A Base-Labile Group for 2′-OH Protection of Ribonucleosides: A Major Challenge for RNA Synthesis
A Base-Labile Group for 2′-OH Protection of Ribonucleosides: A Major Challenge for RNA Synthesis
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DOI：
10.1002/chem.200801392
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发表时间：
2008-01-01
影响因子：
4.3
通讯作者：
Debart, Francoise
中科院分区：
文献类型：
--
作者：
Lavergne, Thomas;Bertrand, Jean-Remi;Debart, Francoise
自从RNA干扰（RNAi）出现以来，对RNA的化学合成产生了极大的兴趣[1]，这是由于生物学研究和治疗应用对大量短RNA分子的迫切需求。[2]与DNA合成相比，RNA的合成更为复杂。在成熟的DNA合成中，所有的亲核功能都是用碱不稳定的保护基团保护的，这些保护基团在延伸过程结束时用碱处理除去。与DNA合成相比，链组装中的偶联产率较低，除此之外，RNA化学的主要困难来自RNA在碱性介质中的不稳定性。一般认为，2 ′-OH保护必须不是碱不稳定的，以避免2 ′-OH对核苷间键的磷原子的亲核攻击，导致在脱保护条件下键的3 ′-5 ′至2 ′-5 ′异构化或3 ′-5 ′裂解。[3]关于在早期工作中报道的对2 '-OH使用酰基（乙酰基或苯甲酰基）保护，这导致非常差的产率。[4]叔丁基二甲基甲硅烷基（TBDMS）基团无疑是最常用于2 '-OH保护的基团。[5]已经提出了几种保护基团[6，7]来代替TBDMS，例如三异丙基甲硅烷基氧基甲基（TOM），[8]双（2-乙酰氧基乙氧基）甲基（ACE），[9]叔丁基二硫甲基（DTM），[10] 1-（2-氰基乙氧基）乙基（CEE），[11] 2-氰基乙氧基甲基（CEM），[12，13] 2-（4-甲苯磺酰基）乙氧基甲基（TEM），[14]和2-氰乙基[15];它们中的大多数，如TBDMS，被氟离子除去。这种脱保护是一个主要障碍，因为它需要通过沉淀或柱纯化进行脱盐步骤，从而导致额外耗时的后处理程序来获得纯的寡核糖核苷酸。在我们寻找一种从根本上改进的合成方法以有效、快速和高纯度地获得RNA的过程中，我们现在报告了一种新的RNA合成策略，该策略基于用碱不稳定的新戊酰氧基甲基（PivOM）基团保护2 '-OH，该基团与5'-OH（DMTr）、磷酸盐（2-氰乙基）和核碱基（酰基）的标准保护相容。这种具有缩醛酯基团的RNA策略的主要优点是在室温下在短时间内（总共3小时）直接的两步全碱基脱保护。它包括1）通过非亲核性强有机碱（1，8-二氮杂双环-ACHTUNGTRENNUNG [5.4]）诱导的β-消除选择性除去磷酸保护基。0]十一碳-7-烯（DBU）或哌啶），和2）通过氨处理同时释放核碱基、2 ′-OH和断裂琥珀酰基或Q-接头，而不发生迁移或链断裂。这种方法只使用碱基不稳定的保护基团，由于众所周知的RNA在碱性介质中的不稳定性而具有挑战性。[3]为了说明这一点，我们报告了使用2 '-O-PivOM亚磷酰胺合成长度达21个核苷酸的RNA寡聚体，并且我们证明了它们以高产率和高纯度获得，而没有链断裂或迁移。
A great interest in the chemical synthesis of RNA has grown up since the advent of RNA interference (RNAi)[1] with the crucial need of a large number of short RNA molecules for biological research and therapeutic applications.[2] Compared to DNA synthesis, RNA production is more complex. In well-established DNA synthesis, all the nucleophilic functions are protected with base-labile protecting groups that are removed at the end of the elongation process with a base treatment. Beside lower coupling yields in chain assembly compared to DNA synthesis, the main difficulty of RNA chemistry results from the instability of RNA in basic media. It is generally admitted that the 2’-OH protection must not be base-labile to avoid the 2’-OH nucleophilic attack on the phosphorus atom of the internucleoside linkages resulting in 3’–5’to 2’–5’isomerisation or 3’–5’cleavage of the linkages under deprotection conditions.[3] With regard to the use of acyl (acetyl or benzoyl) protection for 2’-OH reported in an early work, this resulted in very poor yields.[4] The tert-butyldimethylsilyl (TBDMS) group is certainly the most commonly utilized group for 2’-OH protection.[5] Several protecting groups [6, 7] have been proposed in place of TBDMS, such as triisopropylsilyloxymethyl (TOM),[8] bis (2-acetoxyethyloxy) methyl (ACE),[9] tert-butyldithiomethyl (DTM),[10] 1-(2-cyanoethoxy) ethyl (CEE),[11] 2-cyanoethoxymethyl(CEM),[12, 13] 2-(4-toluylsulfonyl) ethoxymethyl (TEM),[14] and 2-cyanoethyl [15]; most of them, like TBDMS, are removed by fluoride ions. This deprotection is a major hurdle because it requires a desalting step by precipitation or cartridge purification leading to additional time-consuming workup procedures to obtain pure oligoribonucleotides. In our search for a radically improved synthetic method to obtain RNA efficiently, rapidly, and in high purity, we now report a new RNA synthesis strategy based on protecting the 2’-OH with a base-labile pivaloyloxymethyl (PivOM) group compatible with standard protection for 5’-OH (DMTr), phosphates (2-cyanoethyl), and nucleobases (acyl groups). The main advantage of this RNA strategy with an acetal ester group is a straightforward two-step all-base deprotection in a short period of time (3 h total) at room temperature. It consists of 1) the selective removal of the phosphate protecting group by β-elimination induced by a nonnucleophilic strong organic base(1, 8-diazabicyclo-ACHTUNGTRENNUNG [5.4. 0] undec-7-ene (DBU) or piperidine), and 2) the simultaneous liberation of the nucleobases, the 2’-OH, and the rupture of the succinyl or the Q-linker by an ammonia treatment without migration or chain rupture. This approach, which makes exclusive use of base-labile protecting groups, is challenging because of the well-known RNA instability in basic media.[3] To illustrate it, we report on the use of 2’-O-PivOM phosphoramidites to synthesize RNA oligomers up to 21 nucleotides in length and we demonstrate that they were obtained in high yield and high purity without chain rupture or migration.