Syntheses of polycationic dendrimers on lipophilic peptide core for complexation and transport of oligonucleotides

Bioorganic and Medicinal Chemistry Letters

Volumn 12, Issue 18, 2002, Pages 2635-2637

  Wimmer, Norbert ^a   Marano, Robert J ^b   Kearns, Philip S ^a   Rakoczy, Elizabeth P ^b   Toth, Istvan ^a  

^a UNIVERSITY OF QUEENSLAND   (Australia)

^b UNIVERSITY OF WESTERN AUSTRALIA   (Australia)

Author keywords

[No Author keywords available]

Indexed keywords

DENDRIMER; OLIGONUCLEOTIDE; PEPTIDE; POLYCATION; VASCULOTROPIN;

ARTICLE; CALORIMETRY; COMPLEX FORMATION; CONTROLLED STUDY; DRUG SYNTHESIS; ENZYME LINKED IMMUNOSORBENT ASSAY; GENETIC TRANSFECTION; HUMAN; HUMAN CELL; LIPOPHILICITY; MASS SPECTROMETRY; PIGMENT CELL;

CELLS, CULTURED; ENDOTHELIAL GROWTH FACTORS; HUMANS; INTERCELLULAR SIGNALING PEPTIDES AND PROTEINS; LYMPHOKINES; OLIGONUCLEOTIDES; PEPTIDES; POLYAMINES; VASCULAR ENDOTHELIAL GROWTH FACTOR A; VASCULAR ENDOTHELIAL GROWTH FACTORS;

EID: 0037119744     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0960-894X(02)00511-5     Document Type: Article

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22. General experimental procedure for the synthesis of compounds 4-7 in detail: MBHA resin (4-methyl benzhydrylamine, substitution ratio, 0.62 mmol/g, 1000 mg) was swelled in dimethylformamide in a sintered glass peptide synthesis vessel for 90 min. An activation mixture consisting of Boc-amino acid (3 equiv per mol amino-group), HBTU (2-(1H benzotriazole-1-yl)-1,3,3-tetramethyluronium hexafluoro phosphate, 0.5 M in DMF, 3 equiv) and DIEA (0.442 mL, 4 equiv) was shaken with the resin for 12 min. Negative ninhydrin reaction (5 min) showed nearly quantitative coupling (≥99.98%) and the Boc protecting group was subsequently removed using 100% TFA. Between all manipulations the resin was washed intensively with DMF. Coupling and deblocking of Boc protecting groups were done in an analogous manner for peptides 4-7. Upon completion of the synthesis and removal of the terminal Boc groups, the resin was washed with DMF, methanol and DCM. The resin was dried to constant weight over KOH in vacuum. The peptides were cleaved from the resin using a high HF method and p-cresol as scavenger. The cleaved peptide was precipitated using diethyl ether, redissolved in 2.5% aqueous acetic acid and lyophilised to afford an amorphous powder.
23. Analytical RP-HPLC was performed on a Shimadzu instrument (LC-10AT liquid chromatograph, SCL-10A system controller, SPD-6A UV detector, a SIL-6B auto injector with a SCL-6B system controller, and columns C4, C18; 25 cm Vydac C4, C18 column with 5 nm pore size and 4.6 mm internal diameter) in order to optimize the appropriate gradient for the preparative HPLC and to identify the synthesised peptide. The amount of 100 mg of each crude peptide (4-7) was preparative separated on a Waters HPLC system (Model 600 controller, 490E UV detector, F pump, and TSK-GEL C4/C18 columns with 10 nm pore size and 2.5 cm internal diameter) using a acetonitrile/water gradient and characterised by electrospray-MS (Perkin Elmer API 3000 instrument). The resulting peptides were used as diastereomeric mixtures.
24. 16 ITC experiments were performed in a MicroCal VP-ITC microcalorimeter. The oligonucleotide solution (2 μM) was placed in the sample cell and the dendrimer (243 μM) was placed in the syringe. The cell temperature was maintained at 30°C and the dendrimer was added to the solution of oligonucleotide using 25 × 4 μL injections, each injection was 4 min apart.
25. 2) for 24 h after which the media was removed for analysis. 500 μL of media from each of the sample was placed in a microcon-30™ concentrator and centrifuged to a volume of 10 μL. 500 μL of phosphate buffer (pH 7.2) was then applied to wash the sample which was centrifuged to a final volume of 100 μL. The samples were then used in an sandwich ELISA assay as per the manufactures instructions (Cytelisa™ Human VEGF kit, CYTIMMUNE Sciences Inc., Maryland, USA).