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A BIOCATALYTIC APPROACH TO THE DIELSA BIOCATALYTIC APPROACH TO THE DIELS--
ALDER REACTIONALDER REACTION
Mark Evans Ondari
Michigan State University
Wednesday January 24th, 2007
NO
R
R
O N
OTTO PAUL HERMANN DIELSOTTO PAUL HERMANN DIELS KURT ALDERKURT ALDER
THE FACES BEHIND THE NAME…
http://nobelprize.org/nobel_prizes/chemistry/laureates/1950/
OUTLINE
DIELS DIELS -- ALDER : THE BASICSALDER : THE BASICS
BIOMOLECULES IN DIELS- ALDER:
AS ENTROPY TRAPS
AS “DESIGNER CATALYSTS”
CATALYTIC ANTIBODIES
RNA
AS STEREOSELECTIVE CATALYSTS
RNA
DNA- BASED CATALYSIS
THE DIELS-
ALDER…BASICS
A bimolecular [4π + 2π] pericyclic reaction
Reactive (Frontier) molecular orbitals involved
Electron- rich diene/electron poor dienophile (normal electron- demand DA)
Stereospecific; up to four chiral centers can be generated
O
O
OOMe
OO
‡
cb
a
d
e f
hg
cb
a
d
g h
fe
a b
f
h
g
c d
a b
h
fe
c d
g
e
e, g
endo
e, g
endo
a b
e
gh
c d
f
a b
g
ef
c d
h
e, g
exo
e, g
exo
REGIO/STEREOCHEMISTRY
http://academics.keene.edu/rblatchly/OrgoCommon/hand/conjdiene/DARxnStereochem.html#StereoDisc
Another set of four isomers when dienophile approaches diene from the bottom
STEREOSELECTIVITY
Chiral Metal Complexes/Auxiliaries
O
OMe
OOMe
S- VAPOL
Et2AlCl2
-78o C100%, 97% ee (2S)
PhMe
Ph
OHHO
Ph
S- VAPOL
Biomolecules
Heller, D. P.; Goldberg, D. R.; Wu, H.; Wulff, W. D., Can. J. Chem., 2006, 84 (Delayed)
Aldrich, # 675334
OUTLINE
DIELS DIELS -- ALDER : THE BASICSALDER : THE BASICS
BIOMOLECULES IN DIELSBIOMOLECULES IN DIELS-- ALDER REACTIONS:ALDER REACTIONS:
AS ENTROPY TRAPS
AS “DESIGNER CATALYSTS”
CATALYTIC ANTIBODIES
RNA
AS STEREOSELECTIVE CATALYSTS
RNA
DNA
BIOMOLECULES IN DIELS-
ALDER…
AS ENTROPY TRAPS
O
OCH3HO2C
Macrophomic Acid
O
O
OCH3
O
O
OO
O
O
- 2CO2
- H2O
2- Pyrone Oxalacetate
A natural “Diels- Alderase”
Isolated from Macrophoma commelinae fungus
Involved in a complex, multi-step process with high catalytic efficiency:
MACROPHOMATE SYNTHASE
Watanabe, K., Mie, T., Ichihara, I., Oikawa, H., Honma, M. J. Bio. Chem. 2000, 275, 38393-38491
MACROPHOMATE SYNTHASE-
CRYSTAL STRUCTURE
Watanabe, K., et al Nature, 2003, 422, 185-189
O
O
O
O OMg+2
CH3
CO2Me
OCH3
Watanabe, K., et al Nature, 2003, 422, 185-189
PROPOSED MECHANISM: ROLES OF Mg+2, AMINO ACIDS
O-OO-O
OO
O
-O O
OO
O
CH3
O-O
OH
Mg+2
Mg+2
OH
H
OH H
Asp 211Glu 185
H2N
Arg 101NH2
- CO2
OHTyr 169'
Diels- Alder
STEP 1 STEP 2
PROPOSED MECHANISM: ROLES OF Mg+2, AMINO ACIDS
OO
O
CH3
O-O
CH3
O
Mg+2
H2N
Arg 101NH2
OHTyr 169'
H HO
HO2C CH3
O
CH3
Macrophomic Acid
H3C
STEP 3
- CO2- H2O
Diels- Alder
STEP 2
OO
O
CH3
O-O
O-
Mg+2
H2N
Arg 101NH2
OHTyr 169'
Diels- Alder
STEP 2STEP 1
- CO2
Watanabe, K., et al Nature, 2003, 422, 185-189
O
O O
O
O
O
OO
OO
H3COO
O
O
H3COC
O
OH
C
HO
OOCH3
O
CO2
CO2
H2O
OOH
4+2
Oxalacetate
Macrophomic acid
PROPOSED MECHANISM…
SUMMARY
Watanabe, K., et al; Nature, 2003,
422, 185-
189
WHAT ABOUT MICHAEL-ALDOL?
