Determining the Structure of Platinum Streptidine using X-Ray Absorption Spectroscopy Name: Michaëlle Mayalu Project: Determining the Structure of Platinum Streptidine using X-Ray Absorption spectroscopy Michaëlle Mayalu Massachusetts Institute of Technology SULI Program: Stanford Synchrotron Radiation Laboratory, SLAC Mentor: Serena DeBeer George August, 15, 2007

Outline Platinum Anti-Cancer Agents X-Ray Absorption Spectroscopy Data Analysis Conclusion I will talk about Platinum Anti-Cancer Agents (That’s what Pt-Std is) and the background behind them The basics of X-Ray absorption Spectroscopy How you ana the data obtained from abs and Analysis of my data in particular Conclusion of my findings

Platinum Anti-Cancer Agents Background Platinum Streptidine

Background Platinum complexes have been discovered to stop cell division This has useful applications in treating cancer and considerable advances have been made However….. mechanisms are that lead to the drug being taken up by the cell membrane and integrated into the DNA are still unknown the drug is successful in treating some types of cancers but ineffectual treating other types platinum drugs such as cisplatin has been found to be very toxic causing nephrotoxity, neuropathy, ototoxicity, hematological toxicity, neuropathology and seizures. Consequently, the search for improved platinum drugs that treat a wider range of cancers and display fewer toxic side effects still continues. Pt- complexes have been discovered stop cell division and this has had useful applications in cancer However: the mechanisms that lead to the drug being taken up by the cell membrane and integrated into the DNA are unknown It is also very toxic Consequently the search continues for a drug that can have less toxic sides effect cures a wider range of cancers

Platinum Streptidine Streptidine Because the structure and coordination of the drug (especially in solution) is essential to understanding how the drug interacts with molecules in the body, X-ray absorption spectroscopy (XAS) is a powerful tool for determining the local structure of this newly developed drug. main questions to hopefully be answered by analysis of XAS data measured with XAS are: Is it the Pt coordinated to the Streptidine? And if this is the case through which atoms Putative structures (obtained from elemental analysis and nuclear magnetic resonance) Streptidine Here is a diagram of the streptidine complex Although the elements in the Pt-Std complex have been discovered through NMR and elemental analysis, coordination of the streptidine to the platinum is still unknown Since it is important to understand how it works in the body we examined both the dilute and solid Pt-Std XAS is a tool that can help determine coordination Main questions are: Is the Pt Coordinated to the Pt-Std and if so though which atoms Putative structres N. Aliaga-Alcalde and Jan Reedijk

X-Ray Absorption Spectroscopy What Is It Measurements Applications

What is it? continuum X-ray Fluorescence Direct Absorption Auger electron Emitted photo-electron continuum 2p X-ray Fluorescence Direct Absorption (Transmittance) 2s Absorption occurs when an X-ray hitting a sample has a high enough energy to excite a core level electron. The electron can be either excited into a higher energy level or into the continuum. This is what is measured as absorption tranmittance Another way absorption is measured is fluorescence which happens when an electron from a higher energy level falls down to replace the exited electron. As it fall energy is released in the form of a photon. This is what is measured as flourecence Solid use transmission Soln (di-methyl sulfoxide) use fluorescence h h fluorescent photon 1s George DeBeer, S.

What is it? Here is the Bhor atomic model for an atom It takes more energy to remove and electron from a lower energy level Energy it took to remove and electron from the Pt L3 edge: 11550 eV Pt-L3 Edge 11550 eV www.chem.ucalgary/groups/farideh/xas.pdf

Measurements Experimental Hutch X-ray Source Mono slits Double Crystal Monochromator Ta Slits I0 Sample Detector I1 Foil I2 Experimental Hutch Drawing of experimental hutch X-Rays go through slits then a monochomator which through bragg’s law can change the X-Ray to the desired energy The ray then travels trough and ion chamber hits the sample then travels through a second ion chamber The Foil is used for energy calibration Fluorescence detector George DeBeer, S.

