NEW PATHWAY OF ISOPRENOID BIOSYNTHESIS

SIR得到（2S）-N-（2 '-O-苄基-乙醇酰基）硼烷-10，2-磺内酰胺 11，2 - 13 C]乙酸; 20 g最近，一种新的异戊烯基合成途径 焦磷酸盐是由碳水化合物产生的 新陈代谢. 为了探索这一新途径在自然界中的分布， 为了进一步阐明该途径中的中间体，我们目前 合成1-脱氧-D-[2，4 - 13 C2]木酮糖。 起初，一个小 将从乙基溴- [ I， 13 C]乙酸酯和[2- 13 C]丙酮，其已由UW开发 组 在这项工作的过程中，很明显，这条路线 效率不是很高，因此不适合生产较大的 这是一种前体。 因此，第二条路线是 由SIR设计，它有效地利用了标记的标签 他们已经发展。 如果在E. 大肠杆菌与最初合成的前体样品的比较表明， 这种方法的可行性，更大量的化合物将 然后由SIR进行验证。 该前体将被进料至 多种生物系统合成类异戊二烯化合物， 将通过13 C-NMR分析产物中13 C的掺入 13C_13C偶联的出现，是由于 在适当的位置重排前体。 这 将概述新途径的相对重要性 与经典的甲羟戊酸途径相比。 接下来，我们将孵化 具有洗涤的细胞和无细胞提取物的双标记底物 大肠大肠杆菌直接在核磁共振光谱仪和检查 通过1H检测的13 C-NMR定期孵育， 对于双量子跃迁，对于新物种的出现， 两个13 C原子核相互连接。 因为几乎 没有其他代谢途径可以产生广泛的13 C-13 C 偶联，这将是一个非常具体的探针的中间体， 类异戊二烯生物合成的新途径。 的时间过程 新物种的出现和消失将表明它们 反应序列中的相对位置，以及标记 前体在额外的位置将允许分配 这些中间体。 这些体内实验将 需要高灵敏度来检测从非常 少量的瞬时化合物。 这在750上就可以实现 MHz NMR光谱仪在华盛顿大学。

The SIR provided (2S)-N-(2'-O-benzyl-glycoloyl)bomane-10-,2-sultam 11,2- 13C]acetic acid; 20g Recently, a new pathway to isopentenyl pyrophosphate has been discovered which emanates from carbohydrate metabolism. To probe distribution of this new pathway in nature and to elucidate further intermediates in the pathway, we are presently synthesizing 1-deoxy-D-[2,4-13C2]xylulose. Initially, a small quantity for exploratory work will be prepared from ethyl bromo- [ I - I 3C] acetate and [2-13C]acetone, which has been developed by the UW group. In the course of this work it became evident that this route is not very efficient and thus unsuitable for the production of larger quantities of this precursor. A second route has therefore been designed by the SIR, which makes effective use of the labeled synthons they have developed. If experiments on ubiquinone formation in E. coli with the initially synthesized precursor sample demonstrate the feasibility of this approach, a larger quantity of the compound will then be synthsized by the SIR. This precursor will be fed to a variety of biological systems synthesizing isoprenoid compounds and the products will be analyzed by 13C-NMR for the incorporation of 13C and the appearance of 13C_13C coupling, resulting from the rearrangement of the precursor, in the appropriate positions. This will give an overview of the relative importance of the new pathway compared to the classical mevalonate pathway. Next, we will incubate the double labeled substrate with washed cells and cell-free extracts of E. coli directly in the NMR spectrometer and examine the incubations periodically by 1H-detected 13C-NMR, looking specifically for double-quantum transitions, for the appearance of new species in which the two 13C nuclei are connected to each other. Since virtually no other metabolic pathway can give rise to extensive 13C-13C coupling, this will be a very specific probe for the intermediates in the new pathway of isoprenoid biosynthesis. The time course of the appearance and disappearance of new species will indicate their relative position in the reaction sequence, and labeling of the precursor in additional positions will allow the assignment of structures to these intermediates. These in vivo experiments will require high sensitivity to detect multiquantum transitions from very small amounts of transient compounds. This can be achieved on the 750 MHz NMR spectrometer at the Univ. of Wash.