Solvent resistant diafiltration of peptides, pna or oligonucleotides
一种寡核苷酸、肽核酸的技术，应用在制备化合物领域，能够解决复杂性、不合乎需要等问题
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Embodiment 1
[0054] A model peptide having the sequence Tyr-Ala-Tyr-Ala-Tyr was prepared using polymer membrane transmembrane enhanced peptide synthesis (MEPS). This sequence was chosen to contain one of the largest commonly used protected amino acids, Fmoc-Tyr ( t Bu) and one of the smallest protected hydrophobic amino acids, Fmoc-Ala. Chemically cross-linked polyimide membranes (DuraMem TM , MET Ltd, UK, which is believed to be prepared by the technique described in PCT / GB2007 / 050218) for the membrane separation step. The rejection of the MeO-PEG-lnker-peptide was -100% for the coupling step and >99.7% for the deprotection step.
[0055] Methylated amino polyethylene glycol (MeO-PEG-NH 2 )Synthesis
[0056] For the preparation of MeO-PEG-NH 2 The procedure is described in Flow 1:
[0057]
[0058] Scheme 1: Synthesis of methylated amino PEG
[0059] Synthesis of MeO-PEG-tosylate (1). 20g polyethylene glycol monomethyl ether (MeO-PEG, MW~5000gmol -1) were dehydrated in va...
Embodiment 2
[0071] The model peptide sequence Tyr-Ala-Tyr-Ala-Tyr was again prepared as an example of membrane-enhanced peptide synthesis with ceramic membranes. Inopor zirconia-coated membranes (Inopor GmbH, Germany) with a pore size of 3 nm and a hydrophobic surface modified were used in this example.
[0072] Membrane rejection of the MeO-PEG-peptide increased to >99.7% after the ligation of the first and second amino acids and -100% for subsequent ligation.
[0073] Methylated amino polyethylene glycol (MeO-PEG-NH 2 )Synthesis
[0074] By the same route described in Example 1, the MeO-PEG-NH used in this example was prepared 2 .
[0075] Membrane exclusion results
[0076] The rejection data for this membrane is shown in Table 1. The Inopor membrane exhibited 99% rejection of both MeO-PEG and MeO-PEG-HMPA.
[0077] Various Fmoc-amino acids were tested, involving a wide variety of properties - lowest vs highest MW amino acids, acidic, basic and hydrophobic amino acids, an...
Embodiment 3
[0090] In this example, pentapeptide thymopentin (H-Arg-Lys-Asp-Val-Tyr-OH) was prepared by membrane-enhanced peptide synthesis (MEPS) method, and was also prepared by solid-phase peptide synthesis (SPPS) method for use in for comparison.
[0091] Methylated amino polyethylene glycol (MeO-PEG-NH 2 )Synthesis
[0092] The methylated aminopolyethylene glycol synthesized for this example was based on the procedure shown in Scheme 2.
[0093]
[0094] Scheme 2: Synthesis of methylated amino PEG
[0095] Fmoc-Ala was attached to MeO-PEG (4). 10g MeO-PEG was dissolved in 20ml DCM (10ml / mmol MeO-PEG), while 1.2g Fmoc-Ala and 0.54g HOBt (both 2mol / mol MeO -PEG) was dissolved in 8ml DMF (4ml / mmol MeO-PEG), and then the two solutions were mixed together. Then 0.25 g DIC (2 mol / mol MeO-PEG) was added and mixed vigorously for 2 hours at 4°C. The solid impurities were filtered and the product was precipitated with diethyl ether and dried. The coupling step was repeated 3 tim...
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