2’-氧—甲基—核糖核苷的合成研究
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摘要
2′-氧-甲基-核糖核苷,是通过对核苷进行甲基化修饰而得到的。这类2′-氧-甲基-核糖核苷广泛存在于RNA和DNA中,且成为用于第二代基因治疗的寡聚核苷酸序列合成的基本原料组分。含有这样核糖核酸的寡聚核苷酸在体内和体外都表现了较好的稳定性和较好地与目标互补基因的亲和能力。药理作用广泛,具有免疫调节、抗菌、抗炎、抗肿瘤等方面的作用。
本文通过对2′-氧-甲基-核糖核苷的合成路线研究,综述并设计了可行的合成路线及方法。其中2′-氧-甲基-腺嘌呤核苷的合成是在无保护的情况下,直接使用重氮甲烷对腺嘌呤核苷甲基化,最终经过阴离子交换树脂分离得到纯的2′-氧-甲基-腺嘌呤核苷。而2′-氧-甲基-鸟嘌呤核苷的合成是以鸟嘌呤核苷为起始原料,经过五步反应合成出2′-氧-甲基-鸟嘌呤核苷。首先对鸟苷进行乙酰化反应,将鸟苷糖基上的3个羟基保护起来,得到2′,3′,5′-三-氧-乙酰基鸟苷;其次经氯化反应,使鸟苷上的6-位羟基被氯置换,得到化合物2-氨基-6-氯-2′,3′,5′-三-氧-乙酰基鸟苷;然后经NH_3气-MeOH的碱性条件处理,将乙酰基脱去,制得2-氨基-6-氯-鸟苷;之后再使用重氮甲烷对其进行甲基化,得到2-氨基-6-氯-2′-氧-甲基鸟苷,此步也会生成2-氨基-6-氯-3′-氧-甲基鸟苷,最后将2′和3′的异构体水解,使6位上的氯脱掉,最终经过阴离子交换树脂分离得到纯的2′-氧-甲基-鸟嘌呤核苷。
通过熔点测试、红外、质谱、核磁等分析手段对合成的化合物的结构表征,结果表明所得的产物符合目标产物,所合成的化合物的结构、性能指标与设计目标要求一致。
2′-O-Methylribonucleosides are modified by methylation of ribonucleosides. 2′-O-Methylribonucleosides are widely distributed in RNA and DNA, and now have been extensively utilized as a second-generation oligonucleotide constructs due to their favorable in-vitro and in-vivo properties. They provide a better stability and also strong binding capability to the complementary gene targets. They have many pharmacological functions such as immunoregulation, antibacterial, diminish inflammation and kill tumours.
In this dissertation, the synthesis ways of 2′-O-Methylribonucleosides were studied and summarized, and practical and feasible ways were designed. The synthesis of 2′-O-methyl adenosine was directly accomplished by methylation of adenosine with diazomethane without protection, then the isomers were isolated through ion exchange chromatography on Dowex 1-X2(OH~-), getting pure 2′-O-methyl adenosine. The synthesis of 2′-O-methyl guanosine, starting from guanosine through five steps. Firstly, acetylation of guanosine, protecting three hydroxyl of guanosine. Secondly, 2-amino-6-chloro-9-β-D-ribofuranosyl-purine has been prepared via chlorination using phosphorus oxychloride or sulfur dioxide, and deacetylation of 2′,3′,5′-tri-O-acetyl guanosine. Moreover, high regioselective methylation of 2-amino-6-chloro-9-β-D-ribofuranosyl-purine with diazomethane, getting 2-amino-6-chloro-9-[2′-O-methyl-β-D-ribofuranosyl]-purine. Then followed by hydrolysis provided 2′-O-methyl guanosine and 3′-O-methyl guanosine. The isomers were isolated through ion exchange chromatography on Dowex 1-X2(OH~-), getting pure 2′-O-methyl guanosine.
The structure of the synthetic products were characterized by Melting Point(MP), Infrared Spectoscopy(IR), Mass Spectra(MS) and ~1H Nuclear Magnetic Resonance(NMR). The results proved: the structures and performance indicated that the products conformed to the design requirement.
