STRUCTURAL ANALYSIS OF NUCLEIC ACID COMPONENTS BY NMR

在过去的几年里，药物化学实验室一直参与合成糖环上含有各种修饰的核苷类似物。此外，还制备了基于刚性双环[3.1.0]模板的碳环核苷。这些类似物的设计是基于这样的假设，即特定的结构修饰可能会使糖环的构象偏向于特定的环褶皱。为了确定它们的精确构象，我们研究了这些类似物在不同条件下使用NMR光谱。我们发现在2'或3'碳上含有氟原子的脱氧-和/或双脱氧类似物根据该高度电负性原子的位置和立体化学而优选非常不同的糖皱褶。此外，耦合常数和NOE的研究，使我们能够定义的几个[3.1.0]双环核苷类似物的优选环构象和旋转异构体分布。我们发现，某些酶，如腺苷脱氨酶，更喜欢只有3 '-内（北方）糖起皱的类似物。在扩展的构象分析的单磷酸和三磷酸核苷酸的氟化类似物，它表明，磷酸化不影响在溶液中的伪旋转平衡，和环褶皱仍然是由氟原子的位置驱动。我们在分析中包括了19 F和31 P NMR，并使用1H-19 F耦合常数改进了我们的伪旋转计算。使用二维光谱，我们正在表征含有一些上述修饰核苷酸的核酸寡聚体的结构特征。该项目的短期目标是：1）继续研究每个单独类似物的溶液结构，以检查由不同化学修饰产生的特定构象趋势;（2）建立这些趋势的数据库（耦合常数，反应性，伪旋转参数）与未来的合成类似物进行比较，以及3）通过多维和多核NMR光谱和分子动力学模拟使用最新的力场用于核酸结构计算来精确地确定包含这些类似单体的DNA寡聚体的结构。

For the past several years, the Laboratory of Medicinal Chemistry has been involved in the synthesis of nucleoside analogues containing various modifications on the sugar ring. In addition, carbocyclic nucleosides based on a rigid bicyclo[3.1.0] template have also been prepared. These analogues have been designed based on the assumption that the particular structural modification may bias the conformation of the sugar ring to favor a specific ring pucker. In order to define their precise conformations, we have studied these analogues under varying conditions using NMR spectroscopy. We found that deoxy- and/or dideoxy analogues containing a fluorine atom on the 2' or 3'carbon prefer very distinct sugar puckers depending on the position and the stereochemistry of this highly electronegative atom. In addition, coupling constant and NOE studies have allowed us to define the preferred ring conformations and rotamer distributions of several [3.1.0]bicyclo nucleoside analogues. We discovered that certain enzymes, such as adenosine deaminase, prefer analogues with only 3'-endo (Northern) sugar puckers. In extending the conformational analysis to mono- and triphosphate nucleotides of the fluorinated analogues, it was shown that phosphorylation does not affect the pseudorotational equilibrium in solution, and that the ring pucker is still driven by the position of the fluorine atom. We have included 19F and 31P NMR in our analysis and refined our pseudorotational calculations using 1H-19F coupling constants. Using 2-dimensional spectrocopy, we are characterizing the structural features of nucleic acid oligomers containing some of the afoermentioned modified nucleotides. The short term goals of the project are 1) to continue to study the solution structures of each individual analogue to examine specific conformational trends that emerge from distinct chemical modifications; 2) Create a database of these trends (coupling constants, reactivity, pseudorotational parameters)to compare with future synthetic analogues, and 3) to precisely define the structures of DNA oligomers containing these analogue monomers by multidimensional and multinuclear NMR spectroscopy ad molecular dynamics simulations using the most updated force fields for nucleic acid structure calculations.