Transcript Document

DNA-Templated Synthesis:
Principles of Evolution in Organic Chemistry
1
R4 NH2
O
N
O
CO2H
R4'
O
N
HN R3'
O
O
R4
H
R5 NH
O
O
O
O
O
N
N
O
HN R3'
O
O
O
R2 S
HN R1'
HN R6'
R1 NH2
N
R3 NH
O
O
HN R2'
O2N
O
NO2
R3 NH
H
EDC,
Sulfo-NHS,
NaBH3CN
O
O
O
O
HO
N
R1 N
H
R6 NH
R2 SH
O
O
Ph Ph
O
P
HN R5'
O
O
O
R5 NH2
H
HN R1'
HN R2'
R6 NH
O
N
H
R4'
HN R5'
O
HN R6'
O
2
Ph
P
Ph
Ph R1
Ph
P
H
Ph
Ph R2
O
Ph
P
H
Ph
Ph R3
O
O
R
H
One solution
R
R3
R2
R1
3
Introduction to DTS
Organic Reactions in DTS
Fundamental reactions, distance dependence and independence
New, synthetically useful architectures
Example of a Small Molecule Synthesis
Synthetic strategies, linkers, purification
Towards the Multistep Synthesis of Small Molecule Libraries
Conclusions
4
Strategies to Control Reactivity
The chemist’s approach to controlling reactivity
O
O
H 2N
O
OH
N
H
O
OPG
+
S
O
Ph
O
Starting materials
mM – M concentration
N
H
OH
N
H
S
One possible
product
5
Strategies to Control Reactivity
The chemist’s approach to controlling reactivity
O
O
H 2N
O
OPG
OH
N
H
O
+
O
S
Ph
O
Starting materials
mM – M concentration
N
H
OH
N
H
S
One possible
product
Nature’s approach to controlling reactivity:
O
O
NH2
OH HO C
2
H2N
O
Ph
CO2H
NH2
OH
O
O
SH
HO2C
CO2H
nM - M concentration
Many reactants in one solution
O
Macromoleculetemplated synthesis
N
H
OH
N
H
S
Selective product
formation
6
Synthetic Strategies
The chemist’s approach to active molecule discovery:
O
N
N
O
NH OEt
N
N
NH OEt
N
Starting material
N
S
O O
Product
Data: Keq,
ee, IC50, …
7
Synthetic Strategies
The chemist’s approach to active molecule discovery:
O
N
N
O
N
N
NH OEt
NH OEt
N
N
S
O O
Starting material
Product
Data: Keq,
ee, IC50, …
Nature’s approach to active molecule discovery:
O
O
Ph
DNA
RNA
Protein
Selection, Amplification, Diversification
O
N
H
OH
N
H
S
8
The Basics of DNA-Templated Synthesis (DTS)
Reactant for DTS
O
oligonucleotide
HN
O
N
O
reactive
group
linker
General Reaction Scheme
O
O
HN
O
N
HN
O
O
N
"H+"
O
SH
Annealing
SH
Coupling
O
HN
N
O
S
O
9
Selection and Amplification
O
O
NHH
S
O
HO
O
Protein
Ph
N
H
O
S
H2N O
H
N
Protein
NH
HN
H
NH
O
HN
H
NHH
O
NH
S
O
Amplification
PCR
O
N
H
H
N
O
O
NH
Ph
SMe
DNA sequencing
or PAGE
Identity of the active
molecule
10
Polymerase Chain Reaction (PCR)
Sample
Denature
94oC, 30 s
5’
3’
3’
5’
Anneal
primers
55oC, 60 s
Stop
4o C
Extension
75oC, 30 s
5’
3’
3’
5’
Taq polymerase
11
Synthesis of Products Unrelated
to the DNA Backbone
1,4-conjugate addition to carbonyls
O
O
HN
O
N
HN
O
O
N
O
SH
S
Peptide Coupling
NH2
O
H
N
73%
O
DMT-MM
Or EDC / sulfo-NHS
OH
Heck
O
O
HN
NH
O
O
N
H
N
O
I
54%
Na2PdCl4
O
N
H
N
O
O
Gartner, Z. J., Kanan, M. W., Liu, D. R. Angew. Chem. Int. Ed. 2002, 41, 1796
Gartner, Z.J., Liu, D.R. J. Am. Chem. Soc. 2001, 123, 6961.
