1 1 enzymes in organic media tahir rana university of ottawa september 25th 2008
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Enzymes in Organic Media
Tahir RanaUniversity of Ottawa September 25th 2008
22
Outline
Structure and Function Applications of Enzymes Limitations of Enzymes in Aqueous Media Concerns Applications of Enzymes in Organic Media Total Synthesis of Fredericamycin A
33
What are Enzymes ?
Enzymes are proteins
Enzymes catalyze reactions
H2N CH C
R
OH
O
H2N CH C
R
OH
O
R= amino acid side chain
-Phe-Ala-Gly-Tyr-Lys-Ala-
Structure And Function
44
Enzyme Structure
Primary Structure – order of amino acids
Secondary Structure - α-helix, β-sheet
Tertiary Structure - arrangement in 3D
Quaternary Structure- interaction of subunits
-Gly-Ala-Phe-Gly-His-Tyr-
Sakuraba, H. et al. J. Biol. Chem. 2003, 361, 278, 10799-10806.
Structure And Function
55
Catalytic Scheme
Structure And Function
66
Factors Involved in Enzymatic Catalysis
Increase in local concentration
Positioning and enhancement of active site functional groups
Specificity
Introduction of strain into substrate
OH O
O
OH1 000 000 000
Faster
O O
OH
O
+ OH
O
25Cys
159His
S HN NH
Cys His
SHN NH
Thiol Protease
HydrophobicBindng Pocket
Chymostrypsin
NH
OR
RActive Site
Structure And Function
77
Examples: Asymmetric Aldol
Wong, C,H.; Gilsen, H. J. Am. Chem Soc. 1994, 164, 8422-8423.Wong, C.H. Liu, J. J. Angewantde Chemie. 2001, 114, 1462-1465.
O+
O
HO
DERA OH
HO
O
S
N
O
O R
OH
OO OHEpothilone A
O+
O DERA OHO+
OH O
Applications of Enzymes
DERA – Deoxyribose Aldolase
88
Industrial Examples
O
OO
O
OOH
alcohol-NADHoxidoreductase N
RiboseADP
H H NH2
O
NADHO
O NN
H2N
O
Eli Lilly - LY 300164
Anderson, B. et al. J. Am. Chem. Soc. 1995, 117, 12358-12359.Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations. Wiley-VCH, 2005, 117-121.
Applications of Enzymes
99
Industrial Examples
O
O
NH2
subtilisin OH
O
NH2
Coca-Cola - AspartameO
NH
H3N
O
O
O
O
Ricks, E.; Estrada-Valdes, M; Iacobucci, G. Biotech. Prog. 1992. 8, 197-203.Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations, Wiley-VCH. 2005.
Applications of Enzymes
1010
Amide Hydrolysis
O
NH2REnzyme in H2O
PhysiologicalpH and Temp.
O
OHR
O
NH2R
80 % H2SO4
100 °C, 12-18 hrs
O
OHR
O
NH2R
25 % NaOH
100 °C, 9-12 hrsAq. workup
O
OHR
Applications of Enzymes
1111
Limitations of Aqueous Enzymology
Solubility of non-polar substrates
Polymerization of phenols
R = alkyl
horseradishperoxidase
H2O2
Aqueous
Dimers/Trimers
R
OH
R
OH R
HO
Bruno, F.; Ayyagari, S.; Akkara, J. Trends in Biotechnology. 1999, 17, 67-73. Reihmann, M.; Ritter, H. Syn. Of Pol. Using Peroxidases. Adv. Poly. Sci. Springer-Verlag. 2006, 194, 1-49.
1212
Thermal Inactivation in Aqueous Media
Reversible:
Changes in higher order structure
Irreversible: Molecular AggregationDeamidation
R NH
HN
NH
O
O
OR
NH2
O
R NH
NH
OO
RN
O
O
NH3
R NH
HN
NH
O
O
OR
OH
O
H2O H2O
R NH
O
O
OH
NH
O
O
HN
R
Klibanov, A.; Ahern, T. Methods of Biochemical Analysis, 1988, 33, 91-128.