本文通过对2′-氧-甲基-核糖核苷的合成路线研究,综述并设计了可行的合成路线及方法。其中2′-氧-甲基-腺嘌呤核苷的合成是在无保护的情况下,直接使用重氮甲烷对腺嘌呤核苷甲基化,最终经过阴离子交换树脂分离得到纯的2′-氧-甲基-腺嘌呤核苷。而2′-氧-甲基-鸟嘌呤核苷的合成是以鸟嘌呤核苷为起始原料,经过五步反应合成出2′-氧-甲基-鸟嘌呤核苷。首先对鸟苷进行乙酰化反应,将鸟苷糖基上的3个羟基保护起来,得到2′,3′,5′-三-氧-乙酰基鸟苷;其次经氯化反应,使鸟苷上的6-位羟基被氯置换,得到化合物2-氨基-6-氯-2′,3′,5′-三-氧-乙酰基鸟苷;然后经NH_3气-MeOH的碱性条件处理,将乙酰基脱去,制得2-氨基-6-氯-鸟苷;之后再使用重氮甲烷对其进行甲基化,得到2-氨基-6-氯-2′-氧-甲基鸟苷,此步也会生成2-氨基-6-氯-3′-氧-甲基鸟苷,最后将2′和3′的异构体水解,使6位上的氯脱掉,最终经过阴离子交换树脂分离得到纯的2′-氧-甲基-鸟嘌呤核苷。
通过熔点测试、红外、质谱、核磁等分析手段对合成的化合物的结构表征,结果表明所得的产物符合目标产物,所合成的化合物的结构、性能指标与设计目标要求一致。
2′-O-Methylribonucleosides are modified by methylation of ribonucleosides. 2′-O-Methylribonucleosides are widely distributed in RNA and DNA, and now have been extensively utilized as a second-generation oligonucleotide constructs due to their favorable in-vitro and in-vivo properties. They provide a better stability and also strong binding capability to the complementary gene targets. They have many pharmacological functions such as immunoregulation, antibacterial, diminish inflammation and kill tumours.
In this dissertation, the synthesis ways of 2′-O-Methylribonucleosides were studied and summarized, and practical and feasible ways were designed. The synthesis of 2′-O-methyl adenosine was directly accomplished by methylation of adenosine with diazomethane without protection, then the isomers were isolated through ion exchange chromatography on Dowex 1-X2(OH~-), getting pure 2′-O-methyl adenosine. The synthesis of 2′-O-methyl guanosine, starting from guanosine through five steps. Firstly, acetylation of guanosine, protecting three hydroxyl of guanosine. Secondly, 2-amino-6-chloro-9-β-D-ribofuranosyl-purine has been prepared via chlorination using phosphorus oxychloride or sulfur dioxide, and deacetylation of 2′,3′,5′-tri-O-acetyl guanosine. Moreover, high regioselective methylation of 2-amino-6-chloro-9-β-D-ribofuranosyl-purine with diazomethane, getting 2-amino-6-chloro-9-[2′-O-methyl-β-D-ribofuranosyl]-purine. Then followed by hydrolysis provided 2′-O-methyl guanosine and 3′-O-methyl guanosine. The isomers were isolated through ion exchange chromatography on Dowex 1-X2(OH~-), getting pure 2′-O-methyl guanosine.
The structure of the synthetic products were characterized by Melting Point(MP), Infrared Spectoscopy(IR), Mass Spectra(MS) and ~1H Nuclear Magnetic Resonance(NMR). The results proved: the structures and performance indicated that the products conformed to the design requirement.
引文
[1]姚其正.核苷化学合成[M].化学工业出版社,2005.1-32,33-35,85,332-336.
[2]Anne B.Eldrup,Charles R.Allerson,etal.Structure-Activity relationship of purinefibonucleosides inhibition of hepatitis C virus RNA-Dependent RNA polymerase[J].J.Med.Chem,2004,47:2283-2295.
