Schlatterer Jörg C, Stuhlmann Friedrich, Jäschke Andres
Institut für Pharmazie und Molekulare Biotechnologie, Abteilung Chemie, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
Chembiochem. 2003 Oct 6;4(10):1089-92. doi: 10.1002/cbic.200300676.
Development of artificial ribozymes by in vitro selection has so far, mostly been addressed from the viewpoint of fundamental research. However, such ribozymes also have high potential as selective catalysts in practical syntheses. Immobilization of an active and selective ribozyme is an important step towards this end. A 49-nucleotide RNA molecule that was previously found to stereoselectively catalyze Diels-Alder reactions between various anthracene dienes and maleimide dienophiles was quantitatively immobilized on an agarose matrix by periodate oxidation of the 3'-terminal ribose and coupling to a hydrazide moiety. Typical loadings were 45 pmol microL(-1) gel. The specific activity was comparable to that of soluble ribozyme, and high enantioselectivities were obtained in catalyzed cycloadditions. The catalytic matrix was found to be stable and could be regenerated about 40 times with only minimal reduction of catalytic activity. Like the soluble ribozyme, the immobilized catalyst stereoselectively converts various diene and dienophile substrates. By using either natural D-RNA or enantiomeric L-RNA, both product enantiomers were made synthetically accessible with similar selectivities.
到目前为止，通过体外筛选开发人工核酶大多是从基础研究的角度进行的。然而，这类核酶在实际合成中作为选择性催化剂也具有很高的潜力。固定化一种活性且选择性的核酶是朝着这个目标迈出的重要一步。一个先前发现能立体选择性催化各种蒽二烯与马来酰亚胺亲双烯体之间狄尔斯-阿尔德反应的49个核苷酸的RNA分子，通过3'-末端核糖的高碘酸盐氧化并与酰肼部分偶联，被定量固定在琼脂糖基质上。典型的负载量为45 pmol μL(-1)凝胶。比活性与可溶性核酶相当，并且在催化环加成反应中获得了高对映选择性。发现催化基质是稳定的，并且可以再生约40次，催化活性仅有最小程度的降低。与可溶性核酶一样，固定化催化剂能立体选择性地转化各种二烯和亲双烯体底物。通过使用天然的D-RNA或对映体L-RNA，两种产物对映体都能以相似的选择性通过合成获得。