12
Sequence Specificity and Distance Independence
O
Sequence Specificity
O
N
NH
O
H
I
H
N
NH
SH
O
2
O
O
N
O
HS
NH
O
A single base mismatch in the 10-base reagent oligonucleotide slows
the reaction down by a factor of 200
13
Sequence Specificity and Distance Independence
O
Distance Independence
HN
HN
O
H
NR
3
O
O
HN
H R2
O
N
R1
O
N
H
•
Limited ability for diversification
•
Complicated substrate
identification
Coding Region for R1,
R2 and R3
14
Sequence Specificity and Distance Independence
O
Distance Independence
H
NR
HN
HN
O
3
O
O
HN
H R2
O
N
R1
O
N
H
•
Limited ability for diversification
•
Complicated substrate
identification
Coding Region for R1,
R2 and R3
O
HN
Coding Region
for R2
HN
O
Coding Region
for R3
H
NR
3
O
O
HN
H R2
O
N
R1
O
N
H
Coding Region
for R1
•Considerably simplifies the
identification of active
molecules
•Necessary to anneal further
along the template
15
Distance Independence
HS
O
N
X-X-X-X-X-X-X-X-X-X-5’
O
NH
T-G-G-T-A-C-G-A-A-T-T-C-G-A-C-T-C-G-G-G….3’
O
n bases
•As n is varied from 1 to 30, the rate does not significantly change for
Heck couplings, peptide couplings and nucleophilic addition.
•Unfortunately, not all reactions turned out to be distance independent.
16
Distance-Dependent Reactions
1,3-dipolar cycloaddition
O
N
Me
O N HN
53%
O
Me
NH N OO
O
O
N
O
H
Reductive amination
H
NH2
H
N
O
N
H
H
N
NaBH3CN
O
Nitro-Michael
81%
O
O
N
42%
O
pH 8.5 buffer
NH
O
NO2 O
O
N
N
H
O
NO2
Gartner, Z. J., Kanan, M. W., Liu, D. R. Angew. Chem. Int. Ed. 2002, 41, 1796
17
Kinetics of Distance Independance
A
k1
A
k-1
k2
B
A
B
B
In distance independent reactions, k2 >> k1
B
A
n bases
As n increases, k2 decreases.
As long as k2 > k1, reaction rate remains distance
independent.
18
Kinetics of Distance Dependance
A
k1
A
k-1
k2
B
A
B
B
If k2  k1 the coupling reaction becomes rate-determining
B
A
Since k2 decreases as n increases, the rate of the
reaction becomes dependent on the number of
bases between the reagents.
n bases
19
The  Architecture: Overcoming Distance-Dependence
A
B
A
B
Gartner, Z. J., Grubina, R., Calderone, C. T., Liu, D. R. Angew. Chem. Int. Ed. 2003, 42, 1370. 20
The  Architecture: Overcoming Distance-Dependence
A
B
A
B
10-20 base loop
A
B
10-base
coding region
4-5 constant bases
at the reactive end
•Coding-region annealing is the main driving force.
•The constant region forms a secondary structure once the coding region
is annealed.