Limitations
1313
Domination of Hydrolysis
Water is in excess
Cannot use other nucleophiles
O
R' OR''
O
R' OH
Enzymes
Aqueous
O
R' OR''
O
R' NR2
Enzymes
Aqueous,NHR2
X
O
R' OR''
O
R' OR
Enzymes
Aqueous,ROH
X
O
R' OR''
O
R' SR
Enzymes
Aqueous,RSH
X
Limitations
1414
The Solution – Organic Solvents
Increased solubility of non-polar substrates
Bruno, F.; Ayyagari, S.; Akkara, J. Trends in Biotechnology. 1999, 17, 67-73. Reihmann, M.; Ritter, H. Syn. Of Pol. Using Peroxidases. Adv. Poly. Sci. Springer-Verlag. 2006, 194, 1-49.
Overcoming Limitations
R = alkyl
horseradishperoxidase
H2O2
Aqueous
High molecularweight polymers
R
OH
R
OH R
HO
n
15
0
20
40
60
80
100
0 20 40 60
Time (min)
% A
ctiv
ity
Enzyme in Heptanol
Enzyme in Water
15
Suppression of Thermal Inactivation in Organic Sol.
% Activity of Lipase at 100 °C
Klibanov, A.; Zaks, A. Science. 1984, 224, 1249-1251.
Overcoming Limitations
O
Oheptanol
Lipase
O
ORn=5
O
OWater
Lipase
O
OHR
1616
Opportunity for Synthesis
O
R' OR''
O
R' OR
Enzymes
Organic Sol.ROH
O
R' OR''
O
R' NR2
Enzymes
Organic Sol.NHR2
O
R' OR''
O
R' SR
Enzymes
Organic Sol.RSH
Overcoming Limitations
1717
Recap - Advantages of Organic Solvents
Increased solubility of non-polar substrates
Suppression of Thermal Inactivation
Opportunity for synthesis
Overcoming Limitations
1818
Outline
Structure and Function Applications of Enzymes Limitations of Aqueous Enzymology Concerns Regarding Enzymes in Organic
Solvents Applications of Enzymes in Organic Media Total Synthesis of Fredericamycin A
1919
Concerns
Structural Integrity
Mechanistic Integrity
Diminished Activity
2020
Structural Integrity
% Alpha Helix Content of Subtilisin
Klibanov, A.; Griebenow, K. J. Am. Chem. Soc. 1996, 118, 11965-119700.
Concerns Addressed
0
5
10
15
20
25
30
60 % MeCN + 40 % Water (v/v) Water Neat MeCN
Solvent
Alp
ha-
Hel
ix C
on
ten
t (%
)
2121
Structure of Subtilisin in Water and Acetonitrile C Backbone Trace Active Site (Asp-32,His-64,Ser-221)
Heavy lines = MeCNLight lines = water Klibanov, A. et al. Proc. Nat. Acad. Sciences. 1993, 90, 8653-8657.
Concerns Addressed
2222
Mechanism of Transesterification
O
R'O RChymotrypsin +
Organic
Solvent
Chaterjee, S.; Russell, A. Enzyme Microb. Technol. 1993, 15, 1022-1029.
R'OH
Concerns Addressed
2323
Mechanism of Transesterification
Chaterjee, S.; Russell, A. Enzyme Microb. Technol. 1993, 15, 1022-1029.
Concerns Addressed
2424
Mechanistic Integrity
Ping Pong Mechanism
Transesterification in Organic Solvents Ester Hydrolysis in Water
Conclusion: Mechanism is the same
(1) Chaterjee, S.; Russell, A. Enzyme Microb. Technol. 1993, 15, 1022-1029.(2) Klibanov, A. Trends Biochem. Sci. 1989, 14, 141-144.
Concerns Addressed
2525
Diminished Activity
Enzymes have reduced activity in dry organic solvents
Due to lack of: a) conformational mobility
b) transition state stabilization
c) entropy
Klibanov, A. Trends In Biotech. 1997. 15, 97-101.
Concerns Addressed
26
0.0001
0.001
0.01
0.1
1
10
0.1 1 10 100
% Water (v/v)
Ra
te (
mM
/min
)
Ether tAmyl Alcohol
Ethyl Acetate
26
Effect of Water on Activity Activity can be recovered
Klibanov, A. J. Biol. Chem. 1987. 263, 8017-8021.