O
O O
O
OO
OO
O
O
H3COC
O
OH
C
HO
OOCH3
O
CO2
CO2
H2OOOH
O
O O
O
H3C
O
O O
O
O
OO
CH3
Michael
Aldol
Aldol
Oxalacetate
Macrophomic acid
Ose, K. et al Nature, 2003, 422, 185-189
Molecular simulations (QM/MM) suggest Diels-Alder “TS” unstable
Jorgensen, W. L., Blagovich, M., Guimaraes, R. J. Am. Chem. Soc. 2005, 35577
OUTLINE
DIELS DIELS -- ALDER : THE BASICSALDER : THE BASICS
BIOMOLECULES IN DIELS- ALDER:
AS ENTROPY TRAPS
AS “DESIGNER CATALYSTS”
CATALYTIC ANTIBODIES
RNA
AS STEREOSELECTIVE CATALYSTS
RNA
DNA
BIOMOLECULES IN DA…
AS “DESIGNER CATALYSTS”
ANTIBODIES
• Are soluble blood glycoproteins
• Produced by the immune system
in response to antigens (foreign bodies)
http://www.genome.gov//Pages/Hyperion//DIR/VIP/Glossary/Illustration/antibody.cfm
CATALYTIC ANTIBODIES
Generated by immunization against haptens- e.g DA transition state analogs (TSA)
Resulting antibodies are screened for catalytic activity of the reaction
http://faculty.ircc.edu/faculty/TFischer/images/haptens.JPG
http://www.hopkins-arthritis.som.jhmi.edu/edu/mono_anti.html
Haptens Carrier Protein Full antigen
http://www.sigmaaldrich.com/aldrich/bulletin/al_techbull_al207.pdf
EXAMPLE OF A CATALYTIC ANTIBODY
Aldolase Antibody 38C2
Aldrich Catalog # 47,995-0
DIELS-ALDER ANTIBODY IE9
S N
Cl
Cl
Cl
Cl
O
O
O
O
EtAb IE9
TCTD NEM
NO
O
(CH2)5CO2-
Cl
Cl Cl
ClClCl
HAPTEN
N
Cl
ClCl
ClO
O
Et
Jiang Xu, et al, Science, 1999, 282, 2345-2348
NO
O
Et
Cl
Cl Cl
Cl
N
Cl
ClCl
ClO
O
Et1. - SO2
2. [O]
SOO
NCl
Cl
Cl ClO
O-O
OCl
Cl
ANTIBODY IE9: ACTIVE SITE
Jiang Xu, et al, Science, 1999, 282, 2345-2348
S N
Cl
Cl
ClO
O
O
O
Et N
Cl
Cl
ClO
O
Et
O
CO2- O
CO2-
Antibody IE9
ANTIBODY IE9: KINETICS
Jiang Xu, et al, Science, 1999, 282, 2345-2348
Kuncat
= 0.013 M-1
min-1 (second order rate constant)
kcat
= 13 min-1 (molecules of product per minute)
Effective Molarity = 1000 M
ANTIBODY IE9: STERIC VS ELECTRONIC EFFECTS
S N
X
X
XO
O
O
O
Et N
X
X
XO
O
EtAntibody IE9
X XNEM
Volume (Å3) Moment (D) kuncat [M-1min-1] kcat [min-1] KNEM [mM]
X = Cl 146.5 4.600 0.040 1.2 7.6
X = Br 171.1 4.977 0.063 1.8 14
X = Me 153.6 6.902 0.212 -
-
X = H 87.0 6.049 -
-
-
Gozin, Y., Hilvert, D. Helvetica Chimica Acta, 2002, 85, 4328-
4336
Electronic factors more important to catalytic efficiency than are steric
DIELS ALDER ANTIBODY 39-A11
HN
O
OO
O-
N
O
O
NH
O
Ab 39A11N
NHO
O
O
O
NHCOCH3
-O2CNH2
OO-O2C
NO
O
Ar
‡
NH2
OO-O2C
NO
O
NH
O
HAPTEN
Romesberg, F.E.; Spiller, B.; Stevens, R.C.; Schultz, P.G., Science,1998, 279, 1929-1933
DIELS-
ALDERASE ANTIBODY 39-A11
Kcat
= 0.67 sec -1
Kuncat
= 0.17 M-1 sec -1
Romesberg, F.E.; Spiller, B.; Stevens, R.C.; Schultz, P.G., Science, 279, 1929-1933
NNH
NH
O
O-
O
O
O
O
O
ANTIBODY IE9
Snug fit
ANTIBODY 39A11
Loose Fit
COMPARISON
Jiang Xu, et al, Science, 1999, 282, 2345-2348
OUTLINE
DIELS DIELS -- ALDER : THE BASICSALDER : THE BASICS
BIOMOLECULES IN DIELS- ALDER:
AS ENTROPY TRAPS
AS “DESIGNER CATALYSTS”
CATALYTIC ANTIBODIES
RNA
AS STEREOSELECTIVE CATALYSTS
