Catalysis of the Diels Alder Reaction: in vivo and in vitro
Download
Report
Transcript Catalysis of the Diels Alder Reaction: in vivo and in vitro
Biomolecular Catalysis of Diels-Alder
Reactions
Organic Seminar
March 7th, 2002
Lisa Jungbauer
Outline
Introduction
I.
The Diels-Alder Reaction
Biomolecule Catalysts of Diels-Alder Reactions
II.
Catalytic Antibodies (Abzymes)
Ribozymes (Catalytic RNA)
III.
Biocatalysis of Diels-Alder Reactions in Biosynthesis and
Organic Synthesis
IV.
Conclusions
2
The Diels-Alder Reaction
OCH3
OCH3
NC
CH
CN
CH
+
CH2
CH2
Diene
(e- rich)
Dienophile
(e- poor)
Hetero-Diels Alder
Retro-Diels Alder
4+2 Cycloaddition
Concerted
Stereospecific
Inverse Electron Demand Diels-Alder
3
Regiochemistry
OCH3
OCH3
NC
CH
CN
CH
+
CH2
CH2
NC
H3CO
LUMO
NC
H3CO
Major
Regioisomer
of Product
HOMO
H3CO
NC
“ortho”
H3CO
NC
Diene
Dienophile
4
Figure adapted from Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472.
Stereochemistry
Enantiotopic Mirror Plane
endo approach
endo approach
NC
OCH3
OCH3
CN
NC
OCH3
NC
exo approach
exo approach
OCH3
CN
OCH3
OCH3
OCH3
CN
NC
CN
OCH3
5
Figure adapted from Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472.
Products of the Diels-Alder Reaction
OCH3
TS4
TS3
DDG‡
TS2
TS1
DG4‡
CN
OCH3
DG3‡
DG2‡
DG1‡
SM
OCH3
NC
CH
+
CH2
CH
CH2
DG‡ = DH‡ - T DS‡
DS‡ : Rotational and translational entropy
DH‡: Reactivity of substrates
P4
P3
P1
P2
CN
OCH3
CN
OCH3
CN
6
Catalysis
DG‡cat < DG‡uncat
Blackburn, G. M.; Datta, A.; Denham, H.; Paul Wentworth, J. Adv. Phys. Org. Chem. 1998, 31, 249-369.7
Bartlett, P. A.; Mader, M. M. Chem. Rev. 1997, 97, 1281-1301.
Catalysis of Diels-Alder Reactions
Typical Methods for catalysis are
Lewis Acids
ZnCl2, AlCl3, SnCl4, TiCl4, Et2AlCl
Medium Effects (the hydrophobic effect of aqueous solvent) and
pressure also facilitate the reaction
8
Stereochemical Outcome of
Diels-Alder Reactions
Stereoselectivity is influenced by
Chiral auxiliaries
Chiral metal complexes
Lewis acid catalysts typically enhance regioselectivity and
stereoselectivity
Utility of Diels-Alder reactions increases with ability to
influence stereochemical outcome
Catalysts that direct stereoselectivity are valuable tools
9
Biomolecules are Suitable Catalysts for
Diels-Alder Reactions
The Diels-Alder reaction….
Biocatalysts….
1) Large activation entropy
(-30 to -40 cal K-1 mol-1)
1) Compensate for loss in entropy by
binding the substrates in an active site
2) Potential to form stereoisomeric
products
2) Inherent chirality of biomolecules
may direct stereoselectivity
3) No enzymatic example of a
Diels-Alder biocatalyst
3) Expand the limits of biocatalysis
from enzymology to organic synthesis
4) Challenge for catalysis since
there are no ionic intermediates
and little charge separation in
the transition state
4) The binding site should recognize the
transition state based on shape and
structure
Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M.-T. M. J. Am. Chem. Soc. 1989, 111, 9261-9262.
Chen, J.; Deng, Q.; Wang, R.; Houk, K. N.; Hilvert, D. ChemBioChem 2000, 1, 255-261.
10
Outline
Introduction
I.
The Diels-Alder Reaction
II.
Biomolecule Catalysts of the Diels-Alder Reaction
Catalytic Antibodies (Abzymes)
•
•
•
•
Antibody structure, function and production
6 examples of Diels-Alder catalytic antibodies
Limitations
Future directions
Ribozymes (Catalytic RNA)
III.
