Synthetic Approach toward Elucidation of the Structure and Function of Phytochromes

光敏色素是存在于植物中的光感受器，它携带一个胆碱发色团，该发色团与蛋白质共价连接。光致变色剂是生理非活性红光吸收Pr和生理活性远红光吸收Pfr之间的可光可逆分子开关。这种“红光-远红光可逆反应”在感知环境中的光信息方面起着重要作用，并与植物的发育、生长和分化等各种过程有关。在目前的光敏色素研究项目中，我们建立了用光敏色素的脱辅基蛋白组装比林生色团及其空间锁定的衍生物的有效方法。以下是通过合成有机方法阐明光敏色素1的结构和功能所获得的结果。许多细菌光敏色素携带胆绿素(BV)作为天然发色团，以不同的方式与蛋白质偶联。在…中更多的根癌农杆菌的光敏色素Agp1，BV共价连接到N-末端附近的半胱氨酸残基(位置20)。通过测试不同的天然和合成的BV衍生物，发现A环的乙烯基侧链用于生色团的连接。通过新的、有效的方法来阐明光致变色剂中发色团的立体化学结构，完成了具有空间锁定AB环或CD环的BV衍生物的全合成。测试了发色团与农杆菌光敏色素Agp1和Agp2的组装，这两个发色团将BV作为天然发色团。结果表明，Agp1的发色团具有Pr构型的C15=C16Z构型和C14-C15反构象，PfR构型的C15=C16E构型和C14-C15反构象。所有发色团加合物通过大小排除层析和组氨酸激酶活性分析来检测蛋白质构象。在任何一种情况下，15Za加合物的行为类似于Pr，而15Ea加合物的行为类似于Agp1的PfR形式。因此，用锁定的15Ea衍生物取代天然发色团可以在黑暗中带来PFR形式的光敏色素全蛋白。通过这种方式，可以在体内研究PFR的生理作用，并将其与PR/PFR循环和其他光效应分开。农杆菌光敏色素Agp1的N-端光敏模块Agp1-M15与立体锁定的合成BV-衍生物15Za组装得到15Za-Agp1-M15晶体，衍射率为3.4A，属于四方空间群1422，晶胞尺寸a=b=174 A，c=80 A.4。研究了一种新型的亲和层析方法，通过将合成的发色团作为配基固定在载体树脂上，建立了一种分离植物光敏色素的有效方法。合成了双锁定在AB环和CD环的发色团，用于确定PFR的AB环组分的立体化学，并对Agp1的光异构化和信号转导的详细机理进行了分析。为了探索一种制备光学活性光敏色素发色团的有效方法，以酒石酸酯为手性助剂，开发了一种新的高效的对映体选择性反应。此外，还获得了关于“同步效应”的新见解。较少

Phytochromes are photoreceptors existing in plants and carry a bilin chromophore, which is covalently attached to the protein. Phytochromes behave as photoreversible molecular switches between physiologically inactive red light-absorbing Pr and physiologically active far-red light-absorbing Pfr. This "red-far-red lights photoreversible reaction" plays important role to sense light information in the environment and related to various processes such as development, growth, and differentiation of plants. In the present research project on phytochromes, we established the efficient methods for the preparation of bilin chromophores and their sterically locked derivatives, which were assembled with apoproteins of phytochromes. The following are the results obtained by synthetic organic approach toward elucidation of the structure and function of phytochromes1. Many bacterial phytochromes carry biliverdin (BV) as natural chromophore, which is coupled in a different manner to the protein. In … More phytochrome Agpl of Agrobacterium tumefaciens, BV is covalently attached to a cysteine residue close to the N-terminus (position 20). By testing different natural and synthetic BV derivatives, it was found that the ring A vinyl side chain is used for chromophore attachment.2. Total syntheses of BV derivatives with sterically locked AB-or CD-ring component were accomplished via new and efficient methods towards elucidation of the stereochemistry of the chromophore in phytochromes. The chromophores were tested for assembly with Agrobacterium phytochrome Agpl and Agp2, which incorporate BV as natural chromophore. It was found that the chromophore of Agpl adopts a C15=C16 Z configuration and a C14-C15 anti conformation in the Pr form, and a C15=C16 E configuration and a C14-C15 anti conformation in the Pfr form. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agpl. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.3. The N-terminal photosensory module of Agrobacterium phytochrome Agpl, Agp1-M15, was assembled with a sterically locked synthetic BV-derivative 15Za to give crystals of 15Za-Agpl-M15, which diffract to 3.4 A resolution and belong to the tetragonal space group 1422 with unit cell dimensions a=b=174 A, c=80 A.4. A new type of affinity chromatography was investigated to establish an efficient method for the isolation of plant phytochromes by attaching synthetic chromophores as a ligand on carrier resin.5. Chromophores doubly locked at AB-and CD-rings were synthesized to determine the stereochemistry of AB-ring component of Pfr and also toward analysis of the detailed mechanism of photoisomerization of Agpl and signal transduction.6. To exploit an efficient method for the preparation of optically active phytochrome chromophores, new highly efficient and enantioselective reactions were developed utilizing tartaric acid esters as a chiral auxiliary. Furthermore new insights were obtained regarding "syn-effect". Less