Study On The Isolation, Purification And Rigioslective Synthesis Of The Long Effective Riboflavin

As a constituent of two redox coenzymes, namely flavin mononucleotide(FMN) and flavin adenine dinucleotide(FAD), riboflavin, take part in complex oxidation-reduction process and have very important pharmcaological effects. Riboflavin deficiency will bring about all kinds of pathological diseases. Besides traditional riboflavin deficiency diseases, some research in recent year suggest riboflavin have close relation to cancer, heart disease, hypertension, therrmal injury, anti--arthritic and so on. However, as a water-souble vitamins, it is easy for riboflavin to excrete out from body in urine and sweat, so riboflavin bioavalability is very low and it is very difficult for riboflavin to exhibit therapuitic effects and easily causes all kind of diseases as the result of riboflavin deficiency. Academy of Military Medical Science had succeed in developing the oil suspension injection of the long effective riboflavin in the 1960s and was equiped in 1970s and 1990s by PLA, offered an important pharmacology therapy to riboflavin deficiency diseases. In recent years, it was very satifying for the long effective riboflavin to help in treating in leukaemia ,radition therapy, chemical therapy and increased patients endurance to these therapy, which showed wide application prospect. However, as it had no clinical instructions documents, the drug was only confined to be used in the hosptials of PLA, limited to spread it further. The previous research suggested the long effective riboflavin was a mixture of different chemical regio isomers of riboflavin and the 9010- I was a mainly active princple of the mixture, the riboflavin derivative was firstly synthesized by us. Because the long effective riboflavin was a mixture of different chemical regio isomers of riboflavin, which had very similar propteries, low solubility, unstable in water, it was very difficult to isolate the 9010- I from the reaction mixture. The purification methods of TLC (thin layer chromatography) and HPLC used in the past only could get a small amount of 9010-I sample, which was very difficult to enlarge in industry. Therefore, it had been a crucial technology threshold in the new drug research to purify a great quantity of 9010- I sample.The aims in the research emphasis were to solve the crucial technology threshold of 9010- I purification, establish purifiaction process to get a large number of 9010-I .prepare >1kg 9010- I sample according to the first new drug requires, provide raw drug to compelete relevant researchs before clinical practice. At the same time, we explored the possibility to exploit the enzyme high specificity to regioslectively synthesis 9010- I in order to provide the stepping stone of seeking high selectivesynthesis way. Our main research contents and results were as follow.By meams of choosing different chromatography matrix carrier and elution solvents, we established a colunum chromatography way used sephadex LH-20 as matrix and N, N-Dimethylformamide as elution solvent. 9010- I sample isolated from LH-20 chromatography was 90. 59% purity , after it was further recrystallized in acetic ether twice, the sample purity was 99.85% analyed by HPLC, the final yield was 23.70% calculated by the crude materials. The preparation process had many advantages in good repeatance , no absorption of the gel to samplies,high recovery rate, and the elution solvent could be used repeatedly by recover. Besides, the process could enlarge parallelly acording to the scale of production, so it could fit the demands of large scale preparation. Up to date, we have got >lkg 9010- I by use of the process, the compound was verifyed as riboflavin-5'-lauric ester by UV, IR, MS, NMR spectra and 2D nuclear magnetic resonance spectra analysis.209 protease gene was cloned from bacillus pumilus, the DNA sequence of gene was 89.01% homologous to the subtilisin BPN' structure gene, and the translated coding sequence was 97. 38% to the published protein sequence of subtilisin BPN'. Of the forty two codons which distinguished from the subtilisin BPN', thirty two codons of which were synonym codons. Based on coloning 209 protease gene, we constructed different recombitants lacked of some gene fragment, screened a recombitant which was short of the signal sequence, linked with pET3a and could express in escherichia coli BL21(DE3), the expression yield was 47. 27%. The expression enzyme had high catalyze activity, the specific activity was 28U/mg, showed the highest activity in pH=10, the optimum temperature was 60 centigrade. By use of high regioselection of the enzyme catalysis to the compound formum, the enzyme was imibilized on DEAE-cellouse carrier, used lauric ether as acylating agent, Dimethylformamide as a help solvent, we realized regioselectively acylation to riboflavin.All the analysis results based on thin layer chromatography , HPLC and FAB mass spectrometery suggested the product was the aim compound 9010- I .In short, we succeed in establishing a preparation prosess to purify large amount of 9010- I , solved the problem of technical threshold in the new drug research, which provided material and technical base to cotnpelete relevant research before the clinic. We studied the possibility of using high specificity of enzyme catalysis to synthesis 9010- I regioselectively in anhydrous organic solvent and succeed in getting the aim compound. It is promising to set up a new technical way to regioselectively acylate the mutihydroxyl groups compounds by use of enzyme in organic phase.