Biocatalysis of the Diels-Alder Reaction in Biosynthesis
and Organic Synthesis
IV.
Conclusions
11
Brief History of Catalytic Antibodies
1947: Enzyme catalysis achieved by stabilization of the transition state through
binding (Linus Pauling)
1969: Catalytic antibodies were proposed (William P. Jencks)
1986: 1st successful catalytic antibody (reported independently by Lerner et al.
and Schultz et al.)
1989: 1st Antibody catalysis of the Diels-Alder (Hilvert, then Schultz)
1995: First synthetic application: antibody catalysis used to set
stereochemistry in total synthesis of -Multistratin (pheromone)
2002: 16 years of development and detailed studies
Application in pharmaceuticals, total synthesis
One antibody commercially available from Sigma
(aldolase antibody 38C2, Aldrich #47,995-0, $108.70/10 mg)
12
Keinan, E.; Lerner, R. A. Isr. J.Chem. 1996, 36, 113-119. Hasserodt, J. Synlett 1999, 12, 2007-2022.
Many Types of Catalytic Antibodies
Sigmatropic rearrangements
Metal insertion
Hydrolysis
Difficult chemical transformations
and rerouting reaction outcomes
Syn-eliminations
Ester
Exo-Diels-Alder cycloadditions
Amide
6-endo-tet ring closures
Phosphate ester
Glycoside
Conversion of enol ether to
cyclic ketal in water
Redox reactions
Aldol reactions
Michael reactions
Acyl transfer
Cationic olefin cyclization
Over 100 different reactions have been accelerated by antibodies
Hilvert, D. Annu. Rev. Biochem. 2000, 69, 751-793. Stevenson, J. D.; Thomas, N. R. Nat. Prod. Rep.
2000, 17, 535-577.
13
Antibody Structure
Legend
V = variable region
C = constant region
H = heavy chain
L = light chain
CDR =
complementarity
determining region
Davies, D. R.; Chacko, S. Acc. Chem. Res. 1993, 26, 421-427. Hilvert, D.; MacBeath, G.; Shin, J. A. The
Structural Basis of Antibody Catalysis; Hecht, S. M., Ed.; Oxford University Press: New York, 1998,
14
pp 335-366.
Antibody Functions
Antibodies are involved in the immune response, one of the
most important biological defense mechanisms
Antibodies are rapidly produced as advanced, complex
receptors to tightly bind potentially harmful foreign substances
In order to recognize an enormous range of molecules, the
immune system is capable of generating an incredibly diverse
library of antibodies
Blackburn, G. M.; Datta, A.; Denham, H.; Paul Wentworth, J. Adv. Phys. Org. Chem. 1998, 31, 249-369.15
Immunological Methods to Generate Catalytic
Antibodies
Hapten
Hapten = The small
organic molecule to
be bound by the
antibody (transition
state analog, TSA)
6-10
weeks
Affinities for hapten:
(KD=10-4 to 10-10 M)
Hapten
Anti-hapten
antibodies
Screen for
catalytic
activity
16
Burton, D. R. Acc. Chem. Res. 1993, 26, 405-411. Hasserodt, J. Synlett 1999, 12, 2007-2022.
The Diels-Alder Reaction Transition State
Highly ordered cyclic overlap
of electrons
HOMOdiene
Product-like
Boat-like conformation
LUMOdienophile
Partial formation of new
sigma and bonds
17
Hapten Design Strategies for
Diels-Alder Reactions
Transition state analog
Shape complementarity
Must avoid product inhibition
Diels-Alder adduct undergoes further transformation
Conformationally restricted analogs
Conformationally flexible analogs
Hilvert, D. Annu. Rev. Biochem. 2000, 69, 751-793. Hilvert, D.; Hill, K. W.; Nared, K. D.;
Auditor, M.-T. M. J. Am. Chem. Soc. 1989, 111, 9261-9262.
