新型反义药物的研究
|
摘要
反义技术（antisense technology）被认为是继基因工程以后又一项重大的突破，它
是一种新的药物研发方法，利用这一技术研制的药物称反义药物，通常指反义寡核苷酸
（antisense oligonucleotides），包括反义DNA、反义RNA等。根据核酸杂交原理，反义
药物能与特定基因杂交，在基因水平干扰致病蛋白的产生过程，即干扰遗传信息从核酸
向蛋白质的传递。蛋白质在人体代谢中扮演非常重要的角色，几乎所有的人类疾病都是
由蛋白质的异常引起的，无论是宿主疾病（肿瘤等）还是感染疾病（肝炎等）。传统药
物主要是直接作用于致病蛋白本身，反义药物则作用于产生蛋白的基因，因此可广泛应
用于多种疾病的治疗，如传染病、炎症、心血管疾病及肿瘤等，与传统药物相比反义药
物更具有选择性，因此也更高效低毒。迄今，反义药物在许多体外试验中取得了令人鼓
舞的结果。但是，天然的寡核苷酸能被细胞内核酸酶迅速降解，并且不能被动扩散透过
细胞膜，因而不能作为药用。作为第一代的反义药物—硫代寡核苷酸，虽具有较好的抗
酶解活性，并已广泛应用于临床，但它也表现出一定的缺陷。为克服这些缺点，改善反
义药物对靶基因的亲和力以及细胞膜的通透性，而对寡核苷酸结构进行修饰，以期寻找
效果更好的反义药物。根据对寡核苷酸修饰部位的不同，本文独立完成了以下三个主要
部分。
1．含亚甲基缩醛键的寡脱氧核苷酸的研究
对寡脱氧核苷酸（oligodeoxynucleotides，ODNs）进行修饰，其中一个主要的方面
是骨架修饰，目的是增强对核酸酶的稳定性、细胞膜通透性和提高亲和力。ODN中磷
酸二酯键部分用中性的亚甲基缩醛键来取代，目的是不影响寡核苷酸杂交亲和力的前提
下，提高ODN对酶的耐受性。我们以胸苷和脱氧尿苷为基本原料，选择性地对它们的
5'-羟基进行苯甲酰保护，而3'-位用二苯基次膦酸作为离去基团，与另一个5'-位游离的
脱氧核苷在三甲基硅三氟甲磺酸酯（TMSOTf）的条件下进行缩合来引入脱氧核苷内的
亚甲基缩醛键，进而合成了一系列含亚甲基缩醛键的二聚和三聚体脱氧核苷。此合成路
线操作简便，产率高，同时能获得较高纯度的目标化合物。得到的甲缩醛寡聚体3'-位和
5'-位分别经相应保护后应用标准固相DNA合成法掺入到寡核苷酸中，并考察其杂交性
摘 要
质，测定了解链温度几值。结果表明，经亚甲基缩醛键修饰的寡核昔酸的杂交亲和力
大致与对照的天然磷酸二酯键的ODN相当，明显优于磷硫酚修饰的寡核昔酸。亚甲基
缩醛键修饰的ODN应有其广泛的研究前景，提示我们可以进一步对这类修饰的ODN
进行深入的研究。
2．爪烷基5－甲基T－脱氧胞音的合成及其撞人到寡核苦酸中
为了改进修饰寡核昔酸的亲和力和提高抗酶解活性，在脱氧胞昔的5－位引入甲基以
及对小．位进行烷基化修饰。为此发展了一条合成矿烷基6－甲基6’－o汁4’－二甲氧三
苯甲基）．2’．脱氧胞昔．3－O－（2－氰乙基NN二异丙基）氨基磷酸酯的全新方法。以胸苦
为原料，用三甲基氯硅烷对3’－和5’－羟基进行暂时的保护，与三哩反应后脱去三甲基硅
保护基团，一步法得到三陛胸苦。此方法的特点是三哇化胸苦与4，4’－二甲氧三苯甲基氯
反应，然后再用一系列的烷基胺取代Hap基团，最后用（2－氰乙基风NH异丙基）氯化
氨基磷酸酯在3’－位引入磷酸酯键，就成为可以掺入到寡核昔酸中的单体合成子。根据对
这一类修饰ODN杂交亲和力的研究发现，脱氧胞昔5．位甲基的取代能提高寡核昔酸的
杂交亲和力，而小．位烷基的取代却使与互补DNA杂交双螺旋不稳定，这也证实了碱基
啼陡环外炉．烷基的取向与NI成顺式，这对形成稳定的双螺旋不利。
3．5．炔基毛’．脱氧尿苦的合成及其撞人到寡核省酸中
为了增强寡核昔酸的杂交亲和力，以及考察二’．脱氧尿昔5．位修饰后对ODN杂交性
质的影响。首先我们合成了一系列的 5－炔基－2’－脱氧尿昔，用双（三苯基磷）氯化铅（11）
替代价格昂贵且更活泼的四（三苯基磷）把①人偶合末端炔烃与3’，5’－O二苯甲酞碘苦
或5’－O－u，4’－二甲氧三苯甲基）碘昔，并考察催化剂与溶剂对此反应的影响。合成的
5－炔基－5’－o（4，4’－二甲氧三苯甲基）－2’－脱氧尿音3’－乙（2－氰乙基NN二异丙基）氨基
磷酸酯掺入到寡核昔酸中，测定了与靶DNA的杂交亲和力。结果表明，带有5－位炔基
修饰的ODN的解链温度Ti值比对照的天然寡核昔酸有一定幅度的提高，但修饰基团越
大几值升幅越小，而炔烃上连有吸电子基团则解链温度大幅下降。这说明影响寡核昔
酸亲和力有两个因素，一是氢键（常指Watson－Crick碱基对），另一个是所形成双螺旋
2
扬 要
之间的碱基堆集程度。
本文以简单的脱氧核昔为起始原料，经过一系列的反应，其中包括原料和复杂有机