Gartner, Z. J., Grubina, R., Calderone, C. T., Liu, D. R. Angew. Chem. Int. Ed. 2003, 42, 1370. 21
Small Molecule Synthesis: Retrosynthetic Analysis
O
O
NH
HN
O
O
N
O
O
HN
O
N
NH H
O
H
O
O Ph
Ph
P
O
Wittig
O
O
N
O OH
O
HN
HN
O
peptide
coupling
O
O
H
O
O
HN
O
NH
HO
O
OH
NH2
NH
HN
O
OH
NH
HO
oxazolidine
formation
O
OH
NH HN
O
O
O Ph
Ph
P
NH
O
O
NH
HO
O
O OH
HN
22
Multistep Synthesis of Small Molecules
HO
OH
HN
NH2
HO
O
O
OH
HN
O
O
NH2
DMT-MM
OMe
ClN
N
N
N
OMe
HO
O
DMT-MM
HN
O
O
NH
Li, X., Gartner, Z.J., Tse, B.N., Liu, D.R., J. Am. Chem. Soc. 2004, 126, 5090.
23
Multistep Synthesis of Small Molecules
HO
OH
HN
NH2
HO
O
O
OH
HN
O
O
NH2
DMT-MM
OMe
OMe
N
N
O
N
N
N
O
OMe
R
R'
N
N
O
HO
OMe
O
N
R' NH2
O
R
OMe
OH
R
N
H
HO
HN
N
N
OMe
O
O
NH
24
Multistep Synthesis of Small Molecules
HO
OH
HN
NH2
HO
O
O
OH
HN
O
O
NH2
DMT-MM
A
C
X
B
HO
HN
O
O
NH
25
Strategic Linkers
Scarless Linker
O
HN
O
O O
S
reagent
H
B:
O
O
Ph
N
H O
"H+"
NH
Ph
template
H2N
pH 11.8
template
NH
O
>95%
reagent
Gartner, Z.J., Kanan, M.W., Liu, D.R. J. Am. Chem. Soc. 2002, 124, 10304.
+
NH2
26
Strategic Linkers
Scarless Linker
O
HN
O
O
O O
S
reagent
Ph
O
H
N
H
NH
O
Ph
template
H2N
O
pH 11.8
+
>95%
B:
template
NH
NH2
reagent
Useful Scar Linker
O
reagent
HN
OH H
N
OH O
O
O
template
NH
Ph
H
N
H
NaIO4
>95%
O
O
template
NH
Ph
O
+
H
HN
reagent
Gartner, Z.J., Kanan, M.W., Liu, D.R. J. Am. Chem. Soc. 2002, 124, 10304.
O
27
Strategic Linkers
Autocleaving Linker
O
O
O
HN
Ph
P Ph
reagent
template
NH
H
template
R
O
O
O
R
NH
H
+
>95%
O
reagent
HN
Gartner, Z.J., Kanan, M.W., Liu, D.R. J. Am. Chem. Soc. 2002, 124, 10304.
Ph
P Ph
O
28
Wittig Olefination
O
O
HN
Ph
P Ph
reagent
O
O
NH
H
template
reagent
HN
Ph
P Ph
O
O
O
R
R
HN
O
template
O
template
R
O
NH
H
O
reagent
+
O
reagent
HN
HN
Ph
NH
P O
Ph
O
O
R
template
Ph
P Ph
O
29
Multistep Synthesis of Small Molecules
O
1)
HN
NH2
O O
S
O
O
2
OH
HO
O
N
H
O
OH
H2N
O
O
O
NH
3
1
DMT-MM
NH2
2) Cleavage buffer pH = 11.8
?
