1. 1 DNA-Templated Synthesis: Principles of Evolution in Organic Chemistry

2. 2 EDC, Sulfo-NHS, NaBH 3 CN

3. 3 One solution

4. 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. 5 The chemist’s approach to controlling reactivity Starting materials mM – M concentration One possible product Strategies to Control Reactivity +

6. 6 The chemist’s approach to controlling reactivity Starting materials mM – M concentration One possible product Nature’s approach to controlling reactivity: Macromolecule- templated synthesis Strategies to Control Reactivity + Selective product formation nM -  M concentration Many reactants in one solution

7. 7 Synthetic Strategies The chemist’s approach to active molecule discovery: Starting material ProductData: Keq, ee, IC 50, …

8. 8 Synthetic Strategies The chemist’s approach to active molecule discovery: Starting material ProductData: Keq, ee, IC 50, … Nature’s approach to active molecule discovery: Selection, Amplification, Diversification DNARNA Protein

9. 9 The Basics of DNA-Templated Synthesis (DTS) Annealing Coupling oligonucleotide reactive group linker General Reaction Scheme Reactant for DTS SH S

10. 10 Selection and Amplification Protein Amplification PCR DNA sequencing or PAGE Identity of the active molecule

11. 11 Polymerase Chain Reaction (PCR) Denature 94 o C, 30 s Anneal primers 55 o C, 60 s 5’ 3’ 5’ 3’5’ 3’ Extension 75 o C, 30 s Taq polymerase Stop 4 o C Sample

12. 12 Synthesis of Products Unrelated to the DNA Backbone SH 1,4-conjugate addition to carbonyls S Peptide Coupling Heck 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. DMT-MM Or EDC / sulfo-NHS 54% 73% Na 2 PdCl 4

13. 13 Sequence Specificity and Distance Independence SH NH 2 HS Sequence Specificity A single base mismatch in the 10-base reagent oligonucleotide slows the reaction down by a factor of 200

14. 14 Sequence Specificity and Distance Independence Distance Independence Coding Region for R1, R2 and R3 Limited ability for diversification Complicated substrate identification

15. 15 Sequence Specificity and Distance Independence Distance Independence Coding Region for R1, R2 and R3 Limited ability for diversification Complicated substrate identification Coding Region for R1 Coding Region for R2 Coding Region for R3 Considerably simplifies the identification of active molecules Necessary to anneal further along the template

16. 16 Distance Independence T-G-G-T-A-C-G-A-A-T-T-C-G-A-C-T-C-G-G-G….3’ X-X-X-X-X-X-X-X-X-X-5’ HS 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. 17 Distance-Dependent Reactions Reductive amination Nitro-Michael 1,3-dipolar cycloaddition Gartner, Z. J., Kanan, M. W., Liu, D. R. Angew. Chem. Int. Ed. 2002, 41, 1796 53% 81% NaBH 3 CN NH 2 42% pH 8.5 buffer

18. 18 Kinetics of Distance Independance A B A B A B k1k1 k -1 k2k2 In distance independent reactions, k 2 >> k 1 BA n bases As n increases, k 2 decreases. As long as k 2 > k 1, reaction rate remains distance independent.

19. 19 Kinetics of Distance Dependance A B A B A B k1k1 k -1 k2k2 If k 2  k 1 the coupling reaction becomes rate-determining BA n bases Since k 2 decreases as n increases, the rate of the reaction becomes dependent on the number of bases between the reagents.

20. 20 The  Architecture: Overcoming Distance-Dependence Gartner, Z. J., Grubina, R., Calderone, C. T., Liu, D. R. Angew. Chem. Int. Ed. 2003, 42, 1370. B A A B

21. 21 The  Architecture: Overcoming Distance-Dependence Gartner, Z. J., Grubina, R., Calderone, C. T., Liu, D. R. Angew. Chem. Int. Ed. 2003, 42, 1370. B A A B B A 10-20 base loop 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.

22. 22 Small Molecule Synthesis: Retrosynthetic Analysis Wittig peptide coupling oxazolidine formation

23. 23 Multistep Synthesis of Small Molecules NH 2 DMT-MM Li, X., Gartner, Z.J., Tse, B.N., Liu, D.R., J. Am. Chem. Soc. 2004, 126, 5090.

24. 24 Multistep Synthesis of Small Molecules NH 2 DMT-MM

25. 25 Multistep Synthesis of Small Molecules NH 2 DMT-MM C B A X

26. 26 Strategic Linkers Scarless Linker reagent template pH 11.8 >95% reagent NH 2 + Gartner, Z.J., Kanan, M.W., Liu, D.R. J. Am. Chem. Soc. 2002, 124, 10304.