18
Diels-Alder Catalyst Antibody 1E9
O
Cl
S
O
Cl
O
S
+
Cl
O
N Et
Cl
Cl
Cl
2. [ox]
N
Cl
Cl
Et
O
Cl
1. -SO2 Cl
N Et
Cl
O
Cl
O
Cl
Cl
O
Cl
O
Cl
O
Hapten
Cl
O
N
Cl
1st biocatalyst of the Diels-Alder
reaction
Exploited the chemical and
conformational differences between
transition state and product
(CH2)CO2-
O
Hapten mimics the
endo transition state
19
Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M.-T. M. J. Am. Chem. Soc. 1989, 111, 9261-9262.
Diels-Alder Catalyst Antibody 1E9
Uncatalyzed rxn. in H2O
H‡= 15.5 kcal mol-1
S‡= -21.5 cal K-1 mol-1 (e.u.)
Antibody 1E9 catalyzed rxn.
H‡= 11.3 kcal mol-1
S‡= -22.1 cal K-1 mol-1 (e.u.)
Crystal structure of
Fab fragment of 1E9
kuncat = 0.013 M-1min-1
kcat = 13 min-1
KM = 2.4 mM (diene) ;
29 mM (dienophile)
kcat/kuncat = 1000 M
Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M.-T. M. J. Am. Chem. Soc. 1989, 111, 9261-9262.
20
Jian Xu, Q. D., Chem, J., Houk, J. N., Bartek, J., Hilvert, D., Wilson, I. A. Science 1999, 286, 2345-2348.
Diels-Alder Catalyst Antibody 39-A11
O
O
+
N
H
N
N
CH3
O
O
O
HN
O
O
O
-
O-
NH
O
O
N
H
CH3
O
O
O
O
NH CH3
O
N
O
-
O
O
H
NH
O
O
O
Hapten
O
N
O
O
-
O
NH
O
Kinetic Parameters for
Antibody 39-A11
NH
CH3
Locked boat-like
conformation
kuncat = 1.9 M-1 s-1
kcat = 0.67 sec-1
KM = 1.2 mM (diene);
0.74 mM (dienophile)
kcat/kuncat= 0.35 M
O
Braisted, A. C.; Schultz, P. G. J. Am. Chem. Soc. 1990, 112, 7430-7431.
21
Binding Site of 1E9 vs. 39-A11
1E9
Cl
Cl
39A11
Buried surface of
hapten upon
binding (%)
83.6
65.5
Cavities between
hapten and
antibody (Å3)
0
117
121
85
Hapten
Cl
Cl
Cl
1E9
O
N
Cl
(CH2)CO2-
O
39-A11
Van der Waals
contacts
O
Hapten
O
NH
CH3
N
O
O
-
O
NH
O
O
Jian Xu, Q. D., Chem, J., Houk, K. N., Bartek, J., Hilvert, D., Wilson, I. A. Science 1999, 286, 2345-2348.
Hilvert, D. Annu. Rev. Biochem. 2000, 69, 751-793. Chen, J.; Deng, Q.; Wang, R. Houk, K. N.; Hilvert, D.
22
ChemBioChem 2000, 1, 255-261. Golinelli-Pimpaneau, B. Curr. Op. Struct. Biol. 2000, 10, 697-708.
Diels-Alder Antibody 22C8
+
CON(CH3)2
NH
O
CON(CH3)2
CON(CH3)2
NH
O
NH
O
O
O
O
COOMe
COOMe
COOMe
Mixture of endo and exo products
formed in the absence of a catalyst
Gouverneur, V. E.; Houk, K. N.; Pascual-Teresa, B. d.; Beno, B.; Janda, K. D.; Lerner, R. A. Science 23
1993, 262, 204-208.
Diels Alder Antibody 22C8
Endo
Approach
Re face
R1
R2
R2
Endo
Approach
Si face
R1
R2
R2
R1
R1
DDG‡TS =
1.9 kcal/mol
+
R2
R1
Exo
Approach
Si face
R1
R2
R2
Exo
Approach
Re face
R1
R2
R2
R1
R1
Gouverneur, V. E.; Houk, K. N.; Pascual-Teresa, B. d.; Beno, B.; Janda, K. D.; Lerner, R. A. Science 24
1993, 262, 204-208.
