Semi-synthesis of Mycolactone A/B and Conjugable Derivatives for Sec61 Binding Assays

据报道，霉菌内酯A/B(Myca/B)是引起被忽视的热带疾病布鲁里溃疡的唯一致病因子。这种疾病会破坏组织，抑制局部炎症。Myca/B的作用机制包括抑制Sec61的蛋白质转位，Sec61是一个跨膜孔，允许多肽链穿过或进入内质网(ER)膜。我们假设，霉菌内酯与Sec61的结合可以被一个小分子抑制，而不影响易位。这种分子的发现需要开发一种测试方法来测试霉菌内酯与Sec61结合的程度，这就是这个项目的目标。该分析基于生物发光共振能量转移(BRET)，需要：1.对溃烂分枝杆菌进行基因修饰，以产生Sec61-纳米荧光素酶(NLuc)融合蛋白，该融合蛋白通过其底物的反应发光；以及2.荧光团与Myca/B的化学偶联，得到‘示踪剂’配体。当Myca/B示踪剂与Sec61结合时，从近端NLuc发出的光被荧光团吸收，并以不同的可测量波长发射。在抑制剂的存在下，发射的荧光强度与抑制剂的浓度和结合强度成正比。本博士项目致力于Myca/B-荧光团示踪剂的合成。文献合成至少需要Myca/B核心的17个步骤，脂肪酸尾部的17个步骤，以及它们的偶联和去保护的2-3个步骤。此外，溃烂分枝杆菌缓慢和危险的生长阻止了Myca/B的分离。因此，一种新的、有效的Myca/B的半合成将被开发出来，以产生可与荧光素结合的类似物。所提出的半合成首先涉及到替代霉内酯F(MycF，图1)的分离，这是一种由鱼类病原体海洋分枝杆菌产生的脂肪链变体。由于这种细菌对人类的致病性较低，生长速度更快，因此可以扩大其培养规模，并大量分离MycF。以TBS-醚的形式保护醇(方案1)，然后水解烯酮酯，应为核心1提供一个准备好与合成的Myca/B脂肪酸侧链偶联的游离醇。Myca/B脂肪酸侧链的聚合合成(方案2)将使用Burke的迭代MIDA硼酸盐方法用于多烯2和手性池不对称合成三醇前体3。两者将以立体选择性的方式耦合得到4。酯水解然后通过酯化连接到霉菌内酯核心1将以最短的路线得到醇保护的Myca/B。在建立了这条路线后，它将被修改以提供准备好与荧光团连接的胺功能化的Myca/B并开发与Sec61结合的分析。参考文献(1)Chany，A.-C.；Tresse，C.；Casarotto，V.；Blanchard，N.NAT。戳。代表2013，30,1527.(2)Hall，B.S.；Ogbechi，J.；Simmonds，R.E.；Hill，K.；McKenna，M.；High，S.；Willis，A.E.PLoS Path。(3)Ogbechi，J.；Hall，B.S.；Bodman-Smith，K.；Simmonds，R.E.；Ruf，M.-T.；Pluschke，G.；Vogel，M.；Wu，H.-L.；Stainer，A.；Esmon，C.T.；Ahnstrom，J.PLoS Pathog 2015，11，e1005011.(4)Schurmann，M.；Janning，P.；Ziegler，S.；Waldmann，H.Cell Chem。比奥尔。2016年，23,435。(5)布朗，C.A.；阿加瓦尔，V.K.化学。欧元。J·2015，21,13900。(6)王刚；尹宁；Negishi，E.I.化学。欧元。(7)Ranger，B.S.；Mahous，E.A.；Mosi，L.；Adusumilli，S.；Lee，R.E.；Colorni，A.；Rhodes，M.；Small，P.L.C.Infect。伊蒙。2006年，74,6037.(8)Song，F.；Fidanze，S.；Benowitzx，A.B.；Kishi，Y.四面体2007，63,5739.(9)Woerly，E.M.；Roy，J.；Burke，M.D.Nat Chem 2014，6,484.