[3]张礼和.以核糖为作用靶的药物研究[M].科学出版社,1997.195-219.
[4]彭司勋.药物化学[M].中国医药科技出版社,1999.319-322,341-347.
[5]Mansour S,Storer R.Antiviral nucleosides[J].Curr.Pharma.Design,1997,3:227-264.
[6]李子成,陈淑华,蒋宁等.核苷类抗病毒药物的研究进展[J].化学研究与应用,2002,14:15.
[7]黄懿,牛秀珑,张灏.化学合成2'-氧-甲基鸟嘌呤核苷(P6)诱导HeLa细胞凋亡及其机制的研究[J].《现代免疫学》,2007,27(1):59-64.
[8]岳明,倪孟祥.常用的核苷类化合物分离技术及应用[J].药学进展,2006,30(10):442-447
[9]黄红兵,江英桥.赤芝子实体总核苷的含量测定及薄层色谱鉴别[J].植物资源与环境学报,2000,9(3):61-62
[10]徐文豪,薛智,马健民.冬虫夏草的水溶性成份-核苷类物质的研究[J].中药通报,1988,13(4):226-228
[11]邱蔚然.肌苷的快速简易微量测定[J].医药工业杂志,1985,16(2):23-25
[12]吴有光,匡渤海,龙飞.临床化学上氨基酸、有机酸及核苷类的二维薄层色谱多项分离[J].色谱,1996,14(4):259-263
[13]王忠华,周蓉,达世禄.核苷与碱基的苯胺甲基键合硅胶固定相高效液相色谱分离[J].分析测试学报,2003,22(3):28-31
[14]董明,姚恕,林容轩,等.活性炭分离肌苷和次黄嘌呤的研究[J].浙江大学学报(自然科学版),1990,24(2):323-324
[15]李永泉,张进,姚恕,等.活性炭单柱提取肌苷新技术的研究[J].化学反应工程与工艺,1994,10(4):408-412
[16]Jokela J,Leisola M1 Chromatographic separation of nucleosides using a cross-linked xylose isomerase crystal stationary phase[J]1 J Sep Sci,2004,27(17-18):1491-1497
[17]J.D.Smith,D.B.Dunn.Isolation of 2'-O-methyladenosine[J].Biophys Acta,1959,31(573):1591-1597
[18]Cummins L,Owens S,Dan Cook P.et al.Characterization of fully 2'-modified oligoribonucleotide hetero-and homoduplex hybridization and nuclease sensitivity[J].Nucleic Acids Res.1995,23:2019-2026.
[19]Khwaija T.A,Robins R.K.Synthesis of 2'-O-Me-guanosine and adenosine from 2-amino-6-chloropurine riboside[J].J.AM.Chem.Soc.,1966,88:3640-3643.
[20]Morris J.Robins,R.Naik.Nucleic acid related compound,Methylation and transformation of 4-methoxy-2-pyrinidine 1-β-D-ribofuranoside into 2'-O-methyl nucleoside components of ribonucleic acid,their analogs,and derivatives[J]2'-O-Methyl Nucleoside,1971,10(19):3591-3597.
[21]Saroj K.Roy,Jin-yan Tang.Efficient large scale synthesis of 2'-O-Alkyl pyrimidine ribonucleosides[J].Organic Process Research & Development,2000,4:170-171.
[22]Cramer H,Pfleiderer W,Helv Chim Acta.1996,79:2114
[23]Schirmeister H,Himmelsbach F,Pfeiderer W.A simple method for N-15 labelling of exocyclic amino groups in synthetic oligodeoxynucleotides[J].Nucleic Acid Res.1994,22:2982-2989.
[24]Tasneem A.Khwaja,Roland K.Robins.Purine Nucleosides.XVI.Synthesis of the Naturally Occurring 2'-O-Methylpurine Ribonucleosides and Relatet Derivatives[J].Journal of the American Chemical Society.1966,88(55):3640-3643.
