13C 13C CORRELATION SPECTROSCOPY OF U 13C ERYTHROMYCIN

除了下面描述的事态发展外，我们最近 演示了高精度的核间距离测量 CMR7脉冲序列。同核偶联可以测量到 这种方法的精度优于5赫兹，这意味着 核间距离的测量结果优于0。1-0.2 A 精度达到4-5‘A。我们已经开发出一种脉冲序列，用于 多自旋系统中的高效双量子偶极重耦合 在魔角旋转核磁共振下，基于Levitt的C7序列和 同事。对于双自旋系统，C7序列提供了更高的 全极化转移和双量子滤波(DQF) 效率(-73%)高于情节剧序列(-52%)，因为 消除了重耦合干扰对转子相位的依赖。 然而，从实验上讲，C7序列的DQF效率取决于 重要的是，由于交叉项引起的错误 化学位移和射频(RF)场不均匀。许多 应用中需要多自旋的DQ相干激发 系统，它显示了广泛的各向同性和各向异性 化学位移。此外，该序列的实验可靠性 是在低成本条件下成功实施的关键 敏感度和温度。为了满足这些要求，我们有 构造了重新耦合偶极相互作用的脉冲序列 与化学位移无关，通过结合各种不同的CN元素 对称性。去除了C7序列固有的误差项 通过复合MLEV型旋转；因此，我们指的是新的 序列为CMR7(结合MLEV重聚焦和C7)。我们有 用双量子理论证明了这种方法的有效性。 U-“C标记氨基酸的筛选与”C-“C化学位移 U-“C标记抗生素红霉素的相关光谱研究 A.在73%的偏振转移情况下，相声与对角线的比率 强度预计几乎为3：1，在双自旋情况下， 作为U-“C”，N-Gly，我们观察到了比2：1更好的相对 2D光谱中的强度。然而，完整的理论DQF 在多自旋系统和类似的系统中通常不能实现效率 在相关谱中观察到与相声相关的行为 强度。然而，在许多情况下，相声强度超过了 对角线峰值，在有利的情况下， 强度大于2：1，即使在多自旋极限下也是如此。一个 这些效果的说明性示例，以及 在延伸到第二个13C化学位移时观察到的分辨率 维度是由U-“C标记的大环内酯类抗生素提供的 红霉素A(EA)。电针通过与一种蛋白质结合抑制蛋白质合成 细菌核糖体。的实际结构信息。 红霉素-核糖体复合体由于缺乏而无法访问 有效晶体S和溶液核磁共振谱分辨率较差 由于核糖体的缓慢重定向运动。结果, 红霉素-核糖体复合体具有固体核磁共振波谱 即使在溶液中也是如此。因此，为了方便结构调整， 研究EA的自由形式和复杂形式，有必要 在固态下进行明确的化学位移分配。我们 使用CMR7方法完成了化学位移作业 并正根据CMR7进行进一步的结构研究 方法。

In addition to the developments described below, we have recently demonstrated highly precise internuclear distance measurements with the CMR7 pulse sequence. Homonuclear couplings can be measured to a precision of better than 5 Hz with this approach, implying internuclear distance measurements of better than 0. 1-0.2 A precision out to 4-5 'A. We have developed a pulse sequence for efficient double-quantum dipolar recoupling in multiple spin systems under magic-angle-spinning NMR, based on the C7 sequence of Levitt and co-workers. For two-spin systems, the C7 sequence offers higher overall polarization transfer and double-quantum filtration (DQF) efficiency (-73%) than the MELODRAMA sequence (-52%), because the dependence of the recoupled interaction on rotor phase is eliminated. Experimentally, however, DQF efficiency with the C7 sequence depends significantly on the errors that arise from cross terms between chemical shifts and radiofrequency (rf) field inhomogeneity. Many applications require the excitation of DQ coherence in multi-spin systems, which display a wide range of isotropic and anisotropic chemical shifts. Also, the experimental reliability of the sequence is crucial for successful implementation under conditions of low sensitivity and temperature. To meet these requirements, we have constructed a pulse sequenc e that recouples dipolar interactions independent of chemical shifts, by combining Cn elements of various symmetries. The error terms inherent to the C7 sequence are removed by composite MLEV-type rotations; therefore, we refer to the new sequence as CMR7 (Combined MLEV-Refocusing with C7). We have demonstrated the utility of this approach with double-quantum filtration of U-"Clabeled amino acids and "C-"C chemical shift correlation spectroscopy of the U-"C-labeled antibiotic, erythromycin A. With 73% polarization transfer, the ratio of crosspeak to diagonal intensity is expected to be almost 3: 1, and in two-spin cases such as U- "C, "N-Gly, we have observed better than 2: 1 relative intensities in 2D spectra. However, the full theoretical DQF efficiency is usually not realized in multi-spin systems and similar behavior is observed in correlation spectra with respect to crosspeak intensities. Nevertheless, in many cases crosspeak intensities exceed the diagonal peaks, and in favorable instances the ratio of intensities is greater than 2: 1, even in the multi-spin limit. An illustrative example of these effects, and the improvement in resolution observed upon extending to a second 13C chemical shift dimension, is provided by the U-"C-labeled macrolide antibiotic erythromycin A (EA). EA inhibits protein synthesis by binding to a bacterial ribosome. Actual structural information on the erythromycin-ribosome complex has been inaccessible due to the paucity of available crystal s and poor resolution of the solution NMR spectra due to the slow reorientational motion of the ribosome. As a result, the erythromycin-ribosome complex exhibits a solid-state NMR spectrum even in solution. Therefore, in order to facilitate structural studies of EA in both its free and complexed forms, it is necessary to make unambiguous chemical shifts assignments in the solid state. We have accomplished the chemical shift assignments using the CMR7 method and are pursuing further structural studies based upon the CMR7 method.