Novel Natural and Synthetic Brominated and Iodinated Compounds in the Environment

本研究旨在继续利用效应导向分离和鉴定的方法鉴定新型有机溴（OBCs）和有机碘（OICs）化合物，以确定新化合物的生物活性。包括活细胞阵列在内的一系列功能性生物测定将用于识别受最活性化合物影响的分子途径。将开发一种将超高分辨率质谱法与蛋白质组学相结合的“下拉”方法，以确定新型OBCs和OIs的分子靶标。拟议的研究有几个具体目标：1)在一系列海洋和淡水环境中确定和量化新的OBCs和oic；2)使用质量平衡（在Br和I上）方法来确定可能存在哪些额外的OBCs和oic； 3)使用生物测定法来确定新型OBCs和oic的毒性和潜在的有益用途。拟议中的研究计划最初将集中在食物链的基础上，包括海藻和海绵、珊瑚等无脊椎动物。OBCs将通过靶向和非靶向质谱相结合的方式进行鉴定，包括超高分辨率质谱UHR LC/MS （OrbiTrap）和三重四极杆串联质谱（LC-MS/MS）、LC四极杆飞行时间质谱（Q-ToF）和高分辨率GC高分辨率质谱（HRGC-HRMS）。总溴（TBr）和总碘（TI）将通过仪器中子活化分析（INAA）测定粗样品或有机溶剂提取物。精确的质量和公式将由M1确定，并利用M2数据阐明结构。将确定已知合成的阻燃剂和oic以及建议作为替代阻燃剂的化合物的浓度。制备型高效液相色谱将提取和纯化足够数量的未知化合物。然后将浓缩的分离物进行标准方法，如紫外光谱法、红外光谱法和元素分析，以帮助阐明和/或确认结构。一旦确定了结构，就可以开发合成程序，并制造出更多的纯化合物。一旦获得更大质量的材料，将对它们进行一系列生物测定，以确定：1)它们的毒性作用模式和效应阈值，以及第二套生物测定，以确定它们作为治疗剂的潜在效用。除了筛选其毒性和潜在的有益用途外，还将调查其生产的潜在生态和/或生理原因。这将通过使用微生物培养物的逆向工程筛选来完成，基于特定化合物合成途径中酶的存在或不存在。如果确定化合物毒性太大，则不会进一步测试其治疗特性。

The research is designed to continue to identify novel organo-bromine (OBCs) and organo-iodine (OICs) compounds by use of effect-directed fractionation and identification to determine biological activities of novel compounds. A range of functional bioassays including live cell arrays will be used to identify molecular pathways affected by the most active classes of compounds. A “pull-down” approach that will combine ultra-high resolution mass spectrometry with proteomics will be developed to identify molecular targets of novel OBCs and OIs. The proposed research has several specific objectives: 1) identify and quantify novel OBCs and OICs in a range of marine and freshwater environments; 2) use a mass balance (on Br and I) approach to determine what additional OBCs and OICs might be present and 3) use bioassays to determine the toxicity and potential beneficial uses of novel OBCs and OICs. The proposed research program will focus initially on the base of the food chain, including marine algae and invertebrates such as sponges and corals. OBCs will be identified by use of a combination of targeted and un-targeted mass spectrometry, including ultra-high resolution mass spectrometry UHR LC/MS (OrbiTrap) and triple quadrupole tandem mass spectrometry (LC-MS/MS), LC quadrupole-time of flight mass spectrometry (Q-ToF) and high resolution GC High Resolution Mass spectrometry (HRGC-HRMS). Total Br (TBr) and total iodine (TI) will be determined by Instrumental Neutron Activation Analysis (INAA) of either crude samples or organic solvent extracts. Accurate masses and formulas will be determined by M1 and structures elucidated by use of M2 data. Concentrations of known synthetic OBCs and OICs and those compounds that have been suggested as replacement flame retardants will be determined. Sufficient quantities of unknown compounds will be extracted and purified by preparative HPLC. The concentrated isolates will then be subjected to standard methods such as UV and spectrometry and IR spectroscopy and elemental analyses to help elucidate and or confirm structures. Once a structure has been confirmed, a synthesis procedure can be developed and greater quantities of pure compounds made. Once greater masses of material are available they will be subjected to a range of bioassays to determine: 1) their toxic mode of action and threshold for effects and a second set of bioassays to determine their potential utility as therapeutic agents. In addition to screening for toxicity and potential beneficial uses, the potential ecological and or physiological reasons for their production will be investigated. This will be done by use of a reverse-engineering screening of microbial cultures, based on the presence or absence of enzymes in pathways of syntheses of specific compounds. If compounds are determined to be too toxic, they will not be tested farther for therapeutic properties.