Purification
30
Multistep Synthesis of Small Molecules
O O
S
O
1)
HN
NH2
O
OH
HO
O
O
2
N
H
O
OH
O
H2N
O
O
NH
3
1
DMT-MM
2) Cleavage buffer pH = 11.8
Product Purification
O
HN
H
NHH
O
O
NH
S
O O
S
HN
O
HO
O
O
N
H
O
OH
Avidin
Avidin
biotin
R
O
biotin
31
Purification of DNA-Templated Reactions
Purification with scarless or useful scar linker
A
B
A
B
A
B
A
B
B
A
B
Bead-bound
avidin
Biotin
32
Purification of DNA-Templated Reactions
Purification with scarless or useful scar linker
A
B
A
B
A
B
A
B
B
A
Wash with 4M
guanidinium chloride
B
B
B
A
33
Purification of DNA-Templated Reactions
Purification with scarless or useful scar linker
A
B
A
B
A
B
A
B
B
A
Wash with 4M
guanidinium chloride
B
B
A
B
B
A
34
Purification of DNA-Templated Reactions
Purification with autocleaving linkers
A
B
A
B
A
B
A
B
B
A
Wash with 4M
guanidinium chloride
B
B
A
+
A
35
Multistep Synthesis of Small Molecules
1)
O
HN
NH2
O O
S
O
OH
HO
O
O
O
OH
N
H
O
O
2
1
H2N
O
NH
3
DMT-MM
2) Capture with Avidin beads
Wash with 4M guanidinium chloride
3) Cleavage buffer pH = 11.8
HN
OH
O
O
HO HN
OMe
ClN
N
N
N
OMe
O
DMT-MM
O
H
NH
O
O
HN
HO
O
OH
OH
H 2N
NH
HN
5
HN
4
O
O
H
O
O
36
Multistep Synthesis of Small Molecules
"H+"
O
NH
O
O
"H+"
O
HN
HO
O
O
H
NH
OH
OH
H 2N
NH
HN
O
HN
O
O
H
OH
HO
N
HN
NH
O
O
O
HN
OH
HO
NH
O
O
N
"H+"
O
HN
O
OH
HO
HO
NH
5
O
O
O
NH
N
H
O
HN
O
HN
OH
HO
O
NH
6
37
Multistep Synthesis of Small Molecules
O
O
O
O
NH
N
H
O
HN
OH
HO
OH
O
O
OH
HN
HN
O
O
P
Ph
Ph
HN
NH
N
H
O
HN
6
H
N
HO HN
O
NH
O
O Ph
Ph
P
O
HN
8
HN
OH
O
HN
O
P
Ph
Ph
O
N
O OH
O
OH
O
1)
DMT-MM
2)
Avidin beads
33% overall from 3
O
OH
O
HN
OH
O
NH
HN
O
HO
7
O
O
O
HN
38
Multistep Synthesis of Small Molecules
O
HN
O
H
O
O
O
O Ph
Ph
P
O Ph
Ph
P
OH
O
O
N
O OH
O
H
N
O HN
H
N
HO HN
HN
O
O
NaIO4
O
N
O OH
O
HN
HN
9
8
HN
O
pH 8.5
O
N
NH H
O
H
O
O Ph
Ph
P
O
O
N
O OH
O
HN
O
HN
39
Multistep Synthesis of Small Molecules
O
O
N
NH H
O
O
O
HN
O
O
NH
HN
Ph
P Ph
Self-elution
N
OHO
O
H
O
HN
O
O
N
O
O
HN
O
O
10
7% overall yield
Li, X., Gartner, Z.J., Tse, B.N., Liu, D.R., J. Am. Chem. Soc. 2004, 126, 5090.
40
Introduction to DTS
Organic Reactions in DTS
Fundamental reactions, distance dependence and independence
New, synthetically useful architectures
Example of a Small Molecule Synthesis
Synthetic strategies, linkers, purification
Towards the Synthesis of Small Molecule Libraries
Conclusions
41
Synthesis of Libraries of Macrocycles
O
HN
HN
O
H
NR
3
O
O
HN
H R2
O
N
R1
O
N
H
•A library of 65 macrocycles was successfully synthesized and screened
in one solution.
•Each synthetic step carried out in one solution was the same for all
templates, only with different reagents.
•Would it be possible to perform branching syntheses with several
different reaction classes occurring at the same time in the same solution?
Gartner, Z.J., Tse, B.N., Grubina, R., Doyon, J.B., Snyder, T.M., Liu, D.R. Science, 2004, 305, 1601.
42
One-pot, controlled reaction of cross-reactive reagents
NH
NH
O
N
O
NH2
O
N
O
O
HN
O
O
Ph Ph
P
CO2H
NH
HN
O
O
SH
NH
NH
N
O
O
O
H
NH
O
O
O2 N
O
N
O
O
N
O
O
HN
NH
O
O
O
S
HN
O
N
NO2
NH
HN
O
O
Calderone, C.T., Puckett, J.W., Gartner, Z.J., Liu, D.R. Angew. Chem. Int. Ed. Engl. 2002, 41, 4104.
