Synthesis And Studies On Quality Control Of Flutide And Its Modiifcations

This research mainly focuses on synthesis and quality control of a phosphorothioateantisense oligonucleotide flutide and the purification of its modifications. First, anion-pair reversed phased LC method (IP-RPLC) was developed for the analysis ofrelated substances of a phosphorothioate antisense oligonucleotide flutide and routinequality control. The analytical result of IP-RPLC was compared with ion exchangechromatography(IEC) and capillary gel electrophoresis(CGE). The synthesized flutideand its analogs of monphodiester5’(P=O)1as well as deletion sequences5’ n-1and3’n-1were analyzed. The optimized conditions of IP-RPLC were as follows: XTerra MSC18(250mm X4.6mm,3.5μm) column; the mobile phase A was142mol/L1,1,1,3,3,3-hexafluoroisopropanol (HFIP)-36mmol/L triethylamine acetate (TEA)-10%methanol(V/V), the mobile phase B was methanol; The result showed that under theseconditions, flutide, deletion sequences5’ n-1and3’ n-1could be completelly separatedfrom each other and the resolution R of flutide and5’(P=O)1could reach to0.94. Theproposed IP-RPLC method has been applied for the related substances analysis andidentification of crude and purified flutide samples, indicating that this method can beused for the quality control of phoshorothioate antisense oligonucleotide.Second, IP-RPLC method was used as quantitation method of phosphorothioateoligonecleotides flutide. The separation degree of the nearest peak was0.94;theconcentration samples were all reapted three times and precision RSD were0.67%,1.0%,0.56%separately. There was good linear relationship among amount of flutidesand its function was:=26882x-71957, linearity correlation coefficient was0.9999. Thecontent determination range is from20to1200μg/ml.Third, the structure of flutide and its related substances were identified here.Because flutide belonging to oligonucleotides, its nature was different to small molecules. Thecommon identification methods of small molecules could only provide partlyinfomation of oligonucleotides. Cosidering the actural situration, we using ultravioletspectrum, infrared spectrum, mass spectrometry, nuclear magnetic resonancespectrosocopy, atomic absorption spectra and so on together for the first time toidentificate the structure and molecular of flutide and its related substances. In addition,separated by IP-RPLC, the crude and pure flutide samples were collected manually.Then it was identified by ESI-MS. The ESI-MS result showed that both flutide and itsrelated substances5’ n-1,3’ n-1and5’(P=O)1were match to theorical molecular.Then the development of purification method of modified flutide was as following:the crude product of solid-phase synthesis of modified oligonucleotide comprises thefull-length modified phosphorothioate oligonucleotide (n-mer), unmodified full lengthsequence, nucleotide deletion sequences (n-x) and unfully sulfed sequences etc.According to the hydrophobic diferences among sequences, reverse phasechromatography was used to purify modified flutide. Due to good separation ability of5’ n-1,3’ n-1and5’(P=O)1impurities, IP-RPLC method was used to evaluate the purityof collected samples of modified flutide after purification. Then, the purified sampleswere identified by ESI-MS, which showed that the molecular was match to theory. Thusproving that the reverse phase chromatography purification method of modified flutidewas feasible.This experiment developed the purification process of modified flutide.The purification scale reached2.6/time. We also optimized its purification conditionsand established the best elution gradient. Three small scale crude products were purified,the average purity was>95%and method showed high stability among different batchs.