Diels-Alder Antibody 22C8
endo Diels-Alder Antibody
+
R1
R2
R2
R1
R2
R1
endo Hapten A
R2
endo
H
R1
R1
Haptens
exo
H
R2
CON(CH3)2
HN
HN
CON(CH3)2
exo Diels-Alder Antibody
O
+
R2
R
O
O
R
R = CH2CH2CH2CO-O N
R1
R2
R2
O
R1
R1
exo Hapten B
H
R1
R2
R1 R2
H
Gouverneur, V. E.; Houk, K. N.; Pascual-Teresa, B. d.; Beno, B.; Janda, K. D.; Lerner, R. A. Science 25
1993, 262, 204-208.
Exo-Diels-Alder Antibody 22C8
Uncatalyzed rxn. in H2O
Regioselective for ortho product
66:34 endo/exo (toluene)
85:15 endo/exo (aqueous)
Both enantiomers produced
Antibody 22C8 catalyzed rxn.
Regioselective for ortho product
0:100 endo/exo
> 97% ee
kuncat = 1.75 x 10–4 M-1 min-1
kcat = 3.17 x 10-3 min-1
KM = 0.7 mM (diene) ;
7.5 mM (dienophile)
kcat/kuncat = 18 M
Gouverneur, V. E.; Houk, K. N.; Pascual-Teresa, B. d.; Beno, B.; Janda, K. D.; Lerner, R. A. Science 26
1993, 262, 204-208.
Conformationally Unrestricted Hapten
+
CONMe2
CONMe2
NHCO2R'
NHCO2R'
R=CONMe2
R'=NHCO2CH2(C6H4)CO2H
R''=(CH2)3CO2H or 4-carboxybenzyl
ortho-endo
R
CONMe2
Fe
R'
R
ortho-exo
NHCOR''
R'
Yli-Kaukaluoma, J. T.; Ashley, J. A.; Lo, C.-H.; Tucker, L.; Wolfe, M. M.; Janda, K. D. J. Am. Chem. Soc.
27
1995, 117, 7041-7047.
Diels-Alder Antibody 13G5
CONMe2
Haptens
Fe
N
H
CO(CH2)3CO2H
CONMe2
Fe
NH
CO2H
Crystal Structure of Fab with a Ferrocenyl
Hapten Mimic Bound in the Cavity
Yli-Kaukaluoma, J. T.; Ashley, J. A.; Lo, C.-H.; Tucker, L.; Wolfe, M. M.; Janda, K. D. J. Am. Chem. Soc.
28
1995, 117, 7041-7047. Heine, A.; Stura, E. A.; Yli-Kauhaluoma, J. T.; Gao, C. Science 1998, 279, 1934-1940.
Exo-Diels-Alder Antibody Catalyst 13G5
CONMe2
CONMe2
Uncatalyzed Reaction
endo
NHCO2R'
NHCO2R'
Both diastereomers formed
No enantiomeric preference
CONMe2
CONMe2
exo
NHCO2R'
NHCO2R'
Catalyzed Reaction
CONMe2
IgG4D5
endo
NHCO2R'
+
CONMe2
CONMe2
IgG13G5
NHCO2R'
exo
NHCO2R'
kuncat = 1.75 x 10-4 M-1 min-1
kcat = 1.20 x 10-3 min-1
KM = 2.7 mM (diene)
10 mM (dienophile)
kcat/kuncat= 6.9 M
>98 % de; 95% ee
Yli-Kaukaluoma, J. T.; Ashley, J. A.; Lo, C.-H.; Tucker, L.; Wolfe, M. M.; Janda, K. D. J. Am. Chem. Soc.
29
1995, 117, 7041-7047. Heine, A.; Stura, E.A.; Yli-Kauhaluoma, J.T.; Gao, C. Science 1998, 279, 1934-1940.
Hetero-Diels-Alder Catalytic Antibody
O
CH3
N
O
N
NHPr
CH3
+
NHPr
O
CH3
+
O
O
N
R1
Hapten
NHPr
O
N
R2
O
> 95% of targeted regioisomer was formed in 82% ee
CONH(CH2)5CO2H
1
2
5 R =Me, R =H
6 R1=H, R2=Me
kuncat = 7.0 x 10-5 s-1
kcat = 1.83 x 10-1 s-1
KM = 3.94 mM (diene);
kcat / kuncat = 2618
Meekel, A. A. P.; Resmini, M.; Pandit, U. K. Bioorg. Med. Chem. 1996, 4, 1051-1057.