43
One-pot, controlled reaction of cross-reactive reagents
NH2
NH
NH2
O
NH
O
N
O
O
Ph Ph
P
HN
O
SH
O
O
NO2
O
EDC,
Sulfo-NHS,
NaBH3CN
HN
NH
N
NH
NH
O
O
O
NH2
O
N
O
O
HN
H
NH
NH
O
NH
O
O
N
O
HN
O
NH
O
O
S
O
H
N
O2 N
O
HO
O
O
NH
CO2H
NH
O
O
HN
O
H
N
HN
O
O
Calderone, C.T., Puckett, J.W., Gartner, Z.J., Liu, D.R. Angew. Chem. Int. Ed. Engl. 2002, 41, 4104.
44
Diversification by Branching Reaction Pathways
O
NH2
H
N
N
H
O
NH2
O
O
O
N
H
OH
O
H
N
O
N
H
NH2
OH
NH2
O
N
H
O
O
NH
N
S
O
O
N
H
NH2
NHAc
OH
S
H
N
O
O
O
N
H
NHAc
S
H
N
O
NH2
NH
NH2
45
Diversification by Branching Reaction Pathways
O
NH
HO
O
NH2
NH2
N
H
11
NH2
O
O
NH
HO
N
H
SStBu
NH2
NH2
SStBu
12
O
HO
NAc
O
NH2
HN
N
H
O
Ar2P
NH2
NHAc
13
HN
O
Ar2P
Calderone, C., Liu, D.R. Angew. Chem. Int. Ed. 2005, ASAP.
46
Diversification by Branching Reaction Pathways
O
HO
NH2
N
H
O
NH
O
H
N
N
H
11
NH2
O
14
O
S
O
H
NH2
N
H
O
O
OH
N
N
H
SStBu
12
O
O
S
15
O
N
H
NH2
O
H
S
N
H
O
13
16
HN
OH
O
O
O
Ar2P
Calderone, C., Liu, D.R. Angew. Chem. Int. Ed. 2005, ASAP.
NHAc
S
O
O
NH
47
Diversification by Branching Reaction Pathways
O
O
N
H
H
N
NH2
8.3%
14
N
H
O
3.6%
15
O
O
OH
17
N
H
NH2
H
N
NH2
O
O
O
OH
18
OH
S
OH
NHAc
S
O
O
16
NH
OH
O
N
H
16
O
NH2
N
H
N
O
N
H
O
H
N
H
OMe
HN
NH2
14
N
H
N
H
O
H
N
O
O
O
OH
O
O
NH
HO
NHAc
S
O
O
NH
48
Diversification by Branching Reaction Pathways
O
N
H
O
H
N
N
H
NH2
O
O
N
H
O
H
N
NH2
O
O
O
18
14
O
N
H
15
O
HN
2%
S
NH2
O
N
H
19
O
O
O
OH
NH
N
S
NH2
OH
NHAc
S
O
O
16
NH
OH
O
N
H
16
OH
N
O
N
H
OH
NH2
H
N
N
H
NH2
O
NH2
N
H
17
14
O
O
H
N
NHAc
S
O
O
NH
49
Diversification by Branching Reaction Pathways
O
N
H
O
H
N
N
H
NH2
O
N
H
NH2
H
N
N
H
NH2
NH2
O
O
O
18
14
O
N
H
15
O
O
S
S
N
H
1.7%
NH
O
NH
NH
NH2
NHAc
NH
S
H
N
NH2
O
I
S
O
O
O
OH
NHAc
NH
0.8%
OH
S
O
20
O
N
O
NH2
O
O
O
16
HN
O
O
19
OH
16
N
H
O
N
H
O
N
H
OH
N
NHAc
OH
17
O
H
N
NH2
N
H
O
14
O
O
H
N
NHAc
N
H
21
OH
S
H
N
O
NH2 50
Ordered Multistep Synthesis in a Single Solution
Ar
P
Ph
Ph R1
Ar
P
H
Ph
Ph R2
O
Ar
P
H
Ph
Ph R3
O
O
H
One solution
R3
R2
R1
51
Ordered Multistep Synthesis in a Single Solution
O
O
O
O
H
N
H
N
H
H
N
N
H
O
N
H
H
N
N
H
NH2
O
O
O
One solution
O
Ar
Ph P
Ph
O
N
H
O
N
H
biocytin
Ar
Ph P
Ph
O
N
H
N
H
O
O
H
O
Ar
Ph P
Ph
O
N
H
N
H
H
N
H
O
O
H
N
H
O
52
Ordered Multistep Synthesis in a Single Solution
O
