Novel targeting strategy based on multimeric ligands for drug delivery and molecular imaging: Homooligomers of α-MSH

Bioorganic and Medicinal Chemistry Letters

Volumn 14, Issue 1, 2004, Pages 211-215

  Vagner, Josef ^a   Handl, Heather L ^b   Gillies, Robert J ^b   Hruby, Victor J ^a,b  

^a University of Arizona   (United States)

^b UNIVERSITY OF ARIZONA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ALPHA INTERMEDIN; DIMER; ISOPROTEIN; LIGAND; MELANOCORTIN RECEPTOR; MONOMER; OLIGOMER;

ARTICLE; BINDING AFFINITY; BINDING ASSAY; CONTROLLED STUDY; DRUG DELIVERY SYSTEM; EMBRYO; GENETIC TRANSFECTION; HILL REACTION; HUMAN; HUMAN CELL; IMAGING SYSTEM; MOLECULAR DYNAMICS; PROTEIN TARGETING;

EID: 0347517810     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2003.09.079     Document Type: Article

References (17)

1. Mammen M., Choi S.K., Whitesides G.M. Angew. Chem., Int. Ed. 37:1998;2755.
2. Gillies R.J., Hruby V.J. Expert Opin. Ther. Targets. 7:2003;137.
3. Fan E.K., Zhang Z.S., Minke W.E., Hou Z., Verlinde C., Hol W.G.J. J. Am. Chem. Soc. 122:2000;2663.
4. Sharma S.D., Granberry M.E., Jiang J., Leong S.D.L., Hadley M.E., Hruby V.J. Bioconjugate Chem. 5:1994;591.
5. Sharma S.D., Jiang J., Hadley M.E., Bentley D.L., Hruby V.J. Proc. Natl. Acad. Sci. U.S.A. 93:1996;13715.
6. Schwyzer R., Kriwaczek V.M. Biopolymers. 20:1981;2011.
7. Kessler, H.; Schudok, M.; Haupt, A. In Peptides; Jung, G., Bayer, E., Eds.; de Gruyter: Berlin, New York, 1988; p 664.
8. Brandenburg Y., Rose K., Bagutti C., Eberle A.N. J. Recept. Signal Transduction Res. 19:1999;467.
9. Sawyer T.K., Sanfilippo P.J., Hruby V.J., Engel M.H., Heward C.B., Burnett J.B., Hadley M.E. Proc. Natl. Acad. Sci. U.S.A. 77:1980;5754.
10. Hruby, V. J.; Han, G. In The Melanocortin Receptors; Cone, R. D., Ed.; Humana: Totowa, 1997; Chapter 8, p 239.
11. Cook R.M., Adams J.H., Hudson D. Tetrahedron Lett. 35:1994;6777.
12. α-Fmoc amino acid, 3 equiv of HBTU, and 6 equiv of DIEA in DMF) for 3 h. If the second coupling did not result in a negative Kaiser test, the resin was washed with DMF, and the rest of amino groups were capped with 50% acetic anhydride in pyridine for 10 min. When the coupling reaction was finished, the resin was washed with DMF, and the same procedure was repeated for the next amino acid until all the amino acids in the sequence were attached. After the last amino acid was incorporated, the Fmoc group was deprotected and the free amine groups was acetylated with 50% acetic anhydride in pyridine for 10 min, if needed. The resin was washed with DMF, THF and DCM. A cleavage mixture (10 mL per 1 g of the resin) consisting of trifluoroacetic acid (91%), water (3%), 1,2-ethandithiol (3%), and thioanisole (3%) was injected into the resin and the cleavage cocktail was stirred for 3 h at room temperature. The solution was filtered off, and the resin was washed with TFA (2×3 min), concentrated by a stream of nitrogen and the product was precipitated by cold ether. The peptide pellets were washed three times with cold ether, then lyophilized.
13. Purification of the compounds was achieved using a Hewlett-Packard 1100 series HPLC instrument with a reverse-phase column (Vydac, 10 mm × 220 mm, 10 μm, 300 Å). Peptides were eluted with a linear acetonitrile/ 0.1% aqueous TFA gradient at a flow rate of 3.0 mL/min. Separations were monitored at 230 and 280 nm with Hewlett-Packard 110 series UV detector and integrated with Hewlett-Packard 3396 series III integrator. The size exclusion chromatography (SEC) was performed on borosilicate glass column (2.6 × 50 cm, Sigma, St. Louis, MO, USA) filled with medium size Sephadex G-25 (Aldrich, Milwaukee, WI, USA). The compound was eluted with isocratic flow of 1.0 M aqueous acetic acid.
14. Thin-layer chromatography was performed in three different solvent systems and analytical reverse-phase C-18 HPLC using YMC ODS AM032 column (4.6 × 150 mm, 5 μm, 120 Å) at 220 and 280 nm. The compounds were eluted with a linear acetonitrile in 0.1% aqueous TFA gradient.
15. MALDI-TOF (Bruker Reflex-III, α-cyanocinnamic acid as a matrix) or ESI (Finnigan, Thermoquest LCQ ion trap instrument). For internal calibration an appropriate mixture of standard peptides was used with an average resolution of 8000-9000.
16. 50 values and Hill coefficients reported are an average of at least three separate binding assays all done in duplicate. One data point for compound 3 was excluded, as it was an obvious outlier with a Hill coefficient > 8.
17. Motulsky H.J. Analyzing Data with GraphPad Prism. 1999;GraphPad Prism Software, San Diego, CA.