5 hr 95%
Ankudey EG, Peeples TL, Olivo HF Green Chemistry 2006, 8, 923-926
Alkene Epoxide Time Yield
O
161 hr 73%
O
46 hr 85%
O
33 hr 81%
O
46 hr 90%
O
46 hr 86%
O
50 hr
72 hr
77%
96%
Lipases-perhydrolysis-Baeyer-Villiger oxidation
O H
O
O
O
OO
H
O
E tO H H 2O O
O
O
H 2O 2
C an did a an tarctica
lip ase B
Rios MY, Salazar E, Olivo HF Green Chemistry 2007, 9, 459-462
Ankudey EG, Peeples TL, Olivo HF Green Chemistry 2006, 8, 923-926
Cyclohexanone Caprolactone Time Yield O
O
O
6 d 80%
O
Ph
O
O
Ph
8 d 75%
O
O
O
3 d 95%
O
O
O
12 d
19 d
68%
78%
O
O
O
26 d 8%
Ankudey EG, Peeples TL, Olivo HF Green Chemistry 2006, 8, 923-926
Cyclohexanone Caprolactone Time Yield O
O
O
6 d 80%
O
O
O
8 d 75%
O
O
O
3 d 95%
O
O
O
12 d
19 d
68%
78%
O
OO
26 d 8%
Lipase-mediated proline-catalyzed epoxidation
Amrit Goswami. Tetrahedron 2007, 63, 8735.
N CO3H
O2N
NO2
N CO2H
O2N
NO2
O
lipase
UHP
1,2-dichloroethane
O
NO2O
NO2
O2NO
O2N
O2N
O
O2N
ClO
Cl
Cl OCl
Cl
O
Cl
Alkene Epoxide Yield (%) ee (%)
73
80
85
77
79
65
76
75
81
76
80
77
75
80
Lipase from Aspergillus 0%
Lipase from Candida antarctica 0%
Lipase from Candida rugosa 0%
Lipase from Mucor miehei 0%
Lipase from Pseudomonas cepacia 0%
Lipase from Pseudomonas fluorescens 0%
Lipase from Rhizopus arrhizus 0%
Lipase from Rhizopus niveus 0%
Lipase from hog pancreas 0%
N CO3H
O2N
NO2
N CO2H
O2N
NO2
O
lipase
UHP
1,2-dichloroethane
N C O 3H
O 2N
N O 2
N C O 2H
O 2N
N O 2
O
D C C
U H P
dichlo rom ethane
Chemical proline-mediated epoxidation
O
O2N
Cl
OO2N
O
O
O
Cl
O
Alkene Epoxide Yield (%) ee (%)
74
59
57
72
48
12
00
00
00
00
00
00
A Osorio-Lozada and HF Olivo. Organic Letters 2008, 10, 617.
Chemo-enzymatic synthesis of indene
• Microorganisms in Organic Synthesis
Synthesis of epibatidine
Synthesis of modafinil
Synthesis of chiral building blocks
• Enzymes in Organic Synthesis
Resolution of alcohols and acid derivatives
Perhydrolysis of carboxylic acids
Epoxidations
Baeyer-Villiger oxidations
New chiral auxiliares
Summary
Some past members…
Past and Present members
Michael S. Hemenway (PhD ’00)
Francisco Velazquez (PhD ’03)
Dr. Srinivas Pusuluri
Dr. Henrique Trevisan
Dr. Yolanda Rios
Dr. Moises Romero
Dr. Nury Hernandez
Dr. Efrain Barragan
Dr. Ricardo Tovar
Dr. Adrian Ochoa
Dr. Patricia Mendez
Dr. Silvia Balbo
Dr. Suresh Wagmode
Dr. Lemuel Perez
Dr. Luis Hernandez
Dr. Veronica Rivas
Rodolfo Tello (PhD ‘08)
Antonio Osorio (PhD ’08)David A. Colby
Seth Sarduy
Mathis Hodge
Sena Dzakuma
Esdrey Rodriguez
Claudia Rojas
Laura Munive
Dr. Victor Gomez
Ernane De Souza
Gerardo Perez
Alvin De Gall
Moman Nazir
Acknowledgments
Horacio F [email protected]