RNA
DNA- BASED CATALYSIS
Biopolymers Made Up Of Nucleotides:
-
Nitrogenous heterocyclic base
-
Pentose sugar
-
Phosphate groupN
NN
N
NH2
O
HO
OP-OO-
O
DeoxyRibonucleic Acid
(DNA)
R
RiboNucleic Acid
(RNA)
NH
O
ON
O
OHO
OP-OO-
O
R
BIOMOLECULES IN DA…
AS “DESIGNER CATALYSTS”
NUCLEIC ACIDS
Ribozymes are RNAs with catalytic properties
Can form complex secondary structures
Catalysis assisted by nucleobases and/or divalent metals
RIBONUCLEIC ACIDS ENZYMES (RIBOZYMES)
Amanda Y. Chem. Eng. News, 2005, 83, 9
Base
O
OHO
O
P OO
Base
O
OO
O
H
N H
O
NH2
OH
RNA self -cleavage (t rans-esterification)
Mg+2
DIELS-
ALDER RNAs (RIBOZYMES): THE REACTION
N
O
O
R2
R1O
N
O
O
R2
**
OR1
OR1
N
O
O
R2
**
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A. Angew. Chem. Int. Ed. 2000, 39, 4576-4579
ORNA
N
O
OH
HNNH
O
Biotin
ORNA
N
O
O
O
NH
NH*
*
Biotin
DIELS-
ALDER RNAs: REACTION DESIGN
HN NH
S
HH
O
OH
O
Biotin- Stronglybound by Streptavidin
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A. Angew. Chem. Int. Ed. 2000, 39, 4576-4579
GENERATING THE DIELS-
ALDER RNAs
SELEX (IN VITRO SELECTION)
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A. Angew. Chem. Int. Ed. 2000, 39, 4576-4579
SELEX (IN VITRO SELECTION)
Systematic Evolution of Ligands by Exponential Enrichment
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A. Angew. Chem. Int. Ed. 2000, 39, 4576-4579
GENERATING THE DIELS-
ALDER RNA
SELEX (IN VITRO SELECTION)
Systematic Evolution of Ligands by Exponential Enrichment
A combinatorial technique that involves:
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A. Angew. Chem. Int. Ed. 2000, 39, 4576-4579
GENERATING THE DIELS-
ALDER RNA
SELEX (IN VITRO SELECTION)
Systematic Evolution of Ligands by Exponential Enrichment
A combinatorial technique that involves:
Making a large, random library of nucleotides
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A. Angew. Chem. Int. Ed. 2000, 39, 4576-4579
GENERATING THE DIELS-
ALDER RNA
SELEX (IN VITRO SELECTION)
Systematic Evolution of Ligands by Exponential Enrichment
A combinatorial technique that involves:
Making a large, random library of nucleotides
Sequences in the random library subjected to Diels- Alder ligands
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A. Angew. Chem. Int. Ed. 2000, 39, 4576-4579
GENERATING THE DIELS-
ALDER RNA
SELEX (IN VITRO SELECTION)
Systematic Evolution of Ligands by Exponential Enrichment
A combinatorial technique that involves:
Making a large, random library of nucleotides
Sequences in the random library subjected to Diels- Alder ligands
Active sequences (aptamers) separated by affinity chromatography
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A. Angew. Chem. Int. Ed. 2000, 39, 4576-4579
GENERATING THE DIELS-
ALDER RNA
SELEX (IN VITRO SELECTION)
Systematic Evolution of Ligands by Exponential Enrichment
A combinatorial technique that involves:
Making a large, random library of nucleotides