试剂?
Oligonucleotide therapeutics represents a new paradigm for drug discovery. The
paradigm has resulted in substantial enthusiasm because oligonucleotides may display
dramatic increases in affinity and selectivity for their nucleic acid targets compared to
traditional drugs. The widespread occurrence of diseases caused primarily through retrovirus
invasion of a host, such as HIV, has encouraged the development of an ever increasing variety
of potential drugs based on both the antisense and antigene strategy. Furthermore, antisense
technology may facilitate rational drug design. Oligonucleotides are designed to modulate the
information transfer from the gene to protein ?in essence, to alter the intermediary
metabolism of RNA. To date, oligonucleotides have been found to inhibit the growth of a
large number of viruses in tissur culture, the expression of numerous oncogenes, a variety of
normal cellular genes, and a number of transfencted reporter genes controlled by several
regulatory elements. Potent antiviral and antitumor activities have been demonstrated with
oligonucleotides.
There are several obstacles that must be surmounted in order to improve the in vivo
efficacy of oligodeoxynucleotide (ODN) analogs. Unmodified ODNs are rapidly degraded by
intracellular nucleases and are unable to efficiently passively diffuse through cell membranes.
As a first梘eneration antisense oligonucleotides, phosphorthioate modified oligonucleotides
are stable to degradation by nucleases, but in general hybridize to target sequences with a
lesser affinity than a phosphodiester ODN. The ODNs containing this modification are a
mixture of 2~ diastereomers (where n is the number of linkages), and it is possible that an
ODN containing all RP or all SP isomers would hybridize with better affinity. Modification of
the ODN has been shown to impart stability and may allow for enhanced affinity and
increased cellular permeation of ODNs, and for this reason to synthesize more efficient
antisense drugs.
