(2-Hydroxyethyl) Triphenylphosphonium Chloride

Large Molecule/Small Molecule InteractionsRhode Island College

July 14-18, 2008Bailey Sarber, Samantha Barrus

Westerly High School

TPPEO

•Molecular Formula-- HOCH2CH2P(C6H5)3Cl•Molecular Weight—342.79 AMU•Physical State— white, crystalline powder•Solubility in Water– not available•LD 50– not available

ElectrophoresisWell 2 - kb LadderWell 3 - Plasmid DNA sample uncut (5μL) - 6300 base pairsWell 4 - Plasmid DNA sample uncut (5μL) with 1 µL of TPPEO- 6300 base pairsWell 5 - Plasmid DNA sample linearized (5μL) - 6200 base pairsWell 6 - Plasmid DNA sample linearized (5μL) with 1 µL of TPPEO- 6200 base pairs

Possible Conclusions:•The doping agent failed to bind with the DNA samples.•The agent bound, but had no effect.•The agent’s concentration was too low.

Final conclusion: Additional tests yielded the same results despite higher concentrations of TPPEO doping agent (3 µL and 5 µL).

Melting Point of Genomic (Salmon) DNA with Varying Concentrations of TPPEO

Melting pointsControl (DNA + Distilled Water) -77.5°CDNA + 1 μL TPPEO-82.5°CDNA + 3 μL TPPEO-82.5°CDNA + 5 μL TPPEO-82.5°C

45 55 65 75 85 950.1

0.15

0.2

0.25

0.3

Melting Point of Genomic Salmon DNA in Distilled Water

Control

Temperature in Degrees Celsius

Abso

rban

ce

45 55 65 75 85 950.4

0.405

0.41

0.415

0.42

0.425

0.43

0.435

0.44

Melting Point of Genomic Salmon DNA with 1 Microliter of TPPEO

1 microliter

Temperature in Degrees Celsius

Abso

rban

ce

45 55 65 75 85 95 1050.92

0.94

0.96

0.98

1

1.02

1.04

1.06

1.08

Melting Point of Genomic Salmon DNA with 3 Microliters of TPPEO

3 microliter

Temperature in Degrees Celsius

Abso

rban

ce

45 55 65 75 85 951.4

1.421.441.461.48

1.51.521.541.56

Melting Point of Genomic Salmon DNA with 5 Microliters of TPPEO

5 microliter

Temperature in Degrees Celsius

Abso

rban

ce

45 55 65 75 85 95

-0.00500000000000001-0.00300000000000001-0.001000000000000010.000999999999999989

0.002999999999999990.004999999999999990.006999999999999990.00899999999999999

0.0110.013 77.5

DNA Melting Point - Derivitave PlotGenomic Salmon DNA in Distilled

Water

∆A/∆T

Average Temperature in Degrees Celsius

∆A/∆

T

45 55 65 75 85 95

-0.00200000000000001

-0.00100000000000001

-5.20417042793042E-18

0.000999999999999995

0.002

0.003

0.00482.5

DNA Melting Point - Derivitave Plot Genomic Salmon DNA with 1 Micro-

liter of TPPEO

∆A/∆T

Average Temperature in Degrees Celsius

∆A/∆

T

45 55 65 75 85 95

-0.015

-0.01

-0.00500000000000003

-2.60208521396521E-17

0.00499999999999998

0.00999999999999998

82.5

DNA Melting Point - Derivitave Plot Genomic Salmon DNA with 3

Microliters of TPPEO

∆A/∆T

Average Temperaure in Degrees Celsius

∆A/∆

T

45 55 65 75 85 95

-0.015

-0.01

-0.00500000000000003

-2.60208521396521E-17

0.00499999999999998

0.00999999999999998

0.01582.5

DNA Melting Point - Derivitave Plot Genomic Salmon DNA with 5 Micro-

liters of TPPEO

∆A/∆T

Average Temperature in Degrees Celsius

∆A/∆

T

Molecular Modeling

Benzene ring - planar surface may provide the best chance of intercalation within DNA

