STRUCTURAL ANALYSIS OF NUCLEIC ACID COMPONENTS BY NMR

在过去的几年里，药物化学实验室一直致力于在糖环上含有各种修饰的核苷类似物的合成。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，我们使用核磁共振波谱在不同条件下研究了这些类似物。我们发现，在2‘或3’碳上含有氟原子的脱氧和/或二脱氧类似物偏爱非常不同的糖袋，这取决于这个高度电负性原子的位置和立体化学。此外，偶联常数和NOE研究使我们能够确定几种[3.1.0]双环核苷类似物的首选环构象和旋转体分布。我们发现某些酶，如腺苷脱氨酶，更喜欢只有3'-末端（北方）糖袋的类似物。在将构象分析扩展到氟化类似物的单磷酸和三磷酸核苷酸时，表明磷酸化不影响溶液中的假旋转平衡，并且环折叠仍然由氟原子的位置驱动。我们在分析中加入了19F和31P核磁共振，并使用1H-19F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低聚物的结构特征。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，以检查不同化学修饰产生的特定构象趋势；2)建立这些趋势（耦合常数、反应性、伪旋转参数）的数据库，以便与未来的合成类似物进行比较；3)通过多维核磁共振波谱和分子动力学模拟，使用最新的力场进行核酸结构计算，精确定义含有这些类似单体的DNA低聚物的结构。

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.