4
ABSTRACT
1. The study of oligodeoxynucleotides containing methyleneformacetal
The major challenge of ODN analogs is to design backbone modifications which will
increase the nuclease stability and cellular permeability while enhancing affinity. ODNs
partially substituted phosphodiester backbone with neutral methyleneformacetal are designed
to increase the stability against cellular nucleases without disturbing hybridizing affinity of
ODNs. Starting from thymidine and 2?deoxycytidine, 5?hydroxyl group of deoxynucleosides
is protected selectively by benzoyl chloride, also, 3?terminus is used diphenylphosphinic acid
as leaving group. In the presence of trimethylsilyl trifluomethanesulfonate (TMSOTf),
condensation of a nucleoside phosphinate, 3?O-CH2-OP(O)Ph2, with 5?unprotected
deoxynucleoside acceptor affords in most cases the (3挆+S?methylene acetal linked dimers
and trimers. These acetal-linked oligomers, of which 5?hydroxyl group is protected by
4,4?dimethoxytrityl chloride and 3?hydroxyl group is reacted with (2-cyanoethyl
N,N-diisopropyl) chlorophosphoramidite, are incorporated into oligonucleotides by using the
standed solid梡hase DNA chemistry on controlled pore glass (CPG) support with the
phosphoramidite method. The melting temperatures (Tm) of modified oligodeoxynucleotides
with their DNA complements are determined.
是一种新的药物研发方法，利用这一技术研制的药物称反义药物，通常指反义寡核苷酸
（antisense oligonucleotides），包括反义DNA、反义RNA等。根据核酸杂交原理，反义
药物能与特定基因杂交，在基因水平干扰致病蛋白的产生过程，即干扰遗传信息从核酸
向蛋白质的传递。蛋白质在人体代谢中扮演非常重要的角色，几乎所有的人类疾病都是
由蛋白质的异常引起的，无论是宿主疾病（肿瘤等）还是感染疾病（肝炎等）。传统药
物主要是直接作用于致病蛋白本身，反义药物则作用于产生蛋白的基因，因此可广泛应
用于多种疾病的治疗，如传染病、炎症、心血管疾病及肿瘤等，与传统药物相比反义药
物更具有选择性，因此也更高效低毒。迄今，反义药物在许多体外试验中取得了令人鼓
舞的结果。但是，天然的寡核苷酸能被细胞内核酸酶迅速降解，并且不能被动扩散透过
细胞膜，因而不能作为药用。作为第一代的反义药物—硫代寡核苷酸，虽具有较好的抗
酶解活性，并已广泛应用于临床，但它也表现出一定的缺陷。为克服这些缺点，改善反
义药物对靶基因的亲和力以及细胞膜的通透性，而对寡核苷酸结构进行修饰，以期寻找
效果更好的反义药物。根据对寡核苷酸修饰部位的不同，本文独立完成了以下三个主要
部分。
1．含亚甲基缩醛键的寡脱氧核苷酸的研究
对寡脱氧核苷酸（oligodeoxynucleotides，ODNs）进行修饰，其中一个主要的方面
是骨架修饰，目的是增强对核酸酶的稳定性、细胞膜通透性和提高亲和力。ODN中磷
酸二酯键部分用中性的亚甲基缩醛键来取代，目的是不影响寡核苷酸杂交亲和力的前提
下，提高ODN对酶的耐受性。我们以胸苷和脱氧尿苷为基本原料，选择性地对它们的
5'-羟基进行苯甲酰保护，而3'-位用二苯基次膦酸作为离去基团，与另一个5'-位游离的
脱氧核苷在三甲基硅三氟甲磺酸酯（TMSOTf）的条件下进行缩合来引入脱氧核苷内的
亚甲基缩醛键，进而合成了一系列含亚甲基缩醛键的二聚和三聚体脱氧核苷。此合成路
线操作简便，产率高，同时能获得较高纯度的目标化合物。得到的甲缩醛寡聚体3'-位和
5'-位分别经相应保护后应用标准固相DNA合成法掺入到寡核苷酸中，并考察其杂交性
摘 要
质，测定了解链温度几值。结果表明，经亚甲基缩醛键修饰的寡核昔酸的杂交亲和力
大致与对照的天然磷酸二酯键的ODN相当，明显优于磷硫酚修饰的寡核昔酸。亚甲基
缩醛键修饰的ODN应有其广泛的研究前景，提示我们可以进一步对这类修饰的ODN
进行深入的研究。
2．爪烷基5－甲基T－脱氧胞音的合成及其撞人到寡核苦酸中
为了改进修饰寡核昔酸的亲和力和提高抗酶解活性，在脱氧胞昔的5－位引入甲基以
及对小．位进行烷基化修饰。为此发展了一条合成矿烷基6－甲基6’－o汁4’－二甲氧三
苯甲基）．2’．脱氧胞昔．3－O－（2－氰乙基NN二异丙基）氨基磷酸酯的全新方法。以胸苦
为原料，用三甲基氯硅烷对3’－和5’－羟基进行暂时的保护，与三哩反应后脱去三甲基硅
保护基团，一步法得到三陛胸苦。此方法的特点是三哇化胸苦与4，4’－二甲氧三苯甲基氯
