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

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.

Platinum 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: 1. Is it the Pt coordinated to the

Streptidine? 2. And if this is the case through which

atoms

Putative structures (obtained from elemental analysis and nuclear magnetic resonance)

OH7

NH13

NH14H14N

OH8

H9ONH10

H11N

NH11

H12O

H1

H2

H3

H4

H5

H6

•Streptidine

OHNH

NHH2N

OH

ONHN

NH2

HO

PtCl S

OHNH

NHH2N

OH

ON

HO

HN

NHPt

S

OHNH

NHHN

OH

HONH

HOHN

HN

PtCl

S

1 2 3

OH

NHNH

H2N

OHHO

NH HOHN

H2N Pt

ClS

N. Aliaga-Alcalde and Jan Reedijk

X-Ray Absorption Spectroscopy

•What Is It •Measurements•Applications

What is it?

h

continuum

Emitted photo-electron

1s

Auger electron

2p

2s

X-ray FluorescenceX-ray FluorescenceDirect AbsorptionDirect Absorption(Transmittance)(Transmittance)

hfluorescent photon

George DeBeer, S.

What is it?

Pt-L3 Edge 11550 eV

www.chem.ucalgary/groups/farideh/xas.pdf

Measurements

X-ray Source

Monoslits

Double Crystal

Monochromator

Ta Slits

I0

Sample

Detector

I1

Foil

I2

Experimental Hutch

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)

www.ssrl.slac.stanford.edu/dichroism/xas

Transmittance

I0 ISample

X-rays

Ion chamber Ion chamber

A = μt = ln (I0/I1)

μ ,absorption coefficientt sample thickness

t

+

-Ionizing radiation (i.e. X-rays) creates ion pairs in the gas and the sweeping voltage results in a current flow.

X-rays

George DeBeer, S.

Measurements: Fluorescence

Fluorescence detector

sample

X-ray

Soller Slits

filter

George DeBeer, S.

X-Ray Absorption Spectrum (edge + EXAFS)

Pre-edgeand Edge(XANES)

EXAFS (extended x-ray absorption fine structure)

XAS or XAFS

Energy

Abs

orpt

ion

Coe

ffici

ent (

mu)

Applications

George DeBeer, S.

constructive interferenceresults in a maximum

destructive interferenceresults in a minimum

Applications

Extended X-Ray absorption fine structure

George DeBeer, S.

Data Analysis

Processing Data

Analyzing Results

Fitting Data

eV

1.4

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

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

Nor

ma

l

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

Processing Data

George DeBeer, S.

Processing Data

-6

-4

-2

0

2

4

6

4 6 8 10 12(Å-1)k

EX

AF

S*k

3

4 6 8 10 12(Å-1)k

EX

AF

S*k

3 Cu-N

Cu-S

data + fit

Cu-C2/C5 (SS)

Cu-N-C2/C5 (MS)

Cu-C3/N4 (SS)

Cu-N-C3/N4 (MS)

EXAFS data is really a sum of sine waves. The goal of fitting data is to deconvolute the total signal into its components.

A Fourier transformallows you tovisualize the radialdistribution 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.

Getting Information from EXAFS Data

George DeBeer, S.

0.0

5.0

10.0

15.0

0 1 2 3 4 5 6

FT

Mag

nitu

de

R (Å)

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

-2.00E-01

0.00E+00

2.00E-01

4.00E-01

6.00E-01

8.00E-01

1.00E+00

1.20E+00

1.40E+00

1.60E+00

11540 11545 11550 11555 11560 11565 11570 11575 11580

Energy(eV)

Ab

sorb

ance

Analyzing Results

-8.00E+00

-6.00E+00

-4.00E+00

-2.00E+00

0.00E+00

2.00E+00

4.00E+00

6.00E+00

8.00E+00

1.00E+01

Energy(eV)

X(k

)*k^

3

0.00E+00

5.00E-01

1.00E+00

1.50E+00

2.00E+00

2.50E+00

0.00E+00 1.00E+00 2.00E+00 3.00E+00 4.00E+00 5.00E+00 6.00E+00 7.00E+00 8.00E+00

R(A)

Tra

nsf

orm

Mag

nit

ide

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.

Fitting DataInitial Model

Calculation of initial distance & disorder

parameters.

Optimization of distance & disorder

parameters.

Are fit parameters reasonable & is fit

quality good?

Compare with other good fits to determine

best fit.

YesNo

George DeBeer, S.

-10

-8

-6

-4

-2

0

2

4

6

8

10

0 2 4 6 8 10 12 14 16

k(A^-1)

EX

AF

S*k

^3

EXAFS

FIT

Solid Pt-Std 4 Pt-