Properties of fluorophores on solid phase resins; Implications for screening, encoding and reaction monitoring

Bioorganic and Medicinal Chemistry Letters

Volumn 7, Issue 12, 1997, Pages 1567-1572

  Scott, Robin H ^a   Balasubramanian, Shankar ^a  

^a UNIVERSITY OF CAMBRIDGE   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

DYE;

ARTICLE; DRUG SYNTHESIS; FLUORESCENCE; REACTION ANALYSIS; TECHNIQUE;

EID: 0030957160     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0960-894X(97)00263-1     Document Type: Article

References (19)

1. For a review of combinatorial screening methods see Lebl, M.; Krchlñák, V.; Spetov, N.; Seligmann, B.; Strop, B.; Felder, S.; Lam, K.S. Biopolymers, 1995, 37, 177.
2. Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature, 1991, 354, 82.
3. Needels, M.C.; Jones, D. G.; Tate, E. H.; Heinkel, G. L.; Kochersperger, L. M.; Dower, W. J.; Barett, R. W.; Gallop, M. A. Proc. Natl. Acad. Sci. USA, 1993, 90, 10700.
4. Chen, J. K.; Lane, W. S.; Brauer, A. W.; Tanaka, A.; Schreiber, S. L. J. Am. Chem. Soc., 1993, 115, 12591.
5. Meldal, M.; Svendsen, I.; Breddam, K.; Auzanneau, F. Proc. Natl. Acad. Sci. USA, 1994, 91, 3314.
6. th American Peptide Symposium; Hodges, R. S.; Smith, J. A., Ed.; Escom: Leiden, 1994; 1012-1015.
7. Holmes, C. P.; Adams, C. L.; Kochersperger, L. M.; Mortensen, R. B.; Aldwin, L. A. Biopolymers, 1995, 37, 199.
8. Yamashita, D. S.; Weinstock, J. U.S. Patent WO 95/32425, 1995.
9. Felix, A. M.; Jimenez, M. H. Anal. Biochem. 1994, 52, 377.
10. Medvedkin, V. N.; Permyakov, E. A.; Uverskii, V. N.; Gripas, A. F.; Mitin, Y. V. Soviet Journal of Bioorganic Chemistry, 1994, 20, 334.
11. Typical labelling procedure. Dansyl chloride (3) (2.0 μmol, from a 0.50% stock solution in anhydrous DMF, 0.10 eq) and anhydrous pyridine (10 μl, excess) were added to resin b (1.0 mmol/g, 20.0 mg, 20 μmol, 1.0 eq) swollen in anhydrous DMF (1.0 ml). The solution phase lost its colour (yellow) within a few minutes while the resin became stained. The reaction vessel was sealed, excluded from light and allowed to stand at room temperature (3 hr). The resin was washed with DMF (2 × 2 ml), DCM (2 × 2 ml), MeOH (2 × 2 ml) and DCM (2 × 2 ml), and dried under vacuum. Yield was confirmed by sulfur analysis to within 10% of the theoretical yield.
12. Demtröder, W. Laser Spectroscopy, Schäfer, F. P., Ed., Springer: New York, 1981, 116-443.
13. (a) Laser, Ex 527 nm, Em 580-650 nm. (b) Molecular Dynamics Vistra Fluorimager SI, argon-ion laser 488 nm, Em >515nm. The beads are spread out on a glass plate and scanned. Data points are taken from the average of 5 identically labelled beads, (c) Shimadzu RF 5001-PC spectrophotofluorometer. Spectra acquired from a mono-dispersion of resin, maintained by magnetic stirring, in dimethylformamide.
14. Solution analogues were synthesised in situ by acquiring the solution spectrum in DMF in the presence of 5% 2-methoxyethylamine or 5% benzylamine to mimic resins a and b respectively.
15. Spectra are deconvoluted by subtracting a weighted standard spectra of the first fluorophore such that a reference wavelength at which the second fluorophore does not emit is set to zero. Commercial software exists to deconvolute complex spectrum using methods such as 'least squares' if standards are known.
16. Resin a: loading 90 pmol/bead, size 90 μm, site dimension 1.9 nm (dry), 2.8 nm (3 times swollen in solvent). Resin b: loading, 175 pmol/bead, size 55 μm, site dimension 0.94 nm (dry), 1.4 nm (swollen). Resins purchased from Novabiochem.
17. Quantum yields of the 2-methoxyethylamine derivatives of the fluorophores were measured in DMF relative to the dansyl derivative. For methods see Bridges, J. W. The determination of Quantum Yields, Standards in Fluorescence Spectroscopy, Miller, J. N., Ed., Chapman and Hall: London, 1981, 68-79.
18. Wu, P.; Brand, L. Anal. Biochem. 1994, 218, 1.
19. Cheung, Y. W. Unpublished Work.