Fluorescence and Nuclear Magnetic Resonance (NMR) Spectroscopy

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1 Fluorescence and Nuclear Magnetic Resonance (NMR) Spectroscopy Murphy, B. (2017). Fluorescence and Nuclear Magnetic Resonance Spectroscopy: Lecture 3. Lecture presented at PHAR 423 Lecture in UIC College of Pharmacy, Chicago. FLUORESCENCE SPECTROSCOPY Electron is excited by absorption and then emits fluorescence upon relaxation Stokes shift = difference between excited and emitted wavelengths Fluorophore = molecules or functional groups that have the capacity to exhibit fluorescence o Require extended conjugation of pi bonds o More conjugated less energy required for excitement longer wavelength can be used for excitation Fluorescent probes used to identify biological processes o Green fluorescent protein (GFP) fluoresces green light when exposed to light in the blue to UV range Can make its own color using oxygen only Slight modifications can allow for different colors to be emitted. Gives researcher a toolbox of probes for in vivo imaging studies o Can study specific proteins or cellular movements disease states Must be careful too much modification of the protein can impact its natural functioning Protein tagging o Can add the fluorescent probe to the C- or N-terminus. Glycine allows for more flexibility Cellular tagging o Can visualize the G1 phase and the S/G2/M phase Weakness- hemoglobin and melanin can also absorb fluorescent light o Optimal viewing window is near IR region, not visible light region o Near IR probes increase tissue penetration and resolution of image Can use small organic molecules or inorganic nanoparticles Just need a certain degree of conjugation Forster resonance energy transfer (FRET) studying energy transfer between fluorophore molecules allows study of protein interactions in the cell o The excited energy fluorophore passes its energy to the lower energy fluorophore via a dipole-dipole interaction Photosensitizers dyes that can generate reactive oxygen species (ROS) light

2 o Photodynamic therapy using a photosensitizer in tumor cells to kill them with targeted therapy NUCLEAR MAGNETIC RESONANCE (NMR) Involves analyzing nuclear spin of the atom (in the molecule) being studied o Nuclei absorb electromagnetic radiation o Only certain nuclear can exhibit this nuclear spin: 1 H, 13 C, 14 N, 17 O, 19 F o Have to use a deuterated solvent Deuterium = 2 H or D Otherwise the solvent would interfere with the results if we used normal 1 H Electrons shield each nucleus from the magnetic field o For example, oxygen is fairly electronegative so it can pull electrons away from carbon and deshield it. This would give a signal on the spectrum that is more downfield o Signals that appear upfield (to the right) are from nuclei that are more shielded (next to an electron donating group) o Chemical shift electronic environment around a nuclei giving a certain resonance signal on the NMR spectrum Integration area under the peak correlates with how many nuclei there are o Can distinguish between CH3, CH2, CH, etc

3 Will need to use 13 C NMR for molecules that don t have a lot of hydrogens DIFFERENT NMR EXPERIMENTS COSY determines connectivity of 1 H spin systems NOESY will distinguish between stereoisomers (cis vs trans) o Spatial configurations; doesn t say anything about bond connectivity HSQC will determine 13 C- 1 H connectivity HMBC will determine 1 H connectivity to multiple carbons MRI (MAGNETIC RESONANCE IMAGING) The magnetic field at the feet is slightly different than the magnetic field at the head o Can fine tune to focus on different areas of the body

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