Sequences in the random library subjected to Diels- Alder ligands
Active sequences (aptamers) separated by affinity chromatography
Separated, active sequences amplified by polymerase chain reaction
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A. Angew. Chem. Int. Ed. 2000, 39, 4576-4579
GENERATING THE DIELS-
ALDER RNA
SELEX (IN VITRO SELECTION)
Systematic Evolution of Ligands by Exponential Enrichment
A combinatorial technique that involves:
Making a large, random library of nucleotides
Sequences in the random library subjected to Diels- Alder ligands
Active sequences (aptamers) separated by affinity chromatography
Separated sequences amplified by polymerase chain reaction
Amplified sequences subjected to more stringent conditions
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A. Angew. Chem. Int. Ed. 2000, 39, 4576-4579
GENERATING THE DIELS-
ALDER RNA
SELEX (IN VITRO SELECTION)
Systematic Evolution of Ligands by Exponential Enrichment
A combinatorial technique that involves:
Making a large, random library of nucleotides
Sequences in the random library subjected to Diels- Alder ligands
Active sequences (aptamers) separated by affinity chromatography
Separated sequences amplified by polymerase chain reaction
Amplified sequences subjected to more stringent conditions
A pool of Diels-
Alder RNAs (ribozymes) is generated
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A. Angew. Chem. Int. Ed. 2000, 39, 4576-4579
GENERATING THE DIELS-
ALDER RNA
CH2Cl
H O CH2 CH2 OHn
CH2
O CH2 CH2 OHn
CH2
O CH2 CH2 On P O CH2
NiPr iPr
CH2 CN
CH2
O CH2 CH2 On POH
ONH
N
N
O
NH2N
O
OHOH
O
(Ethylcyano)Chlorophosphoamidite
Guanosine (5'-OH)
Initiator Nucleotide- G
MAKING A RANDOM RNA LIBRARY: INITIATOR NUCLEOTIDE
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A., Angew. Chem. Int. Ed. 2000, 39, 4576-4579
CH2
O CH2 CH2 On POH
ONH
N
N
O
NH2N
O
OHOH
ONTP's
CH2
O CH2 CH2 On POH
OO
5' 3'
RNA Polymerase
DNA Template
Initiator Nucleotide- G
T7 Promoter
G RNA LIBRARY
GENERATING A RANDOM RNA LIBRARY (POOL)
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A., Angew. Chem. Int. Ed. 2000, 39, 4576-4579
3' RNA LIBRARY G 5'
NH
N
O
O
CH2
OCH2CH2OPO
OHO
Biotin- Maleimide
O CH2 CH2 O PO
OHO
N
O
O
O
NH
n
n
Product partitioned inStreptavidin agarose
RNA component amplified by RT-PCR
**
OBiotin
Biotin
5' DA RNAG 3'
Seelig, B., Keiper, S., Stuhlmann, F., Jäschke, A., Angew. Chem. Int. Ed. 2000, 39, 4576-4579
ACTIVE DIELS-
ALDER RNA FROM THE RANDOM POOL
HN NH
S
HH
O
OH
O
Biotin- Stronglybound by Streptavidin
ACTIVE DIELS-
ALDER RNAs
Short (49) nucleotides with similar motif
Accelerates Diels- Alder reaction by up to 20,000
Serganov, A.; et al, Nature Struct. Mol. Biol. 2005, 12, 218-224
DIELS-
ALDER RNA : STRUCUTRAL INFORMATION
Serganov, A.; et al, Nature Struct. Mol. Biol. 2005, 12, 218-224
Zhang, X., Bruice, T. C., J. Am. Chem. Soc. 2007, DOI: 10.1021/ja067416i
DIELS-ALDER RNA: CONCERTED OR STEPWISE MECHANISM?