Meekel, A. A. P.; Resmini, M.; Pandit, U. K. J. Chem. Soc., Chem. Comm. 1995, 5, 571-572.
30
Retro-Diels-Alder Catalytic Antibody 9D9
H
N
O
O
OH
OH
O
N
H
N
H
O
retro Diels-Alder
+
H3C
H3C
= 110o
NH
= 180o
H
N
O
O
OH
N
H
H3C
NH(CH2)2COO-
O
OH
CONH(CH2)2COO
CHONH(CH2)2COO-
-
N
H3C
N
CH3
Hapten
N
pK=8.2
H3C
= 140o
Bahr, N.; Guller, R.; Reymond, J.-L.; Lerner, R. A. J. Am. Chem. Soc. 1996, 118, 3550-3555.
31
Retro-Diels-Alder Catalytic Antibody 9D9
Uncatalyzed rxn. in H2O
Spontaneous in aqueous buffer
2% per hour at 20 ºC
t1/2 (substrate) = 36 hr.
Antibody 9D9 catalyzed rxn.
kuncat = 3 x 10-4 min-1
kcat = 0.07 min-1
KM = 0.1 mM
kcat/kuncat = 233
• Unique hapten design to avoid product inhibition
• Antibody as potential prodrug release system
• Successful generation of antibody with hapten
in conformation equilibrium
Bahr, N.; Guller, R.; Reymond, J.-L.; Lerner, R. A. J. Am. Chem. Soc. 1996, 118, 3550-3555.
32
Antibody Catalysts of the Diels-Alder Reaction
Current Limitations
Moderate to low catalytic efficiency
Selected for binding energy not catalytic activity
Expensive and time consuming to produce
High substrate specificity not ideal for practical application
Stevenson, J. D.; Thomas, N. R. Nat. Prod. Rep. 2000, 17, 535-577. Liu, D. R.; Schultz, P. G. Angew. Chem.
Int. Ed. 1999, 38, 36-54. Hilvert, D. Top. Stereochem. 1999, 22, 83-135. Hasserodt, J. Synlett 1999, 12,
33
2007-2022. Hilvert, D. Annu. Rev. Biochem. 2000, 69, 751-793.
Antibody Catalysts of the Diels-Alder Reaction
Advantages and Future Directions
Valuable ability to direct regioselectivity, diastereoselectivity and
enantioselectivity of Diels-Alder reactions
Forthcoming advances in immunological technology, screening and selection to
discover improved catalytic efficiency
Hapten redesign/optimization
Explore broadening of substrate specificity and complexity of substrates
Metallo-antibodies
Gain insight from comparative analysis with “Diels-alderase” enzymes
Stevenson, J. D.; Thomas, N. R. Nat. Prod. Rep. 2000, 17, 535-577. Liu, D. R.; Schultz, P. G. Angew. Chem.
Int. Ed. 1999, 38, 36-54. Hilvert, D. Top. Stereochem. 1999, 22, 83-135. Hasserodt, J. Synlett 1999, 12, 34
2007-2022. Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472.
Outline
Introduction
I.
The Diels-Alder Reaction
II.
Biomolecule Catalysts of the Diels-Alder Reaction
Catalytic Antibodies (Abzymes)
Ribozymes (Catalytic RNA)
•
•
•
Ribozyme Structure, Function and Production
Examples of Diels-Alder Ribozymes
Limitations and Future Directions
“Diels-Alderases” (Enzymes in nature)
III.
Biocatalysis of the Diels-Alder Reaction and Organic
Synthesis
IV.
Conclusions
35
A Brief History of Ribozymes
1989: Sidney Altman and Thomas R. Cech won the Nobel Prize for
their discovery (in 1982) of catalytic properties of RNA
1990: Tuerk & Gold and Szostak independently develop in vitro selection
strategies (SELEX)
1995: First non-phosphate centered reaction catalyzed by RNA
(self-alkylating RNA discovered by Wilson & Szostak)
1997: First Diels-Alder reaction catalyzed by RNA (Bruce Eaton)
2002: Exploration of the scope of ribozyme catalysis—are there
limits to the types of reactions catalyzed by RNA?
