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

据报道，菌内酯A/B （MycA/B）是引起被忽视的热带病布鲁里溃疡的溃疡分枝杆菌的唯一毒力因子。这种疾病导致组织破坏和局部炎症抑制。MycA/B的作用机制涉及通过Sec61抑制蛋白质易位，Sec61是一个跨膜孔，允许多肽链穿过或进入内质网（ER）膜。我们假设真菌内酯与Sec61的结合可以被一个小分子抑制，而不影响易位。发现这种分子需要开发一种检测方法来测试霉菌内酯与Sec61结合的程度，这是本项目的目的。该分析是基于生物发光共振能量转移（BRET），需要：1。对溃疡分枝杆菌进行基因改造以产生Sec61-NanoLuciferase （NLuc）酶融合蛋白，该蛋白通过其底物反应而发光, 2。荧光团与MycA/B的化学偶联得到示踪配体。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出。在抑制剂存在的情况下，发射的荧光与抑制剂的浓度和结合强度成正比。这个博士项目的重点是MycA/ b -荧光团示踪剂的合成。文献合成MycA/B核心至少需要17个步骤，脂肪酸尾部需要17个步骤，它们的偶联和脱保护需要2-3个步骤。此外，溃疡分枝杆菌生长缓慢且危险，妨碍了MycA/B的分离。因此，一种新的、高效的MycA/B半合成将被开发出来，并伴随变化，以产生可与荧光团偶联的类似物。拟议的半合成首先涉及分离替代菌内酯F (MycF，图1)，这是一种由鱼类病原体海洋分枝杆菌产生的脂肪酸链变体。由于它对人类的致病性较低且生长速度较快，因此可以扩大其培养规模并大量分离MycF。将醇保护为tbs醚（方案1），然后水解烯酮酯，可以为核心1提供一个可以与合成的MycA/B脂肪酸侧链偶联的游离醇。聚合合成MycA/B脂肪酸侧链（方案2）将使用Burke的迭代MIDA硼酸法合成多烯2和手性池不对称合成三醇前体3。两者的耦合将以立体选择的方式实现，得到4。酯水解，然后通过酯化作用附着在菌内酯核心1上，以迄今为止最短的途径得到醇保护的MycA/B。随着这一途径的建立，将对其进行修改，以提供胺功能化的MycA/B，准备与荧光基团结合，并开发sec61结合试验。参考文献(1)Chany, a.c；Tresse c;Casarotto诉;布兰查德，n.n .纽约州众议员，2013,30,1527。(2) Hall, b.s.；Ogbechi, j .;西蒙兹，r.e.；山,k;麦肯纳,m;高,美国;李晓明，张晓明。科学与技术学报，2014,32(1):391 - 391。(3) Ogbechi， J.；霍尔，文学士；Bodman-Smith k;西蒙兹，r.e.；革命联合阵线、M.-T;Pluschke g;沃格尔,m;吴、H.-L;色料,a;埃斯蒙，c.t.；李志强，李志强。中国生物医学工程杂志，2015,31(1):391 - 391。(4) Schurmann， M.；1月,p;齐格勒,美国;《细胞化学》生物学报，2016,23,435。(5)布朗，c.a.；Aggarwal， V. K. Chem。欧元。J. 2015,21, 13900。(6)王刚；阴:;根岸英一,e。化学。欧元。[j] . 2011, 17(4): 418 - 418。(7)护林员，学士；马罗斯，e.a.；莫西人,l;Adusumilli,美国;李，r.e.；Colorni, a;罗兹,m;小的，p.l.c.感染。免疫学杂志，2006,74,6037。(8)宋芳；Fidanze,美国;贝诺维茨，a.b.；陈志刚，王志刚。四面体，2007,63,5739。(9)韦利，e.m.；罗伊·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.