Lawson, Terilyn

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Investigation of a New Detection Method for Adenosine:  Binding Studies and Reaction Kinetics Terilyn Lawson  Mentor: Dr. Tom Green UAF Department of Chemistry and Biochemistry  LIF-CE is a powerful analytical separation technique based on the migration of charged molecules under the influence of an applied voltage and electroosmotic flow (EOF).5 Analytes can be identified by migration time and quantified by signal integration.         Adenosine is reacted with trinitrobenzenesulfonic acid (TNBS) to form trinitropheylated-adenosine (TNP-Ado), a stable  Meisenheimer complex. TNP-Ado exhibits two absorbance maxima at 408 nm and 476 nm and a single fluorescence emission maximum in the 530-560 nm range.6        The TNP-Ado derivative experiences enhanced fluorescence in the presence of cyclodextrins. Cyclodextrins are 6, 7 and 8 membered glucose rings with nonpolar cavities which form inclusion complexes with smaller molecules. -Cyclodextrin enhances fluorescence by a factor of 110 compared to the absence of cyclodextrin in water.7       In order to develop a reliable LIF-CE detection method for adenosine, the binding interactions between TNP-Ado and various cyclodextrins were investigated along with the reaction kinetics of adenosine in artificial cerebral spinal fluid (ACSF) and TNBS.   The LIF-CE instrumental parameters were 48 cm total length (28 cm detector) x 50 μm inner diameter bare-fused silica capillary, 20 mM borate buffer pH 9, 10 kV, 50 mbar 3 second injection for the binding studies, 50 mbar 1 second injection for the reaction kinetics studies and 25 mW 405 nm laser.  Adenosine, a purine ribonucleoside, is an important neuromodulator involved with sleep, locomotion, hibernation and is linked to neurological disorders.1-3  Microdialysis techniques are typically used in in vivo adenosine studies and result in small sample sizes (µL) and low analyte concentrations (nM).3   Current adenosine detection methods use reverse phase liquid chromatography coupled with UV detection (HPLC) that require injection volumes of 3-10 μL and can provide low temporal resolution4.   The goal of this research is to develop a new adenosine detection method using laser-induced fluorescence capillary electrophoresis (LIF-CE). LIF-CE is a technique that can analyze lower sample volumes (nL) in high resolution providing a more sensitive detection method for adenosine studies.  Motivation LIF-CE Method Binding Studies Reaction Kinetics  Future Directions and Acknowledgements References Illustration of a LIF-CE capillary depicting EOF and analyte mobility. Reaction scheme of the TNP-Ado derivative.  Structures of -CD, -CD, and -CD. (1) Porkka-Heiskanen, T.; Kalinchuk, A. Sleep Biol Rhythms 2011, 9(1), 18-23.  (2) Quiroz, C. et al.  Exp Neuro 2010, 224, 292-297.  (3) Jinka, T. R. et al. J Neurosci 2011, 31(30), 10752-10758. (4) Chen, L.S.; Fujitaki, J.; Dixon R. J Liq Chromatogr 1993, 16, 2791-2796. (5) Weinberger, R. Ed. Practical Capillary Electrophoresis; Academic Press: San Diego, USA, 2000. (6) Hiratsuka, T. Biochim. Biophys. Acta 1982, 719, 509–517 (7) Green, T. K. et al. J Org Chem 2010, 75, 4048-4055. (8) Azzam, K. M.; Saad, B.; Aboul-Enein, H. Y. Electrophoresis 2010, 31, 2957-2963.   Assuming a 1:1 association of TNP-Ado and cyclodextrin, binding constants can be determined by the electrophoretic mobility and integrated fluorescence of TNP-Ado with respect to the concentration of cyclocextrin.8    Binding studies for TNP-Ado (10 μM) with γ-cyclodextrin were determined at 15, 20, and 25 oC and with β-cyclodextrin at 25 oC by 2 parameter nonlinear curve fitting. Cyclodextrin concentrations ranged from 0 to 10 mM in a 20 mM borate buffer system at pH 9.               From the binding studies, it can be concluded that TNP-Ado has an higher affinity for -CD than β-CD and temperature had no significant effect on the binding constants determined by either electrophoretic mobility or integrated fluorescence. i / fi  = complexed TNP-Ado mobility or             fluorescence  f  / ff  = uncomplexed TNP-Ado mobility               or fluorescence  c / fc  = complexed TNP-Ado mobility or             fluorescence at 10mM cyclodextrin  C  = cyclodextrin concentration  K = binding constant 𝑓𝑖 =𝑓𝑓 + 𝑓𝑐𝐾[𝐶]1 + 𝐾[𝐶] 𝜇𝑖 =𝜇𝑓 + 𝜇𝑐𝐾[𝐶]1 + 𝐾[𝐶] TNP-Ado complexed with -CD The electrophoretic mobility binding constant for TNP-Ado and -CD is approximately 39 orders of magnitude larger than that of TNP-Ado and β-CD.   The derivatization of adenosine in ACSF must be optimized to ensure TNP-Ado formation in future  biological studies. A 100 M sample of adenosine in 0.5 mL ACSF was reacted with 20 L of 1 M NaOH and 10 L of TNBS in H2O and monitored with LIF-CE over a 13 hour period.             Preliminary results show the formation of TNP-Ado in ACSF and a side product, most likely picric acid. It can be concluded that the derivatization of adenosine is possible in ACSF and the LIF-CE method is able to baseline resolve the TNP-Ado signal from side products in ACSF.  Future research will focus on further optimizing the adenosine reaction in ACSF using realistic quantities of adenosine (< 10 M).   An internal standard possibly, N6-cyclohexyladenosine, will be derivatized with TNBS and and used to quantify the TNP-Ado conversion by minimizing error due to injection variability. Other reaction conditions such as temperature, solvent and various bases will also be investigated.  Once the reaction method is optimized in ACSF it will be applied to in vitro and in vivo hibernation studies.   Thank you to Dr. Tom Green, Dr. Bill Simpson and the Department of Chemistry and Biochemistry. Funding source NIH NINDS R15NS074422. Adenosine (100 M) derivatization in ACSF at 2.5 hrs, 4.7 hrs, 6.9 hrs and 9 hrs at 25 oC. TNP-Ado Side Product 2.5 hrs 4.7 hrs 6.9 hrs 9 hrs Temperature(oC) Electrophoretic Mobility K (M-1) Integrated Fluorescence K (M-1)  -Cyclodextrin β-Cyclodextrin -Cyclodextrin β-Cyclodextrin 25 5395 +/- 631 136 +/- 45 1639 +/- 359 297 +/- 142 20 4590 +/- 463 - 1522 +/- 654 - 15 5131 +/- 1201 - 2270 +/- 705 -  Electrophoretic mobility of  TNP-Ado (10 M) as a function of β-CD at 25oC. Electrophoretic mobility of  TNP-Ado (10 M) as a function of -CD at 25oC. 

Investigation of a New Detection Method for Adenosine: Binding Studies and Reaction Kinetics

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Investigation of a New Detection Method for Adenosine: Binding Studies and Reaction Kinetics

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