36
Ribozyme Structure
Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. J. Am. Chem. Soc. 2000, 122, 1015-1021.
Jaschke, A.; Seeling, B.; Keiper, S.; Stuhlmann, F. Angew. Chem. Int. Ed. 2000, 39, 4576-4579.
37
Increasing Catalytic Ability of Ribozymes
Natural ribozymes
Nucleotide ligation
Nucleotide cleavage
Peptide bond formation
Phosphotransfer
Phosphoester hydrolysis
Ribozymes generated in vitro
Aminoacylation
Metallation
Peptide bond formation
Phosphorylation
Acylation
Alkylation
38
Wilson, D. S.; Szostak, J. W. Annu. Rev. Biochem. 1999, 68, 611-647.
Strategies to Isolate New Ribozymes
Selection against transition state analogs
Isolate RNA with affinity for immobilized TSA
Screen for catalytic activity
Direct selection
Self-modified RNA is created by reaction with a
substrate
Screen for catalytic activity
Jaschke, A. Biol. Chem. 2001, 382, 1321-1325. Jaschke, A.; Frauendorf, C.; Hausch, F. Synlett 1999,
6, 825-833. Wilson, D. S.; Szostak, J. W. Annu. Rev. Biochem. 1999, 68, 611-647.
39
Direct Selection of Ribozymes with LinkerCoupled Reactants
Systematic Evolution of Ligands by eXponential Enrichment (SELEX)
Jaschke, A. Curr. Opin. Struct. Biol.. 2001, 11, 321-1326. Jaschke, A. Catalysis of Organic Reactions by
RNA-Strategies for the Selection of Catalytic RNAs; Eggleston, D. S., Prescott, C. D. and Pearson, N. D., 40
Ed.;
Academic Press: San Diego, 1998, pp 179-190.
RNA Binds a Diels-Alder TSA….
Cl
Cl
Cl
1st report of RNA binding a
nonplanar/hydrophobic ligand
Cl
Cl
O
N
Cl
(CH2)5CO2-
Much lower binding affinity
than antibody 1E9
O
• 21 nucleotide consensus sequence in all RNA that bound the ligand
• Predicted to be in a bulge stem loop structure
…..but no catalytic activity
Morris, K. N.; Tarasow, T. M.; Julin, C. M.; Simons, S. L.; Hilvert, D.; Gold, L. Proc. Natl. Acad. Sci.,
USA 1994, 91, 13028-13032.
41
Diels-Alder Reaction is Catalyzed by RNA
O
NH
HN
O
O
+
N
H
O
N
H
N
O
O
4
N
H
H
H
4
S
O
PEG
100N
3'
O
O
N
HN
N
H
O
O
N
H
O
PPPO
O
PEG
O
100N
3'
N
H
O
N
N
O
Biotin
H
H
H
OH
H
OH
pyridyl modified UTP
Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. Nature 1997, 389, 54-57.
42
The First RNA Catalyst of a Diels-Alder Reaction
800-fold rate acceleration ((kcat/Km) / kuncat)
kuncat = 5.42 x 10-3 M-1 s-1
kcat = 0.011 ± 0.002 s-1
KM = 2.3 ± 0.5 mM (dienophile)
kcat / kuncat = 2 M
Modified base
Presence of cupric ion
10 nucleotide consensus sequence
No other sequence/structural homology
Morris, K. N.; Tarasow, T. M.; Julin, C. M.; Simons, S. L.; Hilvert, D.; Gold, L. Proc. Natl. Acad. Sci.,
USA 1994, 91, 13028-13032. Tarasow, T. M.; Tarasow, S. L.; Tu, C.; Kellogg, E.; Eaton, B. E.
J. Am. Chem. Soc. 1999, 121, 3614-3617. Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. J. Am. Chem. Soc.
43
2000, 122, 1015-1021.
Diels-Alder Ribozyme
Jaschke, A. Curr. Opin. Struct. Biol.. 2001, 11, 321-1326.