Distance in Å

Energy in Kcals

1 19.662 95.532 18.744 95.523 17.765 95.504 16.569 95.415 15.712 95.156 15.159 96.627 14.909 95.328 6.831 94.269 1.310 1066.3610 26.157 98.91Optimization 26.216 92.347

Distance in Å Energy in Kcals1 48.939 464.872 42.385 464.873 30.823 464.864 26.837 464.865 23.426 464.866 11.378 464.85

7 7.650 464.728 6.447 464.319 2.569 891.3910 58.730 464.45Optimization 60.553 178.05

Minor Groove – Single Point Energy

Atom - C11 of TPPEOAtom - P193 of DNA

Major Groove –Single Point Energy

Atom - C11 of TPPEOAtom - C3*442

Small Molecule/Large

Molecule Interaction

Attempting to Intercalate TPPEO

within Adenine-Thymine Base Pairs

Optimizing…

TPPEO fails to intercalate, instead causing the DNA molecule to distort

itself away from the drug.

Attempting to Intercalate TPPEO within Cytosine-

Guanine Base Pairs

Optimizing…

TPPEO fails to intercalate, instead causing the DNA to distort and the base pairs to move apart.

Attempting to Bind TPPEO to the Major

Groove of DNA

Optimizing…

TPPEO fails to interact with the major groove, instead causing DNA to distort away from the TPPEO.

Attempting to Bind TPPEO to the Minor

Groove of DNA

Optimizing…

TPPEO appears to interact with the minor groove, causing little

DNA distortion.

Conclusions

Electrophoresis- TPPEO did not affect the migration of the DNA through the gel. The drug either did not bind with DNA or it did bind with the DNA with no affect to the migration through the gel.

Melting Point of Genomic DNA- There was a change in melting temperature between the control sample and those doped with various concentrations TPPEO including 1 μL, 3 μL and 5 μL. The possible shift was 5° Celsius indicating some sort of interaction between the drug and the DNA samples. Further testing is needed to substantiate an actual interaction.

Molecular Modeling- Single point optimization between atoms on TPPEO and DNA revealed that when TPPEO approached the major groove on DNA the optimal energy level was 26.216 Å. This suggests that the interaction based upon these two points is forced.

Optimization on the minor groove had a greater overall distance than that of the major groove, at a distance of 60.553Å. The energy level implies the two atoms do not interact well at the minor groove.

Adenine-Thymine Intercalation- TPPEO failed to intercalate within base pairs containing adenine-thymine sequences. The double hydrogen bonds connecting the two bases were not strong enough to withstand the distortion caused by the attempted intercalation.

Cytosine-Guanine Intercalation-TPPEO again failed to intercalate between base pairs containing cytosine-guanine. These base pairs moved apart but not as far as the adenine-thyamine base pairs because the three hydrogen bonds are stronger then two.

Warning: Hyperchem has its limitations.Some sort of interaction appeared to take place in the minor groove of the

DNA sequence when both molecules were optimized using the Amber program. This occurred despite the single point optimization not supporting the interaction. Single point optimization only configures two atoms but with so many configurations to test it would be realistically impossible to test them all using a program such as this.

Intercalating within a DNA sequence has as many variables; base pair sequences, the size of the DNA molecule, and the location and configurations of the drug yield hypothetical results.

Conclusion: Actual experimentation should be the primary source from which to draw conclusions. Recreating the experiment will quantify/support the data collected and verify the possible binding properties of TPPEO. Hyperchem is a tool, but not to be solely relied on.

Further Research…

•Replicating the experiments •Confirming the assumed minor curve non-covalent bonding nature of TPPEO and DNA using X-Ray Crystallography•Investigate other molecules for possible DNA interaction ex: acetylsalicylic acid , ibuprofen, acetaminophen