Development of a Novel Molecular Recognition Probe for the Detection of

1. CEA-CREST Phosphorus is often found to be the limiting nutrient in freshwater systems. It has been assumed that phosphorus species exist solely as phosphates and other oxidized forms in the environment, thereby failing to undergo redox reactions. However, recent biochemical findings suggest that phosphite and hypophosphite are possible significant alternative sources of phosphorus to organisms. Data on reduced forms of phosphorus and their ultimate products in natural environments is scarce, yet phosphite could play an important role in the phosphorus cycle (1). Phosphite has been measured in the lab using techniques such as ion chromatography, 31P NMR, and ICP-MS. None of these methods are suitable for in situ monitoring. Making measurements directly in the environment is important because the lifespan of phosphite may be too brief (i.e., due to oxidation) for accurate determination in the laboratory (1). 3+ 3+ a a b c c b Bench Top Column LPLC Column Development of a Novel Molecular Recognition Probe for the Detection of Phosphite in Natural Waters Alex Carlton1,3, John A. Moss2, Marc M. Baum2, and Grady Hanrahan1,3. Department of Chemistry & Biochemistry, California State University, Los Angeles, Los Angeles, CA 90032 Department of Chemistry, Oak Crest Institute of Science, 2275 E. Foothill Blvd, Pasadena, CA 91107 Center for Environmental Analysis, California State University, Los Angeles, CA 90032 Terbium and Fluorescence Terbium(III) complexes strongly with oxygen, nitrogen, and sulfur atoms, and exhibits intense fluorescence with long lifetimes (~ms). It is also well known and documented that terbium(III) fluoresces strongly with dipicolinic acid4, which is structurally analogous to the aminophosphonic acid produced by the Mannich reaction. Importance of Phosphite Synthesis Benzylamine phosphonic acid. Phosphorous acid (4.1085 g, .05 mol) and benzylamine (2.8 mL, .025 mol) were dissolved in 5 mL distilled water and 5 mL concentrated HCl. They were refluxed for an hour, when 8 mL of formaldehyde solution was added drop wise. The reaction was refluxed for another hour and left overnight, where the reaction formed a white precipitate. The precipitate was dissolved in a minimum amount of 1 M NaOH solution and the solvent was removed by vacuum leaving a water soluble product. Figure 4. Dipicolinc acid complex with terbium(III) and the analagous complex with the aminophosphonic acid. Figure 5. The fluorescence emission spectrum of terbium(III) and dipicolinic acid showing the expected transitions in the terbium complex. 900 v 700 v Figure 1. Expected structure for benzylamine phosphonic acid. The NMR spectrum for the raw product confirms the structure. Molecular Recognition Figure 2. 1H-NMR spectrum of raw benzylamine phosphonic acid in deuterium oxide. Purification Figure 6. The fluorescence emission spectrum of terbium(III) and benzylamine phosphonic acid showing only three of the expected transitions in the terbium complex. 900 v Molecular recognition combines two separate events: recognition and reporting. The recognition moiety of the probe binds specifically with the relevant chemical species and the reporter unit produces a quantitatively measurable signal using fluorescence spectroscopy, which is sensitive to the detection of small quantities of molecular substrates (2). In our case, the phosphite is bound through a reaction with an amine, where the reaction acts as a recognition event. It then forms a report complex with terbium, as its fluorescent properties change. LPLC System and Column In order to purify these compounds ion exchange chromatography was chosen, because these compounds are pH sensitive and can be deprotonated to give negative ions. Dowex 50X2-400, an anion exchange resin, was used in the columns. Two types of columns were used, a bench top column and one within a low pressure liquid chromatography (lplc) system. The columns were activated by rinsing with .5 N NH4OH solution. The sample was loaded at pH > 8, and products eluted with water and then 5 N NH4OH solution. Finally, in order to remove all remaining substances the columns were rinsed with 1 N HCl. Future Work Aminophosphonic Acids A confirmation of the identity of the peaks with NMR should be made. An examination of the effect on the fluorescence with the terbium(III) using other subsituents also needs to be done. We are planning to look especially at amino acids, such as tyrosine, as their structure may form a complex more easily with the terbium(III). For the molecular recognition method to work, a reaction that uniquely binds reduced phosphorus is necessary. The synthesis of aminophosphonic acids using the Mannich reaction is such a reaction. It reacts primary amines with phosphorous acid in the presence of formaldehyde to give an aminophosphonic acid capable of chelating a metal ion. In particular the literature reaction with benzylamine produced yields up to 85.7%.3 Acknowledgements I gratefully acknowledge CEA-CREST and Oak Crest for their funding and support. We would also like to thank Drs. Foster, Khachikian, and Salmassi for help with the initial reduced phosphorus studies. References • (1) Hanrahan, G; Salmassi, T. M.; Khachikian, C. S.; Foster, K. L. “Reduced Inorganic Phosphorus in the natural Environment: Significance, Speciation and Determination.” Talanta. 66/2. 2005. 435-444 • (2) Moss, J. A.; Yang, J.; Baum, M. M. "Molecular Recognition of Alkenes Using • Luminescent, Bimetallic Coordination-complexes". Inorganic Chemistry, submitted for • publication. • (3) Moedritzer, K.; Irani, R.R. “The Direct Synthesis of a-Aminomethylphosphonic Acids, Mannich-Type Reactions with Orthophosphorous Acid.” Journal of Organic Chemistry. 31. 1966. 1603-7 Figure 3. UV vis spectra of the column separations. The separation is clearly different for the two systems, although the high concentration that is unavoidable in the bench top method may also contribute to this. However, in both systems separation is apparent.