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

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: 1H, 13C, 14N, 17O, 19F

o Have to use a deuterated solvent

▪ Deuterium = 2H or D

▪ Otherwise the solvent would interfere with the results if we used normal 1H

• 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

• Will need to use 13C NMR for molecules that don’t have a lot of hydrogens

DIFFERENT NMR EXPERIMENTS

• COSY – determines connectivity of 1H spin systems

• NOESY – will distinguish between stereoisomers (cis vs trans)

o Spatial configurations; doesn’t say anything about bond connectivity

• HSQC – will determine 13C-1H connectivity

• HMBC – will determine 1H 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