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Engineering microorganisms for synthesis of medicinally
important molecules
Innovación Para el Desarrollo Sostenible, Mérida. Mayo 2014

Engineering microorganisms for synthesis of medicinally important molecules

N
N
O H C
H
O H
OO
O A cH
N
H
N
H
O H
M e O
O
M e O
H O
O
H O
N
M e N
O
O
C O 2 M e
N
O
O
N
H
H
H
OH
O
H
H
HO
O
O
H
H
H
H
O
O H
OO H O H
O H
O H
O H
O
O
OH
H
H
H
OH
N
H
H
OH
H3C
N
O
O
H
Me
O
H
H
O
O
O
O
H
O HH
M e O
O
N H
O
O
M e O
H C l
O
OO H
H O
O H
O H
O H
O
O
OH OH
O
O
NO2
MeO
O
O
O
OO
O O
M e
M e
H H
O
NH2
MeO
OMe
MeO
N
H
N
O
P
O
O
HO
H
O
HO
MeO
OH
OH
OMe

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
H
HO
O
H
H
H
O
OH
H
H
H
O
O
H
H
H
O
OH
OH
O
OHC
H
H
H
HO
OH
O
progesterone estrone testosterone
cortisone aldosterone

Natural sources of steroids
Dioscorea 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
H
A 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 H
M e
p ro g e s te ro n e a n d ro s te n e d io n e e th is te ro n e
te s to s te ro n e m e th y lte s to s te ro n e
h a lo te s tin
2 1 -h y d ro x y p ro g e s te ro n e
Laboratorios Syntex SA (1944)
O
O
O
O
?

Biotransformations
O
O
O
O
HO
Murray and Patterson, J. Am. Chem. Soc. 1952, 74, 1871

Biotransformations
O
O
O
O
HO
O
O
O
O
H O
O
O
O H
O H
O
c o r t is o n e
R h iz o p u s a r r h iz u s

C l
P . p u t id a
m u t a n t s t r a in
C l
O H
O H
David T. Gibson
Tomáš Hudlický
Jack Rosazza

NH
O
O
OH O
OH
OH
OH
HO
OH
OH
X
X
X
O
O
N
OH OH
OH
OH
NH
O
O
H
N
HO
HO
OHH
O
HO
HO
NMe
microorganism
kifunensin
pancratistatin morphine
trihydroxyheliotridane

OH
O
O
O
HO
O
OH
O
OMe
OH
OO
N
HO
erythromycin
N
H
N
O
O
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!
H
N
N Cl
epibatidine
John W. Daly (March 5, 2008)Epipedobates tricolor

Can microorganisms help us shorten the route to
epibatidine?
N
R
N
R
OH
microorganism
H
N
N C l
N
R
O H
N
R
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 ia b a s s ia n a
4 5 - 7 0 %
NCOPh NCOPh
HOBeauveriabassiana
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
O
i v . B . b a s s i a n a
5 6 % , 2 2 % e e
N
O
O H
H
N
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 o
5 - 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 H
1 0 0 C , 3 3 %
x . 6 N H C l
1 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 ia s u lf u r e s c e n s
A T C C - 7 1 5 9
N
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 lla 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.

modafinil
S
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 S
O H
O
i. t r if lu o r o a c e t ic a c id
9 9 %
S
O H
O
S
OH
O
ii.Amycolaptosisorientalis
65%
S
NH2
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 S
O H
O
i. t r if lu o r o a c e t ic a c id
9 9 %
S
O H
O
S
OH
OO
ii.Bacillussubtilis
99%ee,68%
S
NH2
OO
(S)-modafinil
S
OH
O
ii.Beauveriabassiana
99%ee,89%
S
OH
OO
(S)-modafinicacid

Lipases
Candida 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

Lipases
Hydrolysis / Acylation
O
O
O
O
O
O
H 2 O
O
O
O
O H
O
H O
O
lip a s e
+
O
O H
O
O H
E t O H
E t O H
O
O E t
O
O E t
H 2 O
H 2 O
l i p a s e
k 1
l i p a s e
k 2
+
+
+
+
OH
OH
AcOH
AcOH
OAc
OAc
H2O
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
+
+
lip a s e
k 2
lip a s e
k 1
+
+

Reaction mechanism
Asp187
O
O
N
N
His224
H
Ser105
O
H
N
Trp
H
Thr
NH R
O
O
R'
R
*RO
O
R*OH
Asp
O
O
N
N
His
H
Ser
O
H
N
Trp
H
Thr
NHR
O
O
Asp
O
O
N
N
His
H
Asp
O
O
N
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'

R1 OH
O
R1 O
O
R3
H2O
O R1
O
ser
H2O2
R1 N
H
O
R2
R1 O
O
O H
acyl-enzym e interm ediate
R3-OH
R2-NH2
Reaction mechanism
Nucleophile promiscuity

Lipases
-perhydrolysis
H2O2
O
OH
O
O
O
H
H2O
perhydrolase
+
+
Bjorkling, F.; Godtfredsen, S. E.; Kirk, M. L. J. Chem. Soc., Chem. Commun. 1990, 1301

Lipases
-perhydrolysis
-epoxidation
H2O2
O
O
O
O
O
H
EtOH+
perhydrolase
+
O
OH
O
O
O
O
O
H
O
EtOH H2O
H2O2
Candida antarctica
lipase B
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%

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
OH
O
O
O
O
O
H
O
EtOH H2O O
O
O
H2O2
Candida antarctica
lipase B
Rios MY, Salazar E, Olivo HF Green Chemistry 2007, 9, 459-462

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%

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
O
O
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
NO2
O
NO2
O2N O
O2N
O2N
O
O2N
Cl
O
Cl
Cl O
Cl
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 CO3H
O2N
NO2
N CO2H
O2N
NO2
O
DCC
UHP
dichloromethane
Chemical proline-mediated epoxidation

O
O2N
Cl
O
O2N
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

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

Horacio F Olivo
horacio-olivo@uiowa.edu

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Engineering microorganisms for synthesis of medicinally important molecules

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Engineering microorganisms for synthesis of medicinally important molecules