Kanavarioti A, Bernasconi C F, Alberas D J, Baird E E
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
J Am Chem Soc. 1993;115(19):8537-46. doi: 10.1021/ja00072a003.
A kinetic study of oligoguanylate synthesis on a polycytidylate template, poly(C), as a function of the concentration of the activated monomer, guanosine 5'-monophosphate 2-methylimidazolide, 2-MeImpG, is reported. Reactions were run with 0.005-0.045 M 2-MeImpG in the presence of 0.05 M poly(C) at 23 degrees C. The kinetic results are consistent with a reaction scheme (eq 1) that consists of a series of consecutive steps, each step representing the addition of one molecule of 2-MeImpG to the growing oligomer. This scheme allows the calculation of second-order rate constants for every step by analyzing the time-dependent growth of each oligomer. Computer simulations of the course of reaction based on the determined rate constants and eq 1 are in excellent agreement with the product distributions seen in the HPLC profiles. In accord with an earlier study (Fakhrai, H.; Inoue, T.; Orgel, L. E. Tetrahedron 1984, 40, 39), rate constants, ki, for the formation of the tetramer and longer oligomers up to the 16-mer were found to be independent of length and somewhat higher than k3 (formation of trimer), which in turn is much higher than k2 (formation of dimer). The ki (i > or = 4), k3, and k2 values are not true second-order rate constants but vary with monomer concentration. Mechanistic models for the dimerization (Scheme I) and elongation reactions (Scheme II) are proposed that are consistent with our results. These models take into account that the monomer associates with the template in a cooperative manner. Our kinetic analysis allowed the determination of rate constants for the elementary processes of covalent bond formation between two monomers (dimerization) and between an oligomer and a monomer (elongation) on the template. A major conclusion from our study is that bond formation between two monomer units or between a primer and a monomer is assisted by the presence of additional next-neighbor monomer units. This is consistent with recent findings with hairpin oligonucleotides (Wu, T.; Orgel, L. E. J. Am. Chem. Soc. 1992, 114, 317). Our study is the first of its kind that shows the feasibility of a thorough kinetic analysis of a template-directed oligomerization and provides a detailed mechanistic model of these reactions.
本文报道了在聚胞苷酸模板poly(C)上进行的寡聚鸟苷酸合成的动力学研究，该研究考察了活化单体5'-单磷酸鸟苷2-甲基咪唑酯（2-MeImpG）浓度的影响。反应在23℃下，于0.05 M poly(C)存在的条件下，使用0.005 - 0.045 M的2-MeImpG进行。动力学结果与反应方案（式1）一致，该方案由一系列连续步骤组成，每个步骤代表一分子2-MeImpG添加到正在生长的寡聚物上。通过分析每个寡聚物随时间的生长情况，该方案可以计算出每个步骤的二级速率常数。基于所确定的速率常数和式1对反应过程进行的计算机模拟，与HPLC图谱中观察到的产物分布高度吻合。与早期研究（Fakhrai, H.; Inoue, T.; Orgel, L. E. Tetrahedron 1984, 40, 39）一致，发现形成四聚体及更长至16聚体的寡聚物的速率常数ki与长度无关，且略高于k3（三聚体形成），而k3又远高于k2（二聚体形成）。ki（i≥4）、k3和k2值并非真正的二级速率常数，而是随单体浓度变化。提出了与我们的结果一致的二聚化（方案I）和延伸反应（方案II）的机理模型。这些模型考虑到单体以协同方式与模板结合。我们的动力学分析使得能够确定模板上两个单体之间（二聚化）以及寡聚物与单体之间（延伸）共价键形成基本过程的速率常数。我们研究的一个主要结论是，两个单体单元之间或引物与单体之间的键形成受到额外相邻单体单元存在的促进。这与最近关于发夹寡核苷酸的研究结果（Wu, T.; Orgel, L. E. J. Am. Chem. Soc. 1992, 114, 317）一致。我们的研究是同类研究中的首个，展示了对模板导向寡聚化进行全面动力学分析的可行性，并提供了这些反应的详细机理模型。