Zhang, X., Bruice, T. C., J. Am. Chem. Soc. 2007, DOI: 10.1021/ja067416i
STEREOSELECTIVITY USING MIRROR-IMAGE RNAs
Schlaterer, J., Stuhlmann, F., Jäschke
A. ChemBioCHem 2003, 4, 1089-1092
STEREOSELECIVITY THROUGH BACKDOOR ACCESS
N
O
OR
O CH2 CH2 OH
N
O
O
n
**
CH2
OCH2H2Cn
OCH2CH2HO
N
O
O
n
**
HO
R R
R, R S, S* * * *
Zhang, X., Bruice, T. C., J. Am. Chem. Soc. 2007, DOI: 10.1021/ja067416i
Wombacher, R., et al Angew. Chem. Int. Ed. 2006, 45, 2469-2472
STEREOSELECTIVITY THROUGH THE BACKDOOR
TETHERED TO RNA = S,S (OVER 90% ee)
NOT TETHERED = R, R (OVER 90% ee)
Wombacher, R., et al Angew. Chem. Int. Ed. 2006, 45, 2469-2472
OUTLINE
DIELS DIELS -- ALDER : THE BASICSALDER : THE BASICS
BIOMOLECULES IN DIELS- ALDER:
AS ENTROPY TRAPS
AS “DESIGNER CATALYSTS”
CATALYTIC ANTIBODIES
RNA
AS STEREOSELECTIVE CATALYSTS
RNA
DNA
BIOMOLECULES IN DA…
AS STEREOSELECTIVE CATALYSTS
ENANTIOSELECTIVE DNA- BASED CATALYSIS
NH
O
ON
O
HO
OP-OO-
O
Deoxy-Ribonucleic Acid (DNA)
R
http://en.wikipedia.org/wiki/DNA
No known DNA- catalyzed C- C bond formation
N
N
N
N N
N
dppz bipy
SOME ACHIRAL LIGANDS KNOWN TO BIND TO DNA
Ligands have metal binding sites- could be used to transfer chirality from DNA
Roelfes, G., Boersma, J. A., Feringa, B. L. Chem. Commun. 2006, 655, 635-637
Examples
NO
R
R
O N
= Achiral Ligand Complex
ENANTIOSELECTIVE DNA-BASED DIELS-ALDER CATALYSIS
Achiral ligands and Cu2+ used
Reactive Cu2+ brought into closeproximity of the chiral DNA
Chirality transferred directly to the Diels-
Alder reaction
Roelfes, G., Boersma, J. A., Feringa, B. L. Chem. Commun., 2006, 655, 635-637
N
N
N
NCu+2
Example
=
N
N
N
N N
NN
N
N
N N
N
1 2 3 4
Kb (DNA) 8.0 ×
105 M-1 7.2 ×
104
M-1
1.3×
104
M-1
9.4 ×
103
M-1
Endo: exo 96 : 4 95 : 5 96 : 4 a 98 : 2b 97 : 3c 94 : 6
% ee (endo) 49 61 73 a 89b 92c 83
NO
R
R
O N
a : R = Ph
b : R = 4-
Methoxyphenyl
c : R = t-butyl
Roelfes, G., Boersma, J. A., Feringa, B. L. Chem. Commun. 2006, 655, 635-637
N
NH2H2N
Br
Ethidium Bromide
WHY INCREASE IN ee’s
WITH DECREASING Kb(DNA)
?
Used as a nucleic acid stain in bio labs
Known to bind DNA strongly by intercalation, deforming the DNA helix
Does strong ligand
(e.g
dpq) binding = better intercalation into DNA =
deformed helix = decrease/loss of chirality = decrease in Diels-
Alder ee’s??
NN
NN
Dipyridoquinoxaline (dpq)
Vs
Ethidium
bromide:
N
N
N
N N
NN
N
N
N N
N
1 2 3 4
Kb (DNA) 8.0 ×
105 M-1 7.2 ×
104
M-1
1.3 ×
104
M-1
9.4 ×
103
M-1
Endo: exo 96 : 4 95 : 5 96 : 4 a 98 : 2b 97 : 3c 94 : 6
% ee (endo) 49 61 73 a 89b 92c 83
NO
R
R
O N
a : R = Ph
b : R = 4-
Methoxyphenyl
c : R = t-butyl
Roelfes, G., Boersma, J. A., Feringa, B. L. Chem. Commun. 2006, 655, 635-637
N
N
N
NHN
N
NHN
5 6 7
Kb (DNA) 5.2 ×
103
M-1
1.5 ×
104
M-1 1.12 ×
104 M-1
Endo: exo 99 : 1 98 : 2 99 : 1
% ee (endo) 91 a 92 99b 95 99c 90 97
NO
R
R
O Na : R = Ph
b : R = 4-
methoxyphenyl
c : R = t-butyl
Roelfes, G., Boersma, J. A., Feringa, B. L. Chem. Commun., 2006, 655, 635-637
CONCLUSIONS
Biomolecules:
Can, by proximity effect, serve as “entropic traps” to accelerate DA reactions
Provide an opportunity to design catalysts for the reaction of interest
Do catalyze complex C-C bond forming reactions stereoselectively
Challenge: Application in natural product synthesis/ pharmaceuticals
ACKNOWLEDGEMENTS
Dr. Walker
Dr. Wulff
Dr. Borhan
Dr. WalkerDr. Walker’’s Group:s Group:
Amanda, Danielle, Irosha, Karin, Sanjit, Washington, Yemane.
Friends:Friends:
Mercy, Kim, Toyin
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