Human PrRP analogs (see Table 1 for structures) and 1DMe (D-YL(N-Me)FQPQRF-NH2), a stable analog of NPFF, were synthesized and purified as described previously [10 (link)] using Fmoc strategy. The peptide sequences were assembled in a solid-phase synthesizer Liberty Blue (CEM, Mathews, NC, USA) by stepwise coupling of the corresponding Fmoc-amino acids to the growing chain on TENTA GEL S RAM resin (200–400 mesh, 0.25 mmol/g) (IRIS, Biotech GmbH, Marktredwitz, Germany). Fully protected peptide resins were synthesized according to a standard procedure involving (i) cleavage of the Nα- Fmoc protecting group with 20% piperidine in dimethylformamide (DMF), (ii) coupling, mediated by mixtures of coupling reagents diisopropylcarbodiimide ( DIC)/Oxyma in DMF. Lipidization of the PrRP analogs was performed as shown in [16 (link)] on fully protected peptides on resine after the coupling of γ-Glu or 1,13-diamino-4,7,10-trioxatridecan-succinamic acid (TTDS) as the last step. For Lys11, special protecting group of side-chain, N-[1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl] (Dde) was used. Cleavage of Dde was performed by 2% hydrazine monohydrate in N-Methyl-2-pyrrolidone.
On completion of syntheses, the deprotection and detachment of peptides from the resins were carried out simultaneously, using a trifluoroacetic acid (TFA)/H2O/Triisopropylsilane (TIS) (95:2.5:2.5) cleaving mixture. Each of the resins was washed with a dichloromethane and the combined TFA filtrates were evaporated at room temperature. The precipitated residues were triturated with tert-butyl-methylether, collected by suction and dried by lyophilization. The peptides were purified by HPLC using a Waters instrument with Delta 600 pump, 2489 UV/VIS detector (Milford, MA, USA).
The purity and identity of all of the peptides were determined by analytical HPLC and by using a MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Germany) (seeS1 Table ).
Molecular weight was determined by MALDI MS technique (Bruker Daltonics, Germany). In HPLC analyses, retention time in minutes, separation on 25 x 0.46 cm column, 5 μm (Vydac 218TP C18, Separations Group, Hesperia, USA), Waters Alliance instrument, detection at 220 nm. Gradient 2–80% of acetonitrile in 0.1% aqueous TFA, 25 min, 80–100% 2 min, flow 1ml/min.
Human PrRP31 and 1DMe were iodinated at Tyr20 and D-Tyr1, respectively, with Na125I (Izotop, Budapest, Hungary) as described previously [16 (link)].
On completion of syntheses, the deprotection and detachment of peptides from the resins were carried out simultaneously, using a trifluoroacetic acid (TFA)/H2O/Triisopropylsilane (TIS) (95:2.5:2.5) cleaving mixture. Each of the resins was washed with a dichloromethane and the combined TFA filtrates were evaporated at room temperature. The precipitated residues were triturated with tert-butyl-methylether, collected by suction and dried by lyophilization. The peptides were purified by HPLC using a Waters instrument with Delta 600 pump, 2489 UV/VIS detector (Milford, MA, USA).
The purity and identity of all of the peptides were determined by analytical HPLC and by using a MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Germany) (see
Molecular weight was determined by MALDI MS technique (Bruker Daltonics, Germany). In HPLC analyses, retention time in minutes, separation on 25 x 0.46 cm column, 5 μm (Vydac 218TP C18, Separations Group, Hesperia, USA), Waters Alliance instrument, detection at 220 nm. Gradient 2–80% of acetonitrile in 0.1% aqueous TFA, 25 min, 80–100% 2 min, flow 1ml/min.
Human PrRP31 and 1DMe were iodinated at Tyr20 and D-Tyr1, respectively, with Na125I (Izotop, Budapest, Hungary) as described previously [16 (link)].
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