44
True Catalysis of Diels-Alder by RNA
R1=(C2H4O)6-H
R2=(CH2)5COOH
O
N
+
R2
+ Ribozyme
O
OR1
R2
O
O
R2
N
N
O
O
+
R1O
OR1
Uncatalyzed reaction yields racemic products
1100-fold rate acceleration
((kcat/Km) / kuncat)
kuncat = 3.2 M-1min-1
kcat = 21 min-1
KM = 0.37 mM (diene);
8 mM (dienophile)
kcat/kuncat = 6.6 M
6 transformations per minute
Tethering of substrate to RNA is not necessary
Jaschke, A.; Seeling, B.; Keiper, S.; Stuhlmann, F. Angew. Chem. Int. Ed. 2000, 39, 4576-4579.
45
Predicted control of stereochemistry
95 % enantiomeric excess
in both cases
R2
O
O
N
N
O
R1O
R2
R1=(C2H4O)6-H
R2=(CH2)5COOH
O
OR1
Jaschke, A.; Seeling, B.; Keiper, S.; Stuhlmann, F. Angew. Chem. Int. Ed. 2000, 39, 4576-4579. 46
Diels-Alder Ribozymes
Current Limitations
SELEX is a very time consuming process
Substrate specificity not practical
RNA highly susceptible to nucleases
Jaschke, A.; Frauendorf, C.; Hausch, F. Synlett 1999, 6, 825-833. Jaschke, A. Biol. Chem. 2001, 382,
1321-1325. Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472.
47
Diels-Alder Ribozymes
Advantages and Future Directions
Highly stereoselective catalysts
Phenotype is directly linked to genotype
In vitro selection strategies—direct screen for function
Less expensive and smaller in size than antibodies
Easy to create novel features via incorporation of modified nucleotides
Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472. Jaschke, A.; Frauendorf, C.; Hausch,
F. Synlett 1999, 6, 825-833. Jaschke, A. Biol. Chem. 2001, 382, 1321-1325. Golinelli-Pimpaneau, B. 48
Curr. Opin. Struct. Biol. 2000, 10, 697-708.
Outline
I.
Introduction
II.
The Diels-Alder Reaction
Biomolecule Catalysts of the Diels-Alder Reaction
Catalytic Antibodies (Abzymes)
Ribozymes (Catalytic RNA)
•
•
•
III.
Biocatalysis of the Diels-Alder Reaction in Biosynthesis and Organic
Synthesis
•
•
IV.
Ribozyme Structure, Function and Production
Examples of Diels-Alder Ribozymes
Limitations and Future Directions
“Diels-Alderases” (natural enzymes)
Biomolecule catalysts vs. other catalysts of the Diels-Alder reaction
Conclusions
49
Evidence for Diels-Alderases in Biosynthesis
Synthesis of Solanopyrones
Pohnert, G. ChemBioChem 2001, 2, 873-875. Laschat, S. Angew. Chem. Int. Ed. Engl. 1996, 35, 289-291.
Oikawa, H.; Suzuki, Y.; Katayama, K.; Naya, A.; Sakana, C.; Ichihara, A. J. Chem. Soc., Perkin Trans. 1
1999, 1225-1232. Oikawa, H.; Kobayashi, T.; Katayama, K.; Suzuki, Y.; Ichihara, A. J. Org. Chem.
50
1998, 63, 8748-8756.
Evidence for Diels-Alderases in Biosynthesis
Synthesis of benzoate (macrophomic acid)
Diels-Alder
Cyclization
Watanabe, K.; Mie, T.; Ichihara, A.; Oikawa, H.; Honma, M. J. Biol. Chem. 2000, 275, 38393-38401.
Pohnert, G. ChemBioChem 2001, 2, 873-875.
51
Proof of the Existence of a“Diels-Alderase”
Lovastatin Nonaketide Synthase (LNKS)
catalyzes an intramolecular Diels-Alder
reaction of a substrate analog
COR
R=SCH3CH2NHCOCH3
never observed
Formed in 1:1 mixture
in the absence of LNKS
only formed
in presence of
LNKS
Hutchinson, C. R.; Kennedy, J.; Park, C.; Kendrew, S.; Auclair, K.; Vederas, J. Antonie van Leeuwenhoek
52
2000, 78, 287-294. Pohnert, G. ChemBioChem 2001, 2, 873-875.
Diels-Alder Biocatalysts in Total Synthesis?
