The Synthesis and Characterization of Brominated 17α-ethinylestradiol

Paige Brown, 2019

For this project, the goal was to synthesize and characterize a prevalent and potentially environmentally devastating micro-pollutant, brominated 17α-ethinylestradiol (EE2). EE2 is a synthetic estrogen used in contraceptives, and it enters the environment via waste water treatment plants as it is not fully metabolized in the body or fully removed by waste water treatment.1 It has a very high estrogenic activity, so it has a greater effect on the endocrine system of an organism at lower levels then any naturally occurring forms of estrogen.2 Therefore, the levels of EE2 present in wastewater effluent are high enough to effect the surrounding ecosystem.3 EE2 does not necessarily enter the environment in the same form that it is found in contraceptives. In waste water treatment plants, the water is often treated with hypochlorite in order to disinfected it, exposing EE2 to chlorine ions.4 These chlorine ions can react with EE2 and undergo electrophilic aromatic substitution, adding either 1 or 2 chlorine ions to the benzene ring in the molecule. Once this process has occurred, it is possible for these chlorines to be replaced by bromine ions if there are bromine ions present in the water, as is common in communities by the coast where sea water intrudes into the ground water. The final result is three unique species: 2-bromoEE2, 4-bromoEE2, and 2,4-dibromoEE2. These compounds then disperse into the environment, where they can degrade through a variety of pathways. In order to study the brominated forms of EE2, it was necessary to synthesize and characterize these three compounds. The synthesis was performed using purified N-Bromosuccinimide (NBS) as the bromine source. NBS was added to EE2 in proportions of approximately 1.5 mols of NBS to 1 mol of EE2. The reaction was allowed to run for 24 hours, and then Gas Chromatography Mass Spectroscopy (GC-MS) was used to characterize the product. The GC-MS vaporizes the product, and then runs it through a column so that compounds of different masses come out at different times. It then records the mass of these compounds, which makes it possible to compare this mass to the mass of the expected product to see if they match. The chromatograph showed the product was a mixture of 2-bromoEE2, 4-bromoEE2, 2,4-dibromoEE2 and unreacted EE2. Having received confirmation that the desired products were successfully synthesized, the next step was to purify the mixture using High Performance Liquid Chromatography (HPLC). After extensive method development, it was possible to separate the products and to collect them as pure fractions. These fractions were then combined and dried, giving pure 2-bromoEE2, 4-bromoEE2, and 2,4-dibromoEE2 solid products. Due to the stereochemistry of the reaction and the presence of excess bromine ions, the 2,4-dibromoEE2 product was created in highest amount. 4-bromoEE2 was the next most abundant product, while 2-bromoEE2 was created in the lowest amount. 4-bromoEE2 and 2-bromoEE2 are white solids while 2,4-dibromoEE2 is a slightly yellow solid. This suggests that the addition of the two bromine groups allows the molecule to absorb light in the visible range, which is something that the starting material EE2 is not capable of doing. To check the purity of the fractions, GC-MS was used again, and mass was used to distinguish between the identity of the dibrominated product and the two monobrominated products. However, since the monobrominated products, 2-bromoEE2 and 4-bromoEE2, have the same mass, GC-MS was not enough to determine the identity and purity of those fractions. So Nuclear Magnetic Resonance (NMR) was used to identify the location of the hydrogens in each molecule. For each molecule, the hydrogens attached to the benzene ring had a unique chemical shift and splitting. So by examining the location of these hydrogens, it was possible to distinguish which fraction was the 2-bromoEE2 and which was 4-bromoEE2. Therefore, the desired products were successfully synthesized, identified, and purified. Future research will be devoted to studying the photo reactivity of these molecules. Figures

Faculty Mentor: Soren Eustis Funded by the Kufe Family Research Fellowship

OH

HO

EE2OH

HO

2-Bromo-EE2

Br

OH

HO

4-Bromo-EE2

Br

OH

HO

2,4-Bromo-EE2

Br

Br

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