[25]Leonid Beigelman,Peter Haeberli,David Sweedler,Alexander Karpeisky.Improved Synthetic Approaches Toward 2'-O- Methyl-Adenosine and Guanosine and Their Nacyl Derivatives[J].Tetrahedron 56,2000:1047-1056.
[26]J.YANO,L.S.KAN,P.O.P.TSO.A Simple method of the preparation of 2'-O-methyladenosine [J].Biochimica et Biophysica Acta,1980,629:178-183
[27]Morris J.Robins,S.R.Naik,Alan S.K.Lee.Nucleic Acid Related Compounds 12.The Facile and High-Yield Stannous Chloride Catalyzed Monomethylation of the Cis-Glycol System of Nucleosides by Diazomethane[J].J.Org.Chem,1974,39(13):1891-1899
[28]Suetying Chow,Ke Wen,Yogesh S.Sanghvib,Emmanuel A.Theodorakisa.Novel Synthesis of 2'-O-methylguanosine[J].Bioorganic & Medicinal Chemistry letters,2003,13:1631-1634.
[29]Daniel Peter,Claude McGee.Process for the preparation of 2'-O-Alkyl purine phosphoramidttes[P].United States Patens:US005646265A,1997.07.08
[30]Chen X.C.,Kern E.R.,Drach J.C.Structure-activity relationships of(S,Z)-2-amonopurine methylene cyclo propane analogues of nucleosides.Variation of purine-6 substituents and activity against herpesviruses and hepatitis B virus[J].J Med.Chem,2003,48(8):1531-1537.
[31]Zhu Z.X.,Furr J.,Buolamwini J.K.Synthesis and flow cytometic evaluation of novel 1,2,3,4-tetrahydro-isoquinoline con forma tionally aconstrained analogues of nitrobenzyl mercaptopurine riboside(NBMPR)designed for probing.Its conformation when bound to the es nucleoside transporter[J].J Med.Chem,2003,46(5):831-837.
[32]刘启宾,申艳红,渠桂荣,李永.6-氯和6-甲氧基嘌呤核苷的合成[J].精细化工,2004,21(12):941-943.
[33]John F.Gerster,Jesse W.Jones,Roland K.Robins.The Synthesis of 6- Halogenated 9-β-D-Ribofuranosylpurines from Inosine and Guanosine[J].Purine Nucleosides Ⅳ,1962,28:945-948.
[34]Robins M.J.,Uznanski B.Nucleic acid related compounds 33.Conversions of adenosine and guanosine to 2,6-dichloro,2-amino-6-chloro,and derived purine nucleosides[J].Can J Chem.,1981,59(17):2601-2607.
[35]时毅,姜力勋,姚其正.2-氨基-6-氯-9-β-D-呋喃核糖基嘌呤合成方法的改进[J].药学进展,2006,30(9):425-427.
[36]周杰,高吉刚.新的核苷衍生物N~2,N_3-亚乙烯基腺嘌呤核苷的合成[J].Chinese Journal of Organic Chemistry,2004,24(5):498-501.
[37]李庶心,江发龙,赵砚瑾,郭金华,王志清.9-(β-D-2'-脱氧核糖基)-6-甲基嘌呤的合成[J].Chinese Journal of Organic Chemistry,2006,26(10):1394-1397.
[38]S.P.Dutta,C.I.Hong,G.P.Murphy,A.Mittelman,G.B.Chheda.Synthesis and Properties of the Naturally Occurring N-[(9-β-D-Ribofuranosyl purin-6-yl)-N-methylcarbamoyl]-L-threonine(mt~6A)and Other Related Synthetic Analogs[J].Biochemistry,1975,14(14):3143-3151.
[39]刘云,韩广甸.重氮甲烷及其前体试剂在药物合成中的应用[J].中国医药工业杂志,1996,26(9):418-423.
[40]田亚平等.生化分离技术[M].化学工业出版社,2006,99-134.
[41]李校堃,袁辉.药物蛋白质分离纯化技术[M].化学工业出版社,2005,119-135.
