DNA: A Structual Introduction · Department of Physics And Chemistry Nucleic Acid Center University...

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Microwave-Assisted Nucleoside Chemistry and

Major Groove Functionalization of DNA

Ph.D Student Nicolai Krog Andersen

Department of Physics And Chemistry

Nucleic Acid Center

University of Southern Denmark

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Agenda

� DNA: A Structual Introduction

� Double-Headed Nucleosides

� Synthesis of Nucleoside Dimers

� Ring Closing Metathesis

� Dimers via Click Chemistry

� Functionalization of DNA

� Minor Groove Functionalization

� Major Groove Functionalization

� Future Perspectives

� Conclusion

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DNA: A Structual Introduction

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Double-Headed Nucleosides

Pedersen, S. L. and Nielsen, P., Org. Biomol. Chem., 2005, 3, 3570-3575

Wu, T. et. al. Org. Letters, 2004, 6, 51-54

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Jensen, B. and Nielsen, P., Unpublished results.

Pg = Protecting Group

Lg = Leaving Group

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Jensen, B. and Nielsen, P., Unpublished results.

Ichikawa, S. et. al., J. Org. Chem., 1997, 62, 1368-1375

Lin, T. et. al., J. Med. Chem., 1991, 34, 2607-2615

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Shatila, R. S. et. al., Tetrahedron Lett., 2006, 47, 1767-1770

Jensen, B. and Nielsen, P., Unpublished results.

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Synthesis of Nucleoside Dimers

� Stabilizing Secondary DNA and RNA Structures

� Three-way Junctions

� Bulged Duplexes

� Targeting of Secondary Structures

� Artificial Model Systems

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Wang, G. and Middelton, P. J., Nucleosides Nucleotides and Nucleic Acids, 1998, 17, 1033-1051

Nielsen, H. and Nielsen, P., Unpublished results.

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Nielsen, H. and Nielsen, P., Unpublished results.

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Nielsen, H. and Nielsen, P., Unpublished results.

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Ring Closing Metathesis

Liv S. Thomsen, and Poul Nielsen, Unpublished results

Pedersen, S. L. and Nielsen, P., Org. Biomol. Chem., 2005, 3, 3570-3575

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RCM catalysts used

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0:1:1:53015I9.44

1:1:2:23010II9.43

1:101215I9.62

0:10310II9.61

Ratio 9.6:9.7 or

9.4:9.8 : 9.6:9.7

Total rx. Time (min)Amount of

cat. mol%

CatalystDieneExp

Thomsen, L. S. and Nielsen, P., Unpublished results.

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Diene 9.6

Andersen, N. K., Thomsen, L. S. and Nielsen, P., Unpublished results

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Diene 9.4

NH

O

ON

O

ODPS

O

O

O

NH

O

ON

O

O

OR

R = HR = Pixyl

9.6

9.4

Andersen, N. K., Thomsen, L. S. and Nielsen, P., Unpublished results

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Site Specific DNA Functionalization

� Two Examples

� Minor Groove Functionalization

� 2’-Amino-LNA thymine

� Major Groove Functionalization

� Functionalization of the nucleobase C5-position

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Minor Groove Functionalization� Minor groove has been extensively modified

� 2’-Amino-LNA

� 2’-Amino-uridine

Verheyde, J. P. H., et. al., J. Org. Chem., 1971, 36, 250-254.

Sørensen, M. D., Petersen, M. and Wengel, J., Chem. Commun., 2003, 2130.

Babu, B. R. et. al., Chem. Commun., 2005, 1705.Babu, B. R., Sørensen, M. D. and Wengel, J., Unpublished results

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Functionalized 2’-amino-LNA

Sørensen, M. D., Petersen, M. and Wengel, J., Chem. Commun., 2003, 2130.Babu, B. R. et. al., Chem. Commun., 2005, 1705.

Babu, B. R., Sørensen, M. D. and Wengel, J., Unpublished results

Minor Groove

Building Blocks For Nanobiotechnology

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Major Groove Functionalization

� Aim

� To obtain good spatial control of functionalities placed in

the major groove

� Functional Diversity

� Increase duplex stability towards complementary DNA

and RNA

� Good mismatch discrimination

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Introduction of Functionalities via Click Chemistry

� Easy access to a large variety of functionalities

� Tolerance to steric demands

� “Green Chemistry”

� Easy work-up/purification

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The Building Blocks

Requirements:

� High stability

� Extended ππππ−−−−ππππ stacking� Chemoselective functionalization

Kočalka , P. Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press

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Ab-initio Calculations

- The triazole-ring and nucleobaseare found to be co-planar.

- No local minima are found only one global minimum indicating that a

single conformation is dominant.

Kočalka , P. Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press

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Synthesis of key-intermediate

Robins, M. J. and Barr, P. J., J. Org. Chem. 1983, 48, 1854-1862Graham, D., Parkinson, J. A. and Brown, T., J. Chem. Soc., Perkin. Trans. 1, 1998, 1131- 1138

Hurley, D. J. et. al., Org. Lett. 2002, 4, 2305-2308

Kočalka, P., Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press

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Initial Monomers

Kočalka , P. Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press

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Kočalka , P. Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press

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Determination of pKa

NH

O

ON

O

OH

HO

N

N

N

H

Absorption measured at 290nm

Kočalka, P. et. al., ChemBioChem., 2007, in press

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Multiple modified ON against DNA

-15

-10

-5

0

5

10

15

200 210 220 230 240 250 260 270 280 290 300

Wavelength [nm]

CD

[m

de

g]

DNA:DNA

DNA:RNA

RNA:RNA

5'-GTG XXX XGC:DNA

5'-GTG YYY YGC:DNA

5'-GTG ZZZ ZGC:DNA

Kočalka , P. Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press

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Multiple modification ON against RNA

-15

-10

-5

0

5

10

15

200 210 220 230 240 250 260 270 280 290 300

Wavelength [nm]

CD

[m

de

g]

DNA:DNA

DNA:RNA

RNA:RNA

5'-GTG XXX XGC:RNA

5'-GTG YYY YGC:RNA

5'-GTG ZZZ ZGC:RNA

Kočalka , P. Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press

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Kocalka, P., Andersen, N. K., Jensen F. and Nielsen, P., ChemBioChem., 2007, in press

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Kočalka , P. Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press

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Kočalka , P. Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press

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Future Perspectives

� Exploring the potential of fully modified duplex structures

� Possible application in triplex systems

� Increased use of microwave reactions

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Conclusion

� Rate increase for standard reactions

� Good perspectives for reactions with high activation energy

� High temperatures can be used

� Not always the optimal method

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Associate Professor Dr. Poul Nielsen

Associate Professor Dr. Scient. Frank Jensen

Dr. Petr Kočalka

Lucie Spacilova

Liv S. Thomsen

Henrik Nielsen

Brian Jensen

Joan Hansen Funding:

The Danish Research Agency´s Programme for Young Researchers, Nucleic Acid Center

The Danish National Research Foundation,

The NAC-DRUG (Nucleic Acid Based Drug Design Training Center) in the Sixth Framework Programme Marie Curie Host Fellowships for Early

Stage Research Training under contract no. MEST-CT-2004-504018,The Danish Natural Science Research Council,

Danish Center for Scientific Computing and Møllerens Fond.

Acknowledgements