Enzyme Activity as a Function of Water Content
Concerns Addressed
OHSolvent
Oxidase H
O
2727
Concerns Addressed
Structurally intact
Act by the same mechanism
Activity can be recovered
2828
Applications
(1) Wong, C-H.; Koeller, K. Nature. 2001. 409, 232-241(2) Klibanov, A.; Kirchner, G.; Scollar, P. J. Am. Chem. Soc. 1985. 107, 2072-2076.
Problem: Max Conversion = 50 %
O
R1 O H+
R2 R3
OH O
R1 O
R2 R3
O
R1 O
R2 R3
Lipase
Resolution R2 R3
OH
+
Traditonal Resolution
Aqueous
O
R1 O
R2 R3
+
R1, R2 and R3 = different alkyl groups
Resolution in Organic Media
O
R1 O H+
R2 R3
OH OH
R2 R3
O
R1 O
R2 R3
+Lipase
ResolutionEther
Applications in Org. Media
2929
Resolution: Meso Diols
OAc
OAc
LiOH·H2O
MeOH
OH
OH
% Yield: 19
% ee: >97
OH
OH
(±) + meso
(48:52)
OO
H
OAc
OAc
OAc
OH
OH
OH
% Yield: 22 48 22
% ee: >98 >96 >98
Lipase
Hexanes,RT. 10 hrs
Kim, M.J.; Lee, S. Synlett. 1993. 767-768.
Applications in Org. Media
3030
60 % OverallYield
Kim, M.J.; Lee, S. Synlett. 1993. 767-768.
OAc
OH
OAc
OO
O2N
DEAD, PPh3,
pNBA
OAc
OO
O2N
LiOH·H2O
MeOH
OH
OH
% Yield: 40
% ee: >95
Applications in Org. Media
3131
Applications: Desymmetrization
Loss of one or more symmetry elements
Potential for 100 % conversion
Gotor, V. et al. Organic Letters. 2007. 9, 4203-4206.
O O
OLipase
Dioxane
NH2 NH2 N HN O
O
72 % yield and 96 % ee
H2
+
Applications in Org. Media
32
Applications: Total Synthesis of Epoxyquinols A and B
O
O
O
OH
CH3
HO O
OO
CH3
(-) Epoxyquinol A
O
O
O
OH
CH3
HO O
OO
CH3
(-) Epoxyquinol B
Applications in Org. Media
33
Retrosynthesis
O
OH
O
O
O O
OAc
O O
O
O
O
AcOHO
O
O
O
HOHO
H O
O
H
Mehta, G.; Islam, K. Tett. Lett. 2004. 45, 3611-3615.
Applications in Org. Media
O
O
O
OH
CH3
HO O
OO
CH3
O
O
O
OH
CH3
HO O
OO
CH3
+
34
Desymmetrization Step
O
O
O
AcOHO
O
O
O
HOHO
Lipase PS 30, vinyl acetate
tBuOMe, 0 C, 6 h
82 % yield> 99 % ee
Applications in Org. Media
Mehta, G.; Islam, K. Tett. Lett. 2004. 45, 3611-3615.
3535
Outline
Structure and Function Applications of Enzymes Limitations of Aqueous Enzymology Concerns Applications of Enzymes in Organic Media Total Synthesis of Fredericamycin A
3636
Total Synthesis of Fredericamycin A
Isolated from Streptomyceus griseus Antitumor activity 7 Total Syntheses; 5 Racemic, 2 Asymmetric
HN
O
O
O
HO
OMe
OH
HO
O
O
AB
CDEF
3737
Retrosynthesis of 1st Asymmetric Synthesis
HN
O
OO
HO
OMe
OHHO
O
O
HN
O
MeOOMe
MeO
OHC
OMe
OMeHO
O
O
O
MeOOMe
OMe
OO +
MeO
N
MeO MeO
O
O SPhO
AB
CDEF
N
MeO MeO
O
CpCOO
N
OMeCO2Et
Kita. Y. et al. J. Am. Chem. Soc. 2001. 123, 3214-3222.
