DNATemplated Synthesis:

1
DNA-Templated Synthesis Principles of
Evolution in Organic Chemistry
2
EDC, Sulfo-NHS, NaBH3CN
3
One solution
4
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
5
Strategies to Control Reactivity
The chemists approach to controlling reactivity

Starting materials mM M concentration
One possible product
6
Strategies to Control Reactivity
The chemists approach to controlling reactivity

Starting materials mM M concentration
One possible product
Natures approach to controlling reactivity
Macromolecule-templated synthesis
Selective product formation
nM - ?M concentration Many reactants in one
solution
7
Synthetic Strategies
The chemists approach to active molecule
discovery
Starting material
Product
Data Keq, ee, IC50,
8
Synthetic Strategies
The chemists approach to active molecule
discovery
Starting material
Product
Data Keq, ee, IC50,
Natures approach to active molecule discovery
DNA
RNA
Protein
Selection, Amplification, Diversification
9
The Basics of DNA-Templated Synthesis (DTS)
Reactant for DTS
reactive group
oligonucleotide
linker
General Reaction Scheme
SH
Annealing
SH
Coupling
S
10
Selection and Amplification
Protein
Protein
Amplification PCR
DNA sequencing or PAGE
Identity of the active molecule
11
Polymerase Chain Reaction (PCR)
Denature 94oC, 30 s
5
3
Sample
3
5
Anneal primers 55oC, 60 s
Stop 4oC
5
3
Extension 75oC, 30 s
5
3
Taq polymerase
12
Synthesis of Products Unrelated to the DNA
Backbone
1,4-conjugate addition to carbonyls
SH
S
Peptide Coupling
73
DMT-MM Or EDC / sulfo-NHS
Heck
54
Na2PdCl4
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.
13
Sequence Specificity and Distance Independence
Sequence Specificity
NH2
SH
HS
A single base mismatch in the 10-base reagent
oligonucleotide slows the reaction down by a
factor of 200
14
Sequence Specificity and Distance Independence
Distance Independence

• Limited ability for diversification
• Complicated substrate identification

Coding Region for R1, R2 and R3
15
Sequence Specificity and Distance Independence
Distance Independence

• Limited ability for diversification
• Complicated substrate identification

Coding Region for R1, R2 and R3

• Considerably simplifies the identification of
  active molecules
• Necessary to anneal further along the template

Coding Region for R2
Coding Region for R1
Coding Region for R3
16
Distance Independence
HS
X-X-X-X-X-X-X-X-X-X-5
T-G-G-T-A-C-G-A-A-T-T-C-G-A-C-T-C-G-G-G.3
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.

17
Distance-Dependent Reactions
1,3-dipolar cycloaddition
53
Reductive amination
NH2
81
NaBH3CN
Nitro-Michael
42
pH 8.5 buffer
Gartner, Z. J., Kanan, M. W., Liu, D. R. Angew.
Chem. Int. Ed. 2002, 41, 1796
18
Kinetics of Distance Independance
A
k1
A
k2
A
B
B
k-1
B
In distance independent reactions, k2 gtgt k1
B
A
As n increases, k2 decreases. As long as k2 gt
k1, reaction rate remains distance independent.
n bases
19
Kinetics of Distance Dependance
A
k1
A
k2
A
B
B
k-1
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
20
The ? Architecture Overcoming
Distance-Dependence
A
A
B
B
Gartner, Z. J., Grubina, R., Calderone, C. T.,
Liu, D. R. Angew. Chem. Int. Ed. 2003, 42, 1370.
21
The ? Architecture Overcoming
Distance-Dependence
A
A
B
B
10-20 base loop
A
B
4-5 constant bases at the reactive end
10-base coding region

• 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.
22
Small Molecule Synthesis Retrosynthetic Analysis
Wittig
peptide coupling
oxazolidine formation
23
Multistep Synthesis of Small Molecules
NH2
NH2
DMT-MM
DMT-MM
Li, X., Gartner, Z.J., Tse, B.N., Liu, D.R., J.
Am. Chem. Soc. 2004, 126, 5090.
24
Multistep Synthesis of Small Molecules
NH2
NH2
DMT-MM
25
Multistep Synthesis of Small Molecules
NH2
NH2
DMT-MM
B
A
C
X
26
Strategic Linkers
Scarless Linker
template
template
reagent
pH 11.8

gt95
NH2
reagent
Gartner, Z.J., Kanan, M.W., Liu, D.R. J. Am.
Chem. Soc. 2002, 124, 10304.
27
Strategic Linkers
Scarless Linker
template
template
reagent
pH 11.8