Measurements: Transmittance When a beam of monochromatic X-rays goes through matter, it loses its intensity due to interaction with the atoms in the material. The intensity drops exponentially with distance if the material is homogeneous, and after transmission the intensity is: I0=Ie-μt Where: I incident X-ray intensity I0 transmitted X-ray intensity µ absorption coefficient t is the thickness of the sample Absorption can therefore be measured as: A= µt=ln(I0/I) As the X-Ray passes through the sample it’s intensity decreases exponentially. Equation where… Absorption is measured as the ln of the incident X-Ray intensity over the transmitted X-ray intensity. www.ssrl.slac.stanford.edu/dichroism/xas

Transmittance t + - A = μt = ln (I0/I1) μ ,absorption coefficient t sample thickness Ion chamber Ion chamber X-rays I0 Sample I + X-rays               When an X-Ray passes through an ion chamber, which consists 2 oppositely charged plates , the gas inside the chamber ionizes which creates a current which can be measured. - Ionizing radiation (i.e. X-rays) creates ion pairs in the gas and the sweeping voltage results in a current flow. George DeBeer, S.

Measurements: Fluorescence Fluorescence detector sample X-ray Soller Slits filter Fluorescence is measured in much the same manner, a current is detected when the x-Rays pass thorough a diode present in a detector. George DeBeer, S.

X-Ray Absorption Spectrum (edge + EXAFS) Applications X-Ray Absorption Spectrum (edge + EXAFS) EXAFS (extended x-ray absorption fine structure) Pre-edge and Edge (XANES) Absorption Coefficient (mu) Graph of absorption vs. energy Predge: no absorption so its flat Edge: absorption core electron is excited into the continuum EXAFS Energy XAS or XAFS George DeBeer, S.

Applications Extended X-Ray absorption fine structure constructive interference results in a maximum destructive interference results in a minimum EXAFS occur when the emitts photoelectron backscatters off surrounding atoms The scattered wave can interfere with the initial photoelectron wave and either add constructively which creating a local max or destructively creating a local min. George DeBeer, S.

Processing Data Analyzing Results Fitting Data Data Analysis Processing Data Analyzing Results Fitting Data

Processing Data Getting the EXAFS Raw data: This is the way that the XAS transmission mode spectrum looks, right off the beam line. 0.4 0.6 0.8 1.0 1.2 1.4 6800 7000 7200 7400 7600 7800 8000 Raw Getting the EXAFS eV Pre-edge subtraction: A procedure performed to subtract the total absorption from the absorption of the edge in interest. 0.4 0.6 0.8 1.0 1.2 6800 7000 7200 7400 7600 7800 8000 Raw 1.4 Here’s what the absorption signal looks like when if comes off the beam line We subtract the pre-edge is from electrons being released from higher energy levels we’re not interested in looking at. We also do something called splining to remove the atomic background Spline: A method for removing the atomic background from the absorption curve (i.e. the absorption due to the photoabsorber alone, with out any neighboring atoms) . eV 0.0 0.5 1.0 1.5 6800 7000 7200 7400 7600 7800 8000 Normal George DeBeer, S.

Processing Data Getting Information from EXAFS Data 0.0 5.0 10.0 15.0 1 2 3 4 5 6 FT Magnitude R (Å) A Fourier transform allows you to visualize the radial distribution of atoms. Note: k is the photoelectron wave number. k= (2m(E-E0)/ћ2)1/2 EXAFS data are k-weighted to enhance oscillations at high-k. After that the EXAFS region is k weighted to enhance oscillations k is photoelectron wave number and is dependent on energy EXAFS are then Fourier transformed EXAFS data is really a sum of sine waves. The goal of fitting data is to deconvolute the total signal into its components. George DeBeer, S.