27. 27 Strategic Linkers Scarless Linker reagent template pH 11.8 >95% reagent NH 2 + Useful Scar Linker reagent template NaIO 4 >95% template reagent + Gartner, Z.J., Kanan, M.W., Liu, D.R. J. Am. Chem. Soc. 2002, 124, 10304.

28. 28 Strategic Linkers Autocleaving Linker reagent template >95% template reagent + Gartner, Z.J., Kanan, M.W., Liu, D.R. J. Am. Chem. Soc. 2002, 124, 10304.

29. 29 Wittig Olefination reagent template reagent template reagent template reagent +

30. 30 Multistep Synthesis of Small Molecules DMT-MM 2) Cleavage buffer pH = 11.8 Purification ? NH 2 DMT-MM 1) 2 1 3 NH 2

31. 31 Multistep Synthesis of Small Molecules NH 2 DMT-MM 2) Cleavage buffer pH = 11.8 Product Purification biotin Avidin biotin R 1) 2 1 3

32. 32 Purification of DNA-Templated Reactions Purification with scarless or useful scar linker A A A B B B A B A B B Bead-bound avidin Biotin

33. 33 Purification of DNA-Templated Reactions Purification with scarless or useful scar linker A A A B B B A B A B B B A Wash with 4M guanidinium chloride B

34. 34 Purification of DNA-Templated Reactions Purification with scarless or useful scar linker A A A B B B A B A B B B A Wash with 4M guanidinium chloride B A B

35. 35 Purification of DNA-Templated Reactions Purification with autocleaving linkers A A A B B B A B A B B Wash with 4M guanidinium chloride A A + B

36. 36 Multistep Synthesis of Small Molecules NH 2 DMT-MM 1) 2) Capture with Avidin beads Wash with 4M guanidinium chloride 3) Cleavage buffer pH = 11.8 1 2 3 4 5 DMT-MM

37. 37 Multistep Synthesis of Small Molecules 5 6

38. 38 Multistep Synthesis of Small Molecules 1)DMT-MM 2)Avidin beads 33% overall from 3 6 7 8

39. 39 Multistep Synthesis of Small Molecules NaIO 4 9 pH 8.5 8

40. 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. 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. 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. 43 One-pot, controlled reaction of cross-reactive reagents SH NH 2 Calderone, C.T., Puckett, J.W., Gartner, Z.J., Liu, D.R. Angew. Chem. Int. Ed. Engl. 2002, 41, 4104.

44. 44 EDC, Sulfo-NHS, NaBH 3 CN One-pot, controlled reaction of cross-reactive reagents Calderone, C.T., Puckett, J.W., Gartner, Z.J., Liu, D.R. Angew. Chem. Int. Ed. Engl. 2002, 41, 4104. NH 2 SH

45. 45 Diversification by Branching Reaction Pathways NH 2

46. 46 Diversification by Branching Reaction Pathways Calderone, C., Liu, D.R. Angew. Chem. Int. Ed. 2005, ASAP. 12 NH 2 11 13

47. 47 Diversification by Branching Reaction Pathways 14 15 16 12 11 13 Calderone, C., Liu, D.R. Angew. Chem. Int. Ed. 2005, ASAP.

48. 48 Diversification by Branching Reaction Pathways 8.3% 3.6% 17 18 14 15 16 14 16

49. 49 Diversification by Branching Reaction Pathways 17 18 14 15 16 14 16 2% 19

50. 50 Diversification by Branching Reaction Pathways 17 18 14 15 16 14 16 19 0.8% 1.7% 20 21

51. 51 Ordered Multistep Synthesis in a Single Solution One solution

52. 52 Ordered Multistep Synthesis in a Single Solution One solution

53. 53 Ordered Multistep Synthesis in a Single Solution R3R3 R2R2 R1R1 4oC4oC 22 24 23 25 Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed., ASAP.

54. 54 Ordered Multistep Synthesis in a Single Solution 4 to 30 o C Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed., ASAP.

55. 55 DNA Melting Temperature Tm Melting Temperature A T G C

56. 56 Ordered Multistep Synthesis in a Single Solution 4 to 30 o C Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed., ASAP.

57. 57 Ordered Multistep Synthesis in a Single Solution 24% overall yield 4 to 30 o C 30 to 60 o C 26 Snyder, T.M, Liu, D.R. Angew. Chem. Int. Ed., ASAP.

58. 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. 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. 60 Acknowledgements Prof. Keith Fagnou Nicole Blaquiere Louis-Charles Campeau Melissa Leblanc Marc Lafrance Jean-Philippe Leclerc Megan Apsimon Dave Stuart