反应，然后再用一系列的烷基胺取代Hap基团，最后用（2－氰乙基风NH异丙基）氯化
氨基磷酸酯在3’－位引入磷酸酯键，就成为可以掺入到寡核昔酸中的单体合成子。根据对
这一类修饰ODN杂交亲和力的研究发现，脱氧胞昔5．位甲基的取代能提高寡核昔酸的
杂交亲和力，而小．位烷基的取代却使与互补DNA杂交双螺旋不稳定，这也证实了碱基
啼陡环外炉．烷基的取向与NI成顺式，这对形成稳定的双螺旋不利。
3．5．炔基毛’．脱氧尿苦的合成及其撞人到寡核省酸中
为了增强寡核昔酸的杂交亲和力，以及考察二’．脱氧尿昔5．位修饰后对ODN杂交性
质的影响。首先我们合成了一系列的 5－炔基－2’－脱氧尿昔，用双（三苯基磷）氯化铅（11）
替代价格昂贵且更活泼的四（三苯基磷）把①人偶合末端炔烃与3’，5’－O二苯甲酞碘苦
或5’－O－u，4’－二甲氧三苯甲基）碘昔，并考察催化剂与溶剂对此反应的影响。合成的
5－炔基－5’－o（4，4’－二甲氧三苯甲基）－2’－脱氧尿音3’－乙（2－氰乙基NN二异丙基）氨基
磷酸酯掺入到寡核昔酸中，测定了与靶DNA的杂交亲和力。结果表明，带有5－位炔基
修饰的ODN的解链温度Ti值比对照的天然寡核昔酸有一定幅度的提高，但修饰基团越
大几值升幅越小，而炔烃上连有吸电子基团则解链温度大幅下降。这说明影响寡核昔
酸亲和力有两个因素，一是氢键（常指Watson－Crick碱基对），另一个是所形成双螺旋
2
扬 要
之间的碱基堆集程度。
本文以简单的脱氧核昔为起始原料，经过一系列的反应，其中包括原料和复杂有机
试剂?
Oligonucleotide therapeutics represents a new paradigm for drug discovery. The
paradigm has resulted in substantial enthusiasm because oligonucleotides may display
dramatic increases in affinity and selectivity for their nucleic acid targets compared to
traditional drugs. The widespread occurrence of diseases caused primarily through retrovirus
invasion of a host, such as HIV, has encouraged the development of an ever increasing variety
of potential drugs based on both the antisense and antigene strategy. Furthermore, antisense
technology may facilitate rational drug design. Oligonucleotides are designed to modulate the
information transfer from the gene to protein ?in essence, to alter the intermediary
metabolism of RNA. To date, oligonucleotides have been found to inhibit the growth of a
large number of viruses in tissur culture, the expression of numerous oncogenes, a variety of
normal cellular genes, and a number of transfencted reporter genes controlled by several
regulatory elements. Potent antiviral and antitumor activities have been demonstrated with
oligonucleotides.
There are several obstacles that must be surmounted in order to improve the in vivo
efficacy of oligodeoxynucleotide (ODN) analogs. Unmodified ODNs are rapidly degraded by
intracellular nucleases and are unable to efficiently passively diffuse through cell membranes.
As a first梘eneration antisense oligonucleotides, phosphorthioate modified oligonucleotides
are stable to degradation by nucleases, but in general hybridize to target sequences with a
lesser affinity than a phosphodiester ODN. The ODNs containing this modification are a
mixture of 2~ diastereomers (where n is the number of linkages), and it is possible that an
ODN containing all RP or all SP isomers would hybridize with better affinity. Modification of
the ODN has been shown to impart stability and may allow for enhanced affinity and
increased cellular permeation of ODNs, and for this reason to synthesize more efficient
antisense drugs.