gt95
NH2
reagent
Useful Scar Linker
template
template
reagent
NaIO4

gt95
reagent
Gartner, Z.J., Kanan, M.W., Liu, D.R. J. Am.
Chem. Soc. 2002, 124, 10304.
28
Strategic Linkers
Autocleaving Linker
template
template
reagent

gt95
reagent
Gartner, Z.J., Kanan, M.W., Liu, D.R. J. Am.
Chem. Soc. 2002, 124, 10304.
29
Wittig Olefination
template
reagent
reagent
template
template
template
reagent

reagent
30
Multistep Synthesis of Small Molecules
1)
NH2
2
3
DMT-MM
DMT-MM
1
NH2
2) Cleavage buffer pH 11.8
?
Purification
31
Multistep Synthesis of Small Molecules
1)
NH2
2
3
DMT-MM
1
2) Cleavage buffer pH 11.8
Product Purification
Avidin
Avidin
biotin
R
biotin
32
Purification of DNA-Templated Reactions
Purification with scarless or useful scar linker
A
B
A
A
B
B
A
B
B
A
B
Bead-bound avidin
Biotin
33
Purification of DNA-Templated Reactions
Purification with scarless or useful scar linker
A
B
A
A
B
B
A
B
B
A
Wash with 4M guanidinium chloride
B
B
B
A
34
Purification of DNA-Templated Reactions
Purification with scarless or useful scar linker
A
B
A
A
B
B
A
B
B
A
Wash with 4M guanidinium chloride
B
B
B
B
A
A
35
Purification of DNA-Templated Reactions
Purification with autocleaving linkers
A
B
A
A
B
B
A
B
B
A
Wash with 4M guanidinium chloride
B
B
A

A
36
Multistep Synthesis of Small Molecules
1)
NH2
2
3
1
DMT-MM
2) Capture with Avidin beads Wash with 4M
guanidinium chloride 3) Cleavage buffer pH 11.8
4
DMT-MM
5
37
Multistep Synthesis of Small Molecules
5
6
38
Multistep Synthesis of Small Molecules
7
6

• DMT-MM
• Avidin beads
• 33 overall from 3

8
39
Multistep Synthesis of Small Molecules
NaIO4
9
8
pH 8.5
40
Multistep Synthesis of Small Molecules
Self-elution
10
7 overall yield
Li, X., Gartner, Z.J., Tse, B.N., Liu, D.R., J.
Am. Chem. Soc. 2004, 126, 5090.
41
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
42
Synthesis of Libraries of Macrocycles

• 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.
43
One-pot, controlled reaction of cross-reactive
reagents
NH2
SH
Calderone, C.T., Puckett, J.W., Gartner, Z.J.,
Liu, D.R. Angew. Chem. Int. Ed. Engl. 2002, 41,
4104.
44
One-pot, controlled reaction of cross-reactive
reagents
EDC, Sulfo-NHS, NaBH3CN
SH
NH2
Calderone, C.T., Puckett, J.W., Gartner, Z.J.,
Liu, D.R. Angew. Chem. Int. Ed. Engl. 2002, 41,
4104.
45
Diversification by Branching Reaction Pathways
NH2
46
Diversification by Branching Reaction Pathways
NH2
11
NH2
NH2
12
NH2
13
NH2
Calderone, C., Liu, D.R. Angew. Chem. Int. Ed.
2005, ASAP.
47
Diversification by Branching Reaction Pathways
11
14
12
15
13
16
Calderone, C., Liu, D.R. Angew. Chem. Int. Ed.
2005, ASAP.
48
Diversification by Branching Reaction Pathways
8.3
17
14
18
3.6
14
15
16
16
49
Diversification by Branching Reaction Pathways
17
14
18
14
2
19
15
16
16
50
Diversification by Branching Reaction Pathways
17
14
18
14
19
15
1.7
20
16
21
0.8
16
51
Ordered Multistep Synthesis in a Single Solution
One solution
52
Ordered Multistep Synthesis in a Single Solution
One solution
53
Ordered Multistep Synthesis in a Single Solution
22
23
R2
R1
25
24
R3
4oC
Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed.,
ASAP.
54
Ordered Multistep Synthesis in a Single Solution
4 to 30oC
Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed.,
ASAP.
55
DNA Melting Temperature
Tm Melting Temperature
T
A
C
G
56
Ordered Multistep Synthesis in a Single Solution
4 to 30oC
Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed.,
ASAP.
57
Ordered Multistep Synthesis in a Single Solution
4 to 30oC
30 to 60oC
24 overall yield
26
Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed.,
ASAP.
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.

59
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

60
Acknowledgements
Prof. Keith Fagnou Nicole Blaquiere Louis-Charles
Campeau Melissa Leblanc Marc Lafrance Jean-Philip
pe Leclerc Megan Apsimon Dave Stuart