Analyzing Results Pt-Std Edges Solid and Solution As can be seen, the edges of the solid and solution data begin at different energies Because the solid begins at a lower energy, it is more reduced This suggests that the solid structure is surrounded by heavier atoms solid soln Here are the solid and solution edges of Pt-Std As can be seen the solid is more reduced This indicates lighter atoms in the solution

K3 EXAFS and Corresponding Fourier Transforms Analyzing Results K3 EXAFS and Corresponding Fourier Transforms amplitude of the EXAFS slightly decreases from solid to soln this indicate a decrease in coordination number or may also be indicative of lighter atoms present in solution. intensity of FT greatly decreases from solid to solution. Similar to the decrease in EXAFS amplitude, the decrease in peak intensity indicates a decrease in coordination or lighter atoms ligated to the Platinum in solution. Here is the EXAFS and FT for solid and soln. The lower amplitude in the EXAFS as well as the decreased peak intensity in the FT of the soln also indicate lighter atoms in soln

Fitting Data Initial Model Calculation of initial distance & disorder parameters. Optimization of distance & disorder parameters. Are fit parameters reasonable & is fit quality good? Fits are done in a program called EXAFSPAk and calculated by FEFF When you fit it is important to keep in mind if the fit is physically reasonable and compare all fits and pick the best one Compare with other good fits to determine best fit. No Yes George DeBeer, S.

Solid Pt-Std Best Fit: 4 Pt-Cl at 2.30 Å and 1 Pt-N at 2.02 Å Normalized error 0.567 F/(No. pts) Normalized error: 0.547F/(No. pts) Cl Cl Pt Cl Cl Here’s one of the fits for Solid Pt-Std. 4 Cl at 2.3 A It is unlikely that this is the structure because when compared with platinum tetrachloride, the edges are significantly different It is more likely 4 Cl at 2.3 A and 1 N and 2.02 A, errors are relatively the same N Comparison of K2PtCl4 and C8H18N6O7Cl4Pt

Solution Pt-Std 4 Pt-Cl/S at 2.30 Å 1 Pt-N at 2.03 Å Normalized error: 0.359F/(No. pts) Here’s a fit for the soln, note that Pt-Std was dissolved in di-methyl-sulfoxide and it is likely that one of the chlorines was replaced by a sulfur but since data was only usable to k=12, we were unable to distinguish between the sulfur and chlorine. 4 Cl/S at 2.3 A and 1 N at 2.03 A. But Seeing how the previous comparison of the EXAFS indicates that there are lighter atoms present in solution al more likely fit is…

Normalized error: 0.363 F/(No. pts) Solution Pt-Std Best Fit: 3 Pt-Cl/S at 2.30 Å and 2 Pt-N at 2.08 Å Cl/S Normalized error: 0.363 F/(No. pts) Cl/S Cl/S Pt …3 Cl/S and 2 N, the error is quite close N N

Conclusion

Conclusion It can be concluded that Platinum is in fact coordinated to the streptidine through a bond with nitrogen. In the solid… the streptidine is coordinated to the Pt by one nitrogen at 2.02 Å In the solution… the streptidine could be coordinated to 1-2 nitrogens, although with 2 nitrogens at 2.08 Å is more likely. This is shown by analysis and fitting of the EXAFS data of the solid and solution Pt-Std and comparing the edges and EXAFS of the Pt-Std solid and solution Future work should include the determination of how many chlorines and sulfurs are ligated in the solution structure.

Acknowledgments Special Thanks to: My Mentor Serena Debeer George U. S. Department of Energy, Office of Science for giving me the opportunity to participate in the SULI

References Serena DeBeer George, Introduction to Extended X-ray Absorption Fine Structure (EXAFS) Spectroscopy and its Applications (Power Point) XAS Short Course for Structural Molecular Biology Applications www.chem.ucalgary/groups/farideh/xas.pdf www.ssrl.slac.stanford.edu/dichroism/xas N. Aliaga-Alcalde and Jan Reedijk, University of Leiden, the Netherlands, unpublished results, Pt-Std (Platinum-Streptidine complexes)