4
ABSTRACT
1. The study of oligodeoxynucleotides containing methyleneformacetal
The major challenge of ODN analogs is to design backbone modifications which will
increase the nuclease stability and cellular permeability while enhancing affinity. ODNs
partially substituted phosphodiester backbone with neutral methyleneformacetal are designed
to increase the stability against cellular nucleases without disturbing hybridizing affinity of
ODNs. Starting from thymidine and 2?deoxycytidine, 5?hydroxyl group of deoxynucleosides
is protected selectively by benzoyl chloride, also, 3?terminus is used diphenylphosphinic acid
as leaving group. In the presence of trimethylsilyl trifluomethanesulfonate (TMSOTf),
condensation of a nucleoside phosphinate, 3?O-CH2-OP(O)Ph2, with 5?unprotected
deoxynucleoside acceptor affords in most cases the (3挆+S?methylene acetal linked dimers
and trimers. These acetal-linked oligomers, of which 5?hydroxyl group is protected by
4,4?dimethoxytrityl chloride and 3?hydroxyl group is reacted with (2-cyanoethyl
N,N-diisopropyl) chlorophosphoramidite, are incorporated into oligonucleotides by using the
standed solid梡hase DNA chemistry on controlled pore glass (CPG) support with the
phosphoramidite method. The melting temperatures (Tm) of modified oligodeoxynucleotides
with their DNA complements are determined.
引文
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2． Mercola D，Westwick J，Rundell AYK，et al．Analysis of a transformed cell line using antisense c-fos RNA．Gene，1988，72：253-265
3． Matsukura M，Zon G，Shinozaka K，et al．Regulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate oligodeoxynucleotide against rev(art／trs) in chronically infected cells．Proc Natl Acad Sci USA，1989，86：4244-4248
4． Wakita T，Wands JR．Specific inhibition of hepatitis C virus expression by antisense oligodeoxy-nucleotides．J Biol Chem，1994，269：14205-14210
5． 王小红，王开启，毛秉智．反义药物临床前及临床研究进展．国外医学-药学分册，2000，27(1) ： 1-5
6． 陈耀全．化学修饰的寡核苷酸：合成，性质和应用．有机化学，1994，14：456-467
7． 李英，刘克良，恽榴红．肽核酸研究进展．药学学报，1999，34(3) ：235-240
8． Pieken WA，Olsen DB，Benseler F, et al．Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes．Science，1991，253：314-317
9． Crooke ST．Therapeutic applications of oligonucleotides．Annu Rev Pharmacol Toxicol，1992，32： 329-376
10． Debart F，Rayner B，Degols G，et al．Synthesis and base-pairing properties of the nuclease-resistant α-anomeric dodecaribonucleotide α-[r (UCUUAACCCACA)]．Nucleic Acids Res，1992，20：1193-1200
11． Adams AD，Petrie CR，Meyer JRB．Preparation and hybridization properties of oligonucleotides containing 1-α-D-arabinofuranosylthymine．Nucleic Acid Res，1991，19：3647-2651
12． Fuimod S，Shudo K．Enantio-DNA recognizes complementary RNA but not complementary DNA．J Am Chem Soc，1990，112：7436-7438
13． Garbesi A，Capobianco ML，Colonna FP，et al．L-DNAs as potential antimessenger oligonucleotides： a reassessment．Nucleic Acids Res，1993，21：4159-4165
14． Cook PD．Medicinal chemistry of antisense oligonucleotides-future opportunities．Anti-Cancer Drug Des, 1991． 6: 585-607
15． Froehler BC, Wadwani S, Terhorst TJ, er al. Oligodeoxynucleotides containing C-5 propyne analogs of 2'-deoxyuridine and 2'-deoxycytidine. Tetrahedron Lett, 1992, 33: 5307-5310
16． Gewirtz Am, Sokol DL, Ratajczak MZ. Nucleic acid therapeutics: state of the art and future prospects. Blood, 1998, 92(3) : 712-736
17． Branch AD. A good antisense molecule is hard to find. Trends Biochem Sci, 1998, 23(2) : 45-50
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