Total Synthesis of Fredericamycin A
3838
Installation of Spiro Center
NB
O
H PhPh
N
MeO MeOO
BH3Me2SN
MeOMeOHO
92 % 74 % ee
tBuO2HVO(acac)2 N
MeOMeOHO
O
75 % 74 % ee
N
MeOMeO
O
CpCOO
1(S) CpCOOH
PPh3, DEAD
95 % 74 % de
N
MeO MeO
OBF3
BF3OEt
CH2Cl20 °C
CpCOO
N
MeOMeO
O
CpCOO
80 %, 95 % de
Fredericamycin A
33 Steps0.075 % Overall Yield
F E D
Kita. Y. et al. J. Am. Chem. Soc. 2001. 123, 3214-3222.
Total Synthesis of Fredericamycin A
N
MeO MeO
O
CpCOO
3939
Retrosynthesis of 2nd Asymmetric Synthesis
HN
O
OO
HO
OMe
OHHO
O
O
N
MeO
OOMe
MeO
OMe
O
OO
Si
SPh
AB
DEF
N
MeOMeO OHHO
HN
OCO2Et
Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.
F E D
C
B
A
N
MeOMeO
O
O
F E D
Total Synthesis of Fredericamycin A
4040
Synthesis of DEF Ring System
HN
OCOOEt
OCH3
H3C CH3
BF4
DCMN
MeOCOOEt
10 % NaOH 70 %
LDA
O
N
MeOCOEt
OO
72 %
NaH
THF
N
MeO O O
75 %
DDQ
Benzene
N
MeO OH O
76 %
N
MeO MeO OMe2SO4
NaOHBu4N+ Br-
F E D
65 %
Total Synthesis of Fredericamycin A
Clive, D. J. of Heterocyclic Chemistry. 1987, 9, 804-807.
4141
Synthesis of DEF Ring System
N
MeO MeO O
Ph3P+Me Br-, tBuOK N
MeO MeO
THF
83 %
NaOH, H2O2
N
MeO MeO OH
92 %
BH3-THF
OI
O
AcO OAcOAc
N
MeO MeO O
61 %
N
MeOMeO NN
MeON
OMe
NH2
1:1 mixture ofdiastereomers
Total Synthesis of Fredericamycin A
Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.
4242
Synthesis of DEF Ring System
N
MeO MeO NN
MeO
COMe
35 %95 % de(2 steps)
Difficult separation of acyl hydrazone 4.5 % overall yield (from pyridone) Approach abandoned
N
MeO MeO NN
MeO
LDA
AcCl
Total Synthesis of Fredericamycin A
Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.
4343
Synthesis of DEF Ring System- 2
HN
O O1. MeI, Ag2CO3
2. Me3SiOTf, Et3NNBS
N
MeO OBr
83 %
1. NaOMe, MeOH
2. Me2SiCl2, ImidazoleBenzene
HN
O OH R R
87 %
Lipase
Aqueous
HN
O OH R CO2H
HN
O HOCHO
O
X HN
O OH CO2Me
1. NaH
Me3Si
CO2MeCO2Me
2. NaBr, pTSA
HN
O O R R
TMS
81 % R=CO2Me
Total Synthesis of Fredericamycin A
R=CO2Me
Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.
4444
Solution ?
Use the synthetic ability of enzymes in organic solvents
Total Synthesis of Fredericamycin A
4545
Synthesis of DEF Ring System - 2
OOO
EtO
N
MeOMeO O OH
N
MeOMeO OHO
OOO
O
X86 % Yield97 % ee
DEF
1. MeI, Ag2CO3
2. LiAlH4
N
MeOMeOHO OH
61 %
HN
O OH R R
R=CO2Me
Total Synthesis of Fredericamycin A
Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.
N
MeOMeO OHO
O
4646
Total Synthesis – Fredericamycin A
N
MeOMeO O OH
OO
N
MeOMeO OHOTBS1. TBSOTf, Na2CO3
2. K2CO3, MeOH
N
MeOMeO OTBS
DMP
O
1. MeLi
2. TBAF
N
MeOMeO
OH
HO
N
MeOMeO
OOxalyl Chloride
DMSO, Et3N
O
96 % (from alcohol)
30 % Yield (from pyridone)
Total Synthesis of Fredericamycin A
Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.
