Truncated pth peptides with a cyclic conformation
A technology for peptide sequences and analogs, which is applied in the field of preparation of the analogs, and can solve problems such as accumulation
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Embodiment 1
[0361] chemical synthesis
[0362] H-Ac 5 c-Val-Aib-Glu-Ile-Gln-Leu-Met-His-Gln-Har-Ala-Lys()Trp-Leu-Asn-Asp()-NH 2 (Brackets represent side chain cyclization sites) Peptide synthesis on TentaGel S Ram.
[0363] Dried TentaGel S Ram (0.23 mmol / g, 1.2 g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and processed as described above in "Batch peptide synthesis on TentaGel resin" until complete N-terminal Ac 5 Coupling of c. The N-terminal 3 amino acids were coupled as OAt esters using 3 equivalents of HATU and DIEA (2 x 2h), all other amino acids were coupled as OBt esters as above. After the final amino acid coupling is complete, the resin is drained, washed with NMP, Allyl and Aloc are deprotected as described above in "Deprotection of OAll and Aloc", and cyclization with Lys and Asp side chains is performed as described above ” said to cyclize the peptide. After cyclization the final N-terminal Fmoc group was deprotected an...
Embodiment 2
[0368] In vitro testing of PTH analogues using the MC3T3-E1 cAMP potency assay
[0369] Materials and Methods
[0370] MC3T3-E1 subclone 4 (mineralized) (MC3T3-E1) cells (ATCC CRL-2593) in α-MEM (Invitrogen #32571) / 10% fetal bovine serum + penicillin / streptomycin in 95% air / 5%CO 2 grown at 37°C in humidified air.
[0371] Peptides were dissolved in phosphate-buffered saline and further diluted in a solution containing 0.1% alkali-treated casein (ATC) (Livesey and Donald, Clin. Chim. Acta 1982, 123:193-8) and 100 μM isobutyl-methyl yellow. Purines (IBMX, Sigma I5879) in Tyrode's buffer (TB, Sigma, T2145).
[0372] cAMP concentration using [ 125 I]cAMP Assay (Catalog #SMP1001A, Perkin Elmer Life Sciences) assay.
[0373] MC3T3-E1 cells were seeded at a density of 10,000 cells / well in 96-microtiter plates and grown overnight prior to potency assays. On the day of analysis, carefully aspirate the growth medium. Cells were washed once with 200 μl TB / 0.1% ATC. The buffer...
Embodiment 3
[0397] cAMP potency assay in Saos-2 cells
[0398] Research design
[0399] The protocol is from Murrills R.J. et al. (Bone 35:1263-72, 2004), modified to suit this assay. Peptides were dissolved in phosphate buffered saline and further diluted in Tyrode's buffer (TB, Sigma-Aldrich) containing 0.1% BSA and 100 μM isobutyl-methyl-xanthine (IBMX, Sigma-Aldrich). Saos-2 cells were seeded in 96-microtiter plates at 50000 cells / well and grown for 2 days prior to potency assays. On the day of analysis, carefully aspirate the growth medium. Cells were washed once with 200 [mu]l TB / 0.1% BSA. The buffer was replaced with 100 [mu]l of the reaction mixture (± test peptide) and incubated at 37[deg.] C. for 15 minutes. The reaction was terminated by adding 25 μl of ice-cold 0.5M HCl. Cells were then incubated on ice for 60 minutes. 75 µl of acetate buffer, pH 6.2, was added to each well of a 96-well cAMPFlashPlate (Perkin-Elmer). Combine 25 μl of acid cell extract and 100 μl [ 12...
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