PNA solubility enhancers

Tetrahedron Letters

Volumn 39, Issue 40, 1998, Pages 7255-7258

  Gildea, Brian D ^a   Casey, Shelagh ^a   MacNeill, Joan ^a   Perry O'Keefe, Heather ^a   Sørensen, Ditte ^a   Coull, James M ^a  

^a Boston Probes Inc   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL RNA; PEPTIDE NUCLEIC ACID; PURINE; PYRIMIDINE; RIBOSOME RNA;

ARTICLE; DOT HYBRIDIZATION; FLUORESCENCE IN SITU HYBRIDIZATION; HYDROPHILICITY; NONHUMAN; NUCLEIC ACID PROBE; NUCLEIC ACID STRUCTURE; SOLUBILITY;

EID: 0032191513     PISSN: 00404039     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0040-4039(98)01581-0     Document Type: Article

References (19)

1. 1. Nielsen, P.E., Egholm, M.E., Berg, R.H. and Buchardt, O. Science 1991, 254, 1497-1500.
2. 2. Egholm, M.E., Buchardt, O., Nielsen, P.E., and Berg, R.H. J. Am. Chem. Soc. 1992, 114, 1895-1897.
3. 3. Egholm, M.E., Buchardt, O., Christensen, L., Behrens, C., Freier, S. M., Driver, D.A., Berg, R.H., Kim, S.K., Nordén, B., and Nielsen, P.E. Nature 1993, 365, 566-568.
4. 4. Tomac, S., Sarkar, M., Ratilainen, T., Wittung, P., Nielsen, P.E., Nordén, B., Gräslund, A. J. Am. Chem. Soc. 1996, 118 5544-5552.
5. 5. Perry-O'Keefe, H., Yao, X.W., Coull, J.M., Fuchs, M. and Egholm, M.E. Proc. Natl. Acad. Sci. USA 1996, 93, 14670-14675.
6. 6. Stefano, K. and Hyldig-Nielsen, J.J., Chapter 1.2 of Diagnostic Gene Detection & Quantitation Technologies, IBC Library Series publication 1997, #948, 19-37.
7. 7. Demidov, V.V., Potaman, V.N., Frank-Kamenetskii, M.D., Egholm, M.E., Buchardt, O., Sönnichsen, S.S. and Nielsen, P.E. Biochem. Pharm. 1994, 48, 1310-1313.
8. 8. Synthesis Products Catalog, PerSeptive Biosystems, Inc., Guidelines For Design Of PNA Oligomers, p. 45 (1997-1998).
9. 9. Bergman, F., Bannwarth, W. and Tam, S. Tett. Lett. 1995, 36, 6823-6826.
10. 10. Haaima, G., Lohse, A., Buchardt, O. and Nielsen, P.E. Angew. Chem. Int. Ed. Engl. 1996, 35, 1939-1942.
11. 11. Lesnik, E., Hassman, F., Barbeau, J., Teng, K. and Weiler, K. Nucleosides & Nucleotides 1997, 16, 1775-1779.
12. 12. Diglycolic anhydride (500 mmol) in 800 mL of dichloromethane (DCM) was added dropwise with stirring to 1.1 mole of bis(2-methoxyethyl)amine. The mixture was stirred for 2 hours and then 280 mL of 6N HCl was added dropwise. The contents were transferred to a separatory funnel and the product obtained by evaporation of the organic layer. 73.8 g (296 mmole; 59 % yield).
13. 3 was added. After complete mixing, the layers were separated and the product isolated from the organic layer to provide 66.3 g of a thin yellow oil. The crude product was Kugelrohr distilled at 60 °C (200-500 μM Hg) to yield 58.9 g of a clear colorless oil (238 mmol; mmol; 95%).
14. 14. To purified N,N′-(2-methoxyethyl)-glycine-tert-butyl ester was slowly added 12.1 mL of 12N hydrochloric acid. The reaction was stirred overnight and byproducts (e.g. water, HCl, isobutylene) were removed by vacuum evaporation. A 4.4 g portion of the crude product was Kugelrohr distilled at 135-155 °C (100 - 200 μM Hg with rapidly dropping pressure after distillation began). Yield, 4.2 g.
15. 3. The mixture was stirred until all the Fmoc-aeg-OH had dissolved. In a separate flask, 1.15 mmol of 3 or 7 was dissolved in 1.15 mL of dry acetonitrile and 2.2 mmol of N-methylmorpholine and 1.25 mmol of trimethylacetyl chloride were added. This solution was stirred for 30 minute at room temperature and then added dropwise to the Fmoc-aeg-OH solution prepared as described above. The reaction was acidified (pH < 3 for 4, pH ∼ 8 for 8), and the crude product was isolated by extraction with ethyl acetate (DCM for 8). Excess pivalic acid was removed by column chromatography using a C18 stationary phase.
16. 16. Thisted, M., Just, T., Pluzek, K-J., Petersen, K.H., Hyldig-Nielsen, J.J., Godtfredsen, S.E., Cell Vision, 3, 358-363 (1996).
17. 17. Lansdorp, P.M., Verwoerd, N.P., van de Rijke, F.M., Dragowska, V., Little, M-T., Dirks, R.W., Raap, A.K. and Tanke, H.J. Human Mol. Gen. 1996, 5, 685-691.
18. 18. HPLC-purified amine containing PNA was dissolved in DMF/water (1/1, v/v) at a concentration of 330pmol/μL. From this stock, approximately 30 nmole of PNA was combined with 125 μL 0.1 M HEPES (pH 8.5), and enough DMF/water (1/1, v/v) to bring the total volume to 250 μL. This solution was thoroughly mixed and then added to a tube of Cy3 dye (Amersham) according to the manufacturers instructions.
19. 19. Buffer: 100 mM NaCl, 20 mM Sodium Phosphate (pH 7.0); 1:1 PNA:DNA each at 5.1 μmol/L; Perkin-Elmer Lambda 2S fitted with 6 cell holder running Winlab 2.0 and Templab 1.0 software.