NUCLEOTIDE CHEMISTRY .4. SYNTHESIS OF DEOXYOLIGONUCLEOTIDES ON A POLYMER SUPPORT
NUCLEOTIDE CHEMISTRY .4. SYNTHESIS OF DEOXYOLIGONUCLEOTIDES ON A POLYMER SUPPORT
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DOI：
10.1021/ja00401a041
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发表时间：
1981-01-01
影响因子：
15
通讯作者：
CARUTHERS, MH
中科院分区：
文献类型：
--
作者：
MATTEUCCI, MD;CARUTHERS, MH
介绍了一种合成脱氧寡核苷酸的新方法。该合成首先衍生化高效液相色谱级硅胶，使其含有通过3‘’-羟基连接到载体上的羧酸官能团的5‘-O-(二甲氧基三苯基)脱氧核苷。然后将该基质填充到一个柱中，该柱连接到一个泵和一系列阀门上。在载体上加成1个核苷酸的化学步骤如下：在硝基甲烷中用溴化锌进行脱三基反应(30分钟)；5‘-O-(二甲氧基三硝基)脱氧核苷(3’‘-甲氧基四氮基)膦与载体结合的核苷缩合(60分钟)；用二乙氧基三氮唑膦封闭未反应的载体结合的核苷羟基(5分钟)；以及用I2将亚磷酸盐氧化成磷酸盐(5分钟)。用硫酚和氢氧化铵依次处理，反相色谱纯化，80%醋酸处理，分离得到完整的脱氧寡核苷酸。该方法非常快速(每个核苷酸加成周期需要2.5h)，每次缩合的产率超过95%，最终产物的分离是简单的一步柱纯化。进行了d(C-G-T-C-A-C-A-A-T-T)和d(A-C-G-C-T-C-A-C-A-A-T-T)的合成。载体结合的脱氧寡核苷酸的产率分别为和55%，分离的脱氧核苷酸的产率为30%。根据目前测试的所有标准，这两种合成产品都是均一的，具有生物活性。
The development of a new method for synthesizing deoxyoligonucleotides is described. The synthesis begins by derivatizing high-performance liquid chromatography grade silica gel to contain 5''-O-(dimethoxytrityl)deoxynucleosides linked through the 3''-hydroxyl to a carboxylic acid functional group on the support. This matrix is then packed into a column which is attached to a pump and a series of valves. The chemical steps for the addition of 1 nucleotide to the support are as follows: detritylation using ZnBr2 in nitromethane (30 min); condensation of a 5''-O-(dimethoxytrityl)deoxynucleoside (3''-methoxytetrazoyl)phosphine with the support-bound nucleoside (60 min); blocking unreacted, support-bound nucleoside hydroxyl groups with diethoxytriazolylphosphine (5 min); and oxidation of phosphites to phosphates with I2 (5 min). Completed deoxyoligonucleotides are isolated by sequential treatment with thiophenol and ammonium hydroxide, purification by reverse-phase chromatography and treatment with 80% acetic acid. The method is extremely fast (< 2.5 h are needed for each nucleotide addition cycle), yields in excess of 95% per condensation are obtained and isolation of the final product is a simple 1-step column purification. The syntheses of d(C-G-T-C-A-C-A-A-T-T) and d(A-C-G-C-T-C-A-C-A-A-T-T) were carried out as a test of this method. Yields of support-bound deoxyoligonucleotides were 64 and 55%; the isolated yield of deoxydecanucleotide was 30%. Both synthetic products were homogeneous and biologically active by every criteria so far tested.