4747
Total Synthesis – Fredericamycin A
N
MeOMeO OO
SPh
LiHMDS
OBn
OMeOMe
Cl
O
-78 °C
N
MeOMeO OO
SPh
O OBn
OMeOMe
F E D
67 %
A
1. Co2(CO)8
2. BCl3N
MeO MeO OO
O
SPh
HO
OMe
85 %
OMe
N
MeOMeO
OMe
OMe
SPh
O
O
OSiO
tButBu1.Me2SiCl2, Et3N,
Chloranil
2. (tBu)2Si(OTf)2
Et3N, DMF
81 %
BC
DEF
A
1. LiHMDS
2. DMP
N
MeOMeO OO
O
SPh
BnO
OMe
73 %
F E D
A OMe
Total Synthesis of Fredericamycin A
Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.
4848
Total Synthesis – Fredericamycin A
N
MeO MeO
OMe
OMe
SPh
O
O
OSiO
tButBu
mCPBA
O
76 %
N
MeO MeO
OMe
OMe
O
O
OSiO
tButBu
OCOCH2Clcat. pTSOH
OEtOCl
O
86 %
PPh3Br
1. SeO2
2. BuLi, -78 °C
3. BBr3, H2OHN
O
OO
OMe
OHHO
O
OHO
35 %
Et3N; (tBu)2Si(OTf)2;
MeI
HN
OMeO
OMe
OMe
O
O
OSiO
tButBu
OCOCH2Cl
69 %
28 Linear Steps0.75 % Overall Yield
Total Synthesis of Fredericamycin A
Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.
4949
Comparison of Syntheses
Lewis Acid: 4 steps to establish chirality at spiro center
Enzymatic: 1 step to establish chirality at spiro center
Enzymatic: 28 yield steps, 0.75 % yield, Lewis Acid: 33 steps, 0.075 %
Total Synthesis of Fredericamycin A
5050
Summary
Enzymes are valuable tools for organic synthesis
Enzymes can be used in organic solvents
There are clear advantages to using enzymes in organic media
Application to the total synthesis of Fredericamycin A
5151
AcknowledgementsDr. Robert BenTaz CheemaPawel CzechuraLiz von MoosJohn TrantJennifer ChaytorSandra FerreiraWendy CampbellRuoying GongRoger TamJackie TokarewTaline BoghossianDr. Michael SouwehaDr. Mathieu Leclere
5252
Enzyme Preparations
Enzymes are insoluble in organic solvents
Enzyme powders
Suspension of enzymes in bulk solvent or on solid supports
Covalent modifications, e.g. PEG; surfactants
5353
pH Memory Affect
Rate of transesterification with pH adjusted subtilisin 75x that of bottled enzyme
Enzymatic activity in organic solvent depends upon pH of the last aqueous solution enzyme was exposed to.
5454
Structural Integrity of Enzymes
Explanation ? Enzymes possess reduced mobility in pure organic media
Evidence: Structurally rigid e.g. decrease in motion of lipase Tyr 123
acetonitrile than in water
Conclusion: Denaturation is thermodynamically favourable, yet
conformational flexibility is lacking
Ref. Burke + Klibanov
5555
Total Synthesis of Fredericamycin A
5 Racemic
1. Kelly 1986
2. Clive 1992
3. Rao 1993
4. Julia 1993
5. Boger 1995
2 Asymmetric
1. Kita 2001
2. Kita 2005
5656
Reversal of Chemoselectivity
Ref. Ebert
Conditions Ratio of Products A B
Benzene, tAmyl Alcohol 7 1
Pyridine 8 1
tAmyl Alcohol w/lipase 10 1
Pyridine w/ lipase 1 10
Benzene w/ lipase 3 1
Benzene (2 % pyridine) w/ lipase
3 1
NH
O
H2N
OHNH
O
HN
OHO
Cl
O
NH
O
H2N
O
O
A B
++
Conditions
5757
Reversal of Regioselectivity
Conditions Ratio of Products
A B
Acetonitrile w/ KCN 2 1
Toluene w/ lipase and nBuOH
2 1
Acetonitrile w/ lipase and nBuOH
1 2
O
OO
O
n=5
HO
O
O
n=5
O
HOO
n=5
Conditions
A B
+
Ref.
5858
Reversal of Regioselectivity
OO
O
O
SerOH
Toluene
SerOHOO
O
O
Acetonitrile
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