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“CTP” cinnamyltriphenylphosphonium chloride Cytotoxin Molecular Weight: 414.906221 g/mol Structure: Three benzene rings and cinnamyl group attached to phosphorous central atom. Chloride anion. Physical Properties: Melting Point: 225 º – 227 º C Appearance: White to Off-White Powder or Crystalline Powder IR Spectrum of CTP If a is being absorbed, it means that energy (E = h) is being absorbed by the cmpd. Energies in the IR region correspond to the energies involved in bond vibrations (stretches/bends). 3100 - 3000 =C-H (aryl) stretch 2000 - 1665 weak overtones of aromatic 1600 - 1585, 1500 -1400 C=C stretch (in ring) 3000 - 2850 C-H (alkyl) stretch 1470 - 1450 C-H (alkyl) bend 1000 - 650 =C-H (alkene) bend DNA Melting Curve dsDNA 2 ssDNA Tm = Melting Point of DNA Half of DNA has uncoiled and remains single-stranded when the melting temperature is reached A shift in Melting Temperature is a strong indicator of the presence of an interaction between the DNA and any small molecule, such as any arylphosphonium salt. Sample Melting Temp. (ºC) Native Salmon DNA + 3µL Salmon DNA + 5µL Salmon DNA (15.2µg/150µL DMSO) (15.2µg/150µL DMSO) CTP CTP 64.6 67.2 67.8 DNA/CTP Melting Curve Experiment Summary 120 100 D Absorbance 80 60 40 Native Salmon DNA 20 Salmon DNA + TCP (3 microliters) Salmon DNA + TCP (5 microliters) 0 40 50 60 70 -20 Temperature (degrees Celsius) 80 90 100 DNA/CTP Melting Point Experiment Summary - Derivative Plots 0.09 0.08 0.07 delta A/delta T 0.06 0.05 0.04 Native Salmon DNA Salmon DNA + TCP (3 microliters) Salmon DNA + TCP (5 microliters) 0.03 0.02 0.01 0 40 50 60 70 80 -0.01 Average Temperature (degrees Celsius) 90 100 Gel Electrophoresis Lane 2: (Control) Digested DNA Lane 3: (Experimental) Digested DNA w/ 1ml CTP Gel Electrophoresis: Digested Plasmid DNA Movement and Determination of Base Pairs 10000 # of Base Pairs ~ 5000 base pairs (qualitative trial) 1000 20 25 30 35 40 45 50 55 Distance from Well (mm) 60 65 70 HyperChem Predictions • CTP would bind effectively with the DNA through intercalation. This was based upon the flat, characteristic structure of the cinnamyl group. • CTP might also bind with DNA in its major groove, based on the relative size of the benzene groups and the electrostatic attraction of the phosphorous backbone of DNA. HyperChem Computations Minor groove interactions were most favorable with cinnamyl group in closest proximity to DNA: Distance of two closest atoms = 3.8913 Å E = 14.699580 kcal Gd = 0.099771 HyperChem Predictions HyperChem Computations Intercalation and Major Groove Interactions of CTP and DNA were of greater energy: Major Groove Triphenyl group of closest proximity to DNA E = 894.84832 kcal Cinnamyl group E = 898.00171 kcal Intercalation – (Interaction optimized with CTP passing through nitrogen base of cytosine) Triphenyl group of closest proximity to DNA E = 1553.85107 kcal Cinnamyl group through base pairs in major groove E = 923.01587 kcal Further Research • Nature of DNA Cooling after denaturation and possible restructuring of DNA • DNA Structure effects on Movement through gel • Potential Applications of the CTP Molecule in DNA of organisms • Further HyperChem Computations • Ultimate objective: X-ray crystallography (orientation and type of interaction)