Mycolactone A/B (MycA/B) is reportedly the sole virulence factor of Mycobacterium ulcerans which causes the neglected tropical disease Buruli ulcer. This disease causes tissue destruction and suppression of local inflammation. The mechanism of action of MycA/B involves inhibition of protein translocation by Sec61, a transmembrane pore which permits passage of polypeptide chains across or into endoplasmic reticulum (ER) membranes. We hypothesise that binding of mycolactone to Sec61 can be inhibited, by a small molecule, without affecting translocation. Discovery of such a molecule requires the development of an assay to test the extent to which mycolactone is binding to Sec61 and this is the aim of this project. The assay is based on Bioluminescence Resonance Energy Transfer (BRET) and requires:1. Genetic modification of M. ulcerans to produce a Sec61-NanoLuciferase (NLuc) enzyme fusion protein which emits light through reaction of its substrate; and,2. Chemical conjugation of a fluorophore to MycA/B to give a 'tracer' ligand. When the MycA/B tracer binds to Sec61, the light emitted from proximal NLuc is absorbed by the fluorophore and emitted at a different, measurable wavelength. In the presence of inhibitors, there will be less emitted fluorescence in proportion to the concentration and binding strength of the inhibitor.This PhD project focuses on synthesis of the MycA/B-fluorophore tracer. Literature syntheses require at least 17 steps for the MycA/B core, 17 for the fatty acid tail and 2-3 more for their coupling and deprotection. In addition, the slow and hazardous growth of M. ulcerans precludes isolation of MycA/B. Hence, a novel, efficient semi-synthesis of MycA/B will be developed along with variations to produce analogues amenable to conjugation with the fluorophore.The proposed semi-synthesis firstly involves isolation of alternative mycolactone F (MycF, Figure 1), a fatty acid chain variant produced by the fish pathogen M. marinum. Since this is less pathogenic to humans and grows at a faster rate, its culture can be scaled up and MycF isolated in larger quantities. Protection of the alcohols as TBS-ethers (Scheme 1) followed by hydrolysis of the enone ester should provide the core 1 with one free alcohol ready for coupling with synthetic MycA/B fatty acid side chain. A convergent synthesis of the MycA/B fatty acid side chain (Scheme 2) will use Burke's iterative MIDA boronate methodology for the polyene 2 and chiral pool asymmetric synthesis of the triol precursor 3. Coupling of the two will be achieved in a stereoselective manner to give 4. Hydrolysis of the ester followed by attachment to the mycolactone core 1 by esterification will give alcohol-protected MycA/B in the shortest route to date. With this route established, it will be modified to provide amine-functionalised MycA/B ready for conjugation to a fluorophore and the development of a Sec61-binding assay.References(1) Chany, A.-C.; Tresse, C.; Casarotto, V.; Blanchard, N. Nat. Prod. Rep. 2013, 30, 1527.(2) Hall, B. S.; Ogbechi, J.; Simmonds, R. E.; Hill, K.; McKenna, M.; High, S.; Willis, A. E. PLoS Path. 2014, 10, e1004061.(3) Ogbechi, J.; Hall, B. S.; Bodman-Smith, K.; Simmonds, R. E.; Ruf, M.-T.; Pluschke, G.; Vogel, M.; Wu, H.-L.; Stainer, A.; Esmon, C. T.; Ahnstrom, J. PLoS Pathog 2015, 11, e1005011.(4) Schurmann, M.; Janning, P.; Ziegler, S.; Waldmann, H. Cell Chem. Biol. 2016, 23, 435.(5) Brown, C. A.; Aggarwal, V. K. Chem. Eur. J. 2015, 21, 13900.(6) Wang, G.; Yin, N.; Negishi, E.-i. Chem. Eur. J. 2011, 17, 4118.(7) Ranger, B. S.; Mahrous, E. A.; Mosi, L.; Adusumilli, S.; Lee, R. E.; Colorni, A.; Rhodes, M.; Small, P. L. C. Infect. Immun. 2006, 74, 6037.(8) Song, F.; Fidanze, S.; Benowitzx, A. B.; Kishi, Y. Tetrahedron 2007, 63, 5739.(9) Woerly, E. M.; Roy, J.; Burke, M. D. Nat Chem 2014, 6, 484.