[2]Anne B.Eldrup,Charles R.Allerson,etal.Structure-Activity relationship of purinefibonucleosides inhibition of hepatitis C virus RNA-Dependent RNA polymerase[J].J.Med.Chem,2004,47:2283-2295.
[3]张礼和.以核糖为作用靶的药物研究[M].科学出版社,1997.195-219.
[4]彭司勋.药物化学[M].中国医药科技出版社,1999.319-322,341-347.
[5]Mansour S,Storer R.Antiviral nucleosides[J].Curr.Pharma.Design,1997,3:227-264.
[6]李子成,陈淑华,蒋宁等.核苷类抗病毒药物的研究进展[J].化学研究与应用,2002,14:15.
[7]黄懿,牛秀珑,张灏.化学合成2'-氧-甲基鸟嘌呤核苷(P6)诱导HeLa细胞凋亡及其机制的研究[J].《现代免疫学》,2007,27(1):59-64.
[8]岳明,倪孟祥.常用的核苷类化合物分离技术及应用[J].药学进展,2006,30(10):442-447
[9]黄红兵,江英桥.赤芝子实体总核苷的含量测定及薄层色谱鉴别[J].植物资源与环境学报,2000,9(3):61-62
[10]徐文豪,薛智,马健民.冬虫夏草的水溶性成份-核苷类物质的研究[J].中药通报,1988,13(4):226-228
[11]邱蔚然.肌苷的快速简易微量测定[J].医药工业杂志,1985,16(2):23-25
[12]吴有光,匡渤海,龙飞.临床化学上氨基酸、有机酸及核苷类的二维薄层色谱多项分离[J].色谱,1996,14(4):259-263
[13]王忠华,周蓉,达世禄.核苷与碱基的苯胺甲基键合硅胶固定相高效液相色谱分离[J].分析测试学报,2003,22(3):28-31
[14]董明,姚恕,林容轩,等.活性炭分离肌苷和次黄嘌呤的研究[J].浙江大学学报(自然科学版),1990,24(2):323-324
[15]李永泉,张进,姚恕,等.活性炭单柱提取肌苷新技术的研究[J].化学反应工程与工艺,1994,10(4):408-412
[16]Jokela J,Leisola M1 Chromatographic separation of nucleosides using a cross-linked xylose isomerase crystal stationary phase[J]1 J Sep Sci,2004,27(17-18):1491-1497
[17]J.D.Smith,D.B.Dunn.Isolation of 2'-O-methyladenosine[J].Biophys Acta,1959,31(573):1591-1597
[18]Cummins L,Owens S,Dan Cook P.et al.Characterization of fully 2'-modified oligoribonucleotide hetero-and homoduplex hybridization and nuclease sensitivity[J].Nucleic Acids Res.1995,23:2019-2026.
[19]Khwaija T.A,Robins R.K.Synthesis of 2'-O-Me-guanosine and adenosine from 2-amino-6-chloropurine riboside[J].J.AM.Chem.Soc.,1966,88:3640-3643.
[20]Morris J.Robins,R.Naik.Nucleic acid related compound,Methylation and transformation of 4-methoxy-2-pyrinidine 1-β-D-ribofuranoside into 2'-O-methyl nucleoside components of ribonucleic acid,their analogs,and derivatives[J]2'-O-Methyl Nucleoside,1971,10(19):3591-3597.
[21]Saroj K.Roy,Jin-yan Tang.Efficient large scale synthesis of 2'-O-Alkyl pyrimidine ribonucleosides[J].Organic Process Research & Development,2000,4:170-171.
[22]Cramer H,Pfleiderer W,Helv Chim Acta.1996,79:2114
[23]Schirmeister H,Himmelsbach F,Pfeiderer W.A simple method for N-15 labelling of exocyclic amino groups in synthetic oligodeoxynucleotides[J].Nucleic Acid Res.1994,22:2982-2989.
[24]Tasneem A.Khwaja,Roland K.Robins.Purine Nucleosides.XVI.Synthesis of the Naturally Occurring 2'-O-Methylpurine Ribonucleosides and Relatet Derivatives[J].Journal of the American Chemical Society.1966,88(55):3640-3643.