O
Ar
Ph P
Ph
22
O
N
H
N
H
biocytin
Ar
Ph P
Ph
O
N
H
23
N
H
O
O
Ar
Ph P
Ph
H
H
N
H
O
R2
R1
25
O
O
N
H
N
H
O
H
N
H
O
24
4oC
R3
Ph
O
Ph P
Ar R3
H
O
Ph Ar
Ar Ph
P
O P Ph
Ph
H R2
R1
Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed., ASAP.
H
53
Ordered Multistep Synthesis in a Single Solution
Ph
O
Ph P
Ar R3
H
O
O
H
Ar Ph
Ph Ar
O P Ph
Ph P
R1
H R2
H
4 to 30oC
Ar
Ph
P O
Ph
Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed., ASAP.
Ph Ar
Ph P
Ar Ph
P Ph
R3
R2
H
R1
O
54
DNA Melting Temperature
Tm
Melting Temperature
H NH
A
H
O
N
N
N
R
NR
O
N
H
G
N
N
N
N
R
T
O
N
H
N
C
NR
N
H
N
H
O
H
55
Ordered Multistep Synthesis in a Single Solution
Ph
O
Ph P
Ar R3
H
O
O
H
Ar Ph
Ph Ar
O P Ph
Ph P
R1
H R2
H
4 to 30oC
Ar
Ph
P O
Ph
Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed., ASAP.
Ph Ar
Ph P
Ar Ph
P Ph
R3
R2
H
R1
O
56
Ordered Multistep Synthesis in a Single Solution
Ph
O
Ph P
Ar R3
H
O
H
4 to 30oC
O
H
Ar Ph
Ph Ar
O P Ph
Ph P
R1
H R2
Ar
Ph
P O
Ph
Ph Ar
Ph P
Ar Ph
P Ph
R3
R2
H
R1
O
Ar
30 to 60oC
Ph
R3
R2
R1
P O
Ph
O
24% overall yield
H
26
Ar
Ph
Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed., ASAP.
P O
Ph
R1
Ar Ph
P
Ph
R3
R2
57
Conclusions
•DNA-templated synthesis is sequence-specific and compatible with
a wide variety of reaction conditions
•Reactions otherwise incompatible can be run in one pot, without
detectable side-products, enabling the synthesis of large small
molecule libraries
•Multistep syntheses can be performed selectively in one solution.
•This technique is still limited by compatibility with DNA backbone
and aqueous conditions.
58
Conclusions
•DNA-templated synthesis is sequence-specific and compatible with
a wide variety of reaction conditions
•Reactions otherwise incompatible can be run in one pot, without
detectable side-products, enabling the synthesis of large small
molecule libraries
•Multistep syntheses can be performed selectively in one solution.
•This technique is still limited by compatibility with DNA backbone
and aqueous conditions.
•Recently, DTS has been performed in THF, DMF, MeCN and DCM
with minimal amounts of water
59
Acknowledgements
Prof. Keith Fagnou
Nicole Blaquiere
Louis-Charles Campeau
Melissa Leblanc
Marc Lafrance
Jean-Philippe Leclerc
Megan Apsimon
Dave Stuart
60