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1
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
2
4
5
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Jensen, B. and Nielsen, P., Unpublished results.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
3
7
Jensen, B. and Nielsen, P., Unpublished results.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Jensen, B. and Nielsen, P., Unpublished results.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Nielsen, H. and Nielsen, P., Unpublished results.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Nielsen, H. and Nielsen, P., Unpublished results.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Nielsen, H. and Nielsen, P., Unpublished results.
5
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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Liv S. Thomsen, and Poul Nielsen, Unpublished results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Thomsen, L. S. and Nielsen, P., Unpublished results.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
9
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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
27
Kočalka , P. Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press
10
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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
29
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
30
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
11
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Kocalka, P., Andersen, N. K., Jensen F. and Nielsen, P., ChemBioChem., 2007, in press
32
Kočalka , P. Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press
33
Kočalka , P. Andersen, N. K., Jensen, F. and Nielsen, P., ChemBioChem., 2007, in press
12
34
35
Future Perspectives
� Exploring the potential of fully modified duplex structures
� Possible application in triplex systems
� Increased use of microwave reactions
36
Conclusion
� Rate increase for standard reactions
� Good perspectives for reactions with high activation energy
� High temperatures can be used
� Not always the optimal method
13
37
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