No examples in synthesis
An important pursuit for valuable catalysts of Diels-Alder
Stereoselectivity, redirect reaction route
Many complex natural products
OMe
O
CHO
H
HO
O
O
OMe
MeO
HO
H
O
OMe
O
Heliocide H1
Alflabene
MeO
O
O
O
Carpanone
OMe
OH
HO
O
O
O
O
O
O
O
OMe
O
OH
O
H
H
O
O
H
O
NH
Mevinolin
H
Nargenicin
HN
Secodaphniphylline
53
Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472.
Other Diels-Alder Catalysts
Molecularly Imprinted
Polymers (MIPs)
Chiral Lewis Acids
Metallo-Porphyrin Systems
Zn2+
H3C
SbF6-
Ru+
(C6F5)2P
Zn2+
Zn2+
COMe2
O
P(C6F5)2
O
H3C
H3C
CH3
Liu, X.-C.; Mosbach, K. Macromol. Rapid Commun. 1997, 18, 609-615. Walter, C. J.; Sanders, J. K. M.
Angew. Chem. Int. Ed. Engl. 1995, 34, 217-219. Nakash, M.; Clyde-Watson, Z.; Feeder, N.; Davies, J. E.;
Teat, S. J.; Sanders, J. K. M. J. Am. Chem. Soc. 2000, 122, 5286-5293. Otto, S.; Bertoncin, F.;
Engberts, J. B. F. N. J. Am. Chem. Soc. 1996, 118, 7702-7707. Kundig, E. P.; Saudan, C. M.; Alezra, V.; 54
Viton, F.; Bernardinelli, G. Angew. Chem. Int. Ed. 2001, 40, 44814485.
Catalyst
Reaction
Kinetics
O
Antibody
22C8
O
MeOOC
+
(H3C)2NOC
HN
H2O
O
Ribozyme
(untethered)
+
N R2
H2O
O
OR1
O
H2O
N
Selectivity
kcat = 3.17 x 10-3 min-1
kcat/kuncat = 18 M
Exo product
> 97% ee
kcat = 21 min-1
kcat/kuncat = 6.6 M
Exo product
> 95% ee
k2= 4.02 x 10-3 M-1s-1
84 endo :16 exo
k2= 1.11 M-1s-1
93 endo : 7 exo
kcat = 4.0 M-1s-1
kcat/kuncat = 1030
Exo product
(no turnover)
+
H2O
O
Lewis Acid
(10 mM
Cu(NO3)2)
Porphyrin
System
N
H2O
N
+
N
N
O
CH2Cl2
CHO
Chiral metal
compound
MIP
+
+
CH2Cl2
O
Cl
Cl
O
S
+
O
O
Cl
Cl
71 exo : 29 endo
85% ee
CH3CN
O
kcat/kuncat = 270
kcat = 3.82 x 10-2 min-1
(no turnover)
endo
55
Future Directions of Biomolecular Catalysis of
Diels-Alder Reactions
Focus on biomolecules as catalysts used to direct the
regiochemistry, stereochemistry and enantioselectivity of DielsAlder reactions
Learn from enzyme “Diels-Alderases”
Expand substrate complexity
Broaden substrate range
Improve catalytic efficiency
Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472. Hilvert, D. Annu. Rev. Biochem. 56
2000, 69, 751-793.
Conclusions
Remarkable selectivity provided by biomolecules should
drive the pursuit of optimized catalytic efficiency
Biomolecular Diels-Alder catalysts demonstrate low
catalytic efficiency not necessarily due to their inherent
catalytic inabilities but to suboptimal selection and
screening
Diels-Alder catalytic antibodies and ribozymes possess
excellent features that may be exploited for the creation of
tailored biocatalysts to be used in synthesis and
pharmaceuticals
Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472. Jaschke, A.; Frauendorf, C.; Hausch, F.
57
Synlett 1999, 6, 825-833.
Acknowledgements
Dr. Silvia Cavagnero
Susie Martins
Ken Nikolas
Jason Pontrello
Konstantin Levitsky
Margaret Biddle
Whitney Erwin
Matt Hinderaker
Courtney Bakke
Charles Chow
Eric Fulmer
Val Keller
Sena Rajagopalan Mike Birkeland
Brenda Foster Jason Ellefson
Thank you!
Sarah Maifeld
Clement Chow
58