[25]Leonid Beigelman,Peter Haeberli,David Sweedler,Alexander Karpeisky.Improved Synthetic Approaches Toward 2'-O- Methyl-Adenosine and Guanosine and Their Nacyl Derivatives[J].Tetrahedron 56,2000:1047-1056.
[26]J.YANO,L.S.KAN,P.O.P.TSO.A Simple method of the preparation of 2'-O-methyladenosine [J].Biochimica et Biophysica Acta,1980,629:178-183
[27]Morris J.Robins,S.R.Naik,Alan S.K.Lee.Nucleic Acid Related Compounds 12.The Facile and High-Yield Stannous Chloride Catalyzed Monomethylation of the Cis-Glycol System of Nucleosides by Diazomethane[J].J.Org.Chem,1974,39(13):1891-1899
[28]Suetying Chow,Ke Wen,Yogesh S.Sanghvib,Emmanuel A.Theodorakisa.Novel Synthesis of 2'-O-methylguanosine[J].Bioorganic & Medicinal Chemistry letters,2003,13:1631-1634.
[29]Daniel Peter,Claude McGee.Process for the preparation of 2'-O-Alkyl purine phosphoramidttes[P].United States Patens:US005646265A,1997.07.08
[30]Chen X.C.,Kern E.R.,Drach J.C.Structure-activity relationships of(S,Z)-2-amonopurine methylene cyclo propane analogues of nucleosides.Variation of purine-6 substituents and activity against herpesviruses and hepatitis B virus[J].J Med.Chem,2003,48(8):1531-1537.
[31]Zhu Z.X.,Furr J.,Buolamwini J.K.Synthesis and flow cytometic evaluation of novel 1,2,3,4-tetrahydro-isoquinoline con forma tionally aconstrained analogues of nitrobenzyl mercaptopurine riboside(NBMPR)designed for probing.Its conformation when bound to the es nucleoside transporter[J].J Med.Chem,2003,46(5):831-837.
[32]刘启宾,申艳红,渠桂荣,李永.6-氯和6-甲氧基嘌呤核苷的合成[J].精细化工,2004,21(12):941-943.
[33]John F.Gerster,Jesse W.Jones,Roland K.Robins.The Synthesis of 6- Halogenated 9-β-D-Ribofuranosylpurines from Inosine and Guanosine[J].Purine Nucleosides Ⅳ,1962,28:945-948.
[34]Robins M.J.,Uznanski B.Nucleic acid related compounds 33.Conversions of adenosine and guanosine to 2,6-dichloro,2-amino-6-chloro,and derived purine nucleosides[J].Can J Chem.,1981,59(17):2601-2607.
[35]时毅,姜力勋,姚其正.2-氨基-6-氯-9-β-D-呋喃核糖基嘌呤合成方法的改进[J].药学进展,2006,30(9):425-427.
[36]周杰,高吉刚.新的核苷衍生物N~2,N_3-亚乙烯基腺嘌呤核苷的合成[J].Chinese Journal of Organic Chemistry,2004,24(5):498-501.
[37]李庶心,江发龙,赵砚瑾,郭金华,王志清.9-(β-D-2'-脱氧核糖基)-6-甲基嘌呤的合成[J].Chinese Journal of Organic Chemistry,2006,26(10):1394-1397.
[38]S.P.Dutta,C.I.Hong,G.P.Murphy,A.Mittelman,G.B.Chheda.Synthesis and Properties of the Naturally Occurring N-[(9-β-D-Ribofuranosyl purin-6-yl)-N-methylcarbamoyl]-L-threonine(mt~6A)and Other Related Synthetic Analogs[J].Biochemistry,1975,14(14):3143-3151.
[39]刘云,韩广甸.重氮甲烷及其前体试剂在药物合成中的应用[J].中国医药工业杂志,1996,26(9):418-423.
[40]田亚平等.生化分离技术[M].化学工业出版社,2006,99-134.
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