1. 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

2. 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

3. Platinum Anti-Cancer Agents
Background
Platinum Streptidine

4. 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

5. 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

6. X-Ray Absorption Spectroscopy
What Is It
Measurements
Applications

7. 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.

8. 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: eV
Pt-L3 Edge eV

9. 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.

10. 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
I 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. Processing Data Analyzing Results Fitting Data
Data Analysis
Processing Data
Analyzing Results
Fitting Data

16. 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
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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
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8000
Normal
George DeBeer, S.

17. 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.

18. 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

19. 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

20. 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.

21. Solid Pt-Std Best Fit: 4 Pt-Cl at 2.30 Å and 1 Pt-N at 2.02 Å
Normalized error 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

22. 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…

23. 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: F/(No. pts)
Cl/S
Cl/S Pt
…3 Cl/S and 2 N, the error is quite close N N

24. Conclusion

25. 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.

26. 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

27. 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
N. Aliaga-Alcalde and Jan Reedijk, University of Leiden, the Netherlands, unpublished results, Pt-Std (Platinum-Streptidine complexes)