A Photoactive Isoprenoid Diphosphate Analogue Containing a Stable Phosphonate Linkage: Synthesis and Biochemical Studies with Prenyltransferases

A number of biochemical processes rely on isoprenoids, including the post-translational modification of
signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have
been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and prenyltransferases. While these initial analogues proved to be excellent structural analogues with good cross-linking
capability, they lack the stability needed when the goals include isolation of cross-linked species, tryptic
digestion, and subsequent peptide sequencing. Here, the synthesis of a benzophenone-based farnesyl
diphosphate analogue containing a stable phosphonophosphate group is described. Inhibition kinetics,
photolabeling experiments, as well as X-ray crystallographic analysis with a protein prenyltransferase
are described, verifying this compound as a good isoprenoid mimetic. In addition, the utility of this new
analogue was explored by using it to photoaffinity label crude protein extracts obtained from Hevea
brasiliensis latex. Those experiments suggest that a small protein, rubber elongation factor, interacts
directly with farnesyl diphosphate during rubber biosynthesis. These results indicate that this benzophenone-based isoprenoid analogue will be useful for identifying enzymes that utilize farnesyl diphosphate as a
substrate.

诸多生化过程依赖类异戊二烯（isoprenoids），涵盖信号蛋白的翻译后修饰以及多种化合物的生物合成。科研人员已开发出光活化类似物（photoactivatable analogues），用于研究利用类异戊二烯的酶类，例如类异戊二烯合酶（isoprenoid synthases）与异戊烯转移酶（prenyltransferases）。尽管这类早期类似物作为结构类似物表现优异，且具备良好的交联能力，但当研究目标为分离交联产物、进行胰蛋白酶消化（tryptic digestion）及后续肽测序时，其稳定性仍无法满足需求。本文报道了一种带有稳定膦酰磷酸基团的二苯甲酮修饰法尼基二磷酸（farnesyl diphosphate）类似物的合成方法。通过抑制动力学实验、光标记实验以及与蛋白质异戊烯转移酶的X射线晶体学分析，验证了该化合物为优良的类异戊二烯模拟物。此外，本文通过将该新型类似物用于光亲和标记巴西橡胶树（Hevea brasiliensis）乳胶提取的粗蛋白样品，探究了其应用价值。实验结果表明，小分子蛋白质橡胶延伸因子在橡胶生物合成过程中可直接与法尼基二磷酸相互作用。上述结果显示，这种二苯甲酮修饰的类异戊二烯类似物可用于识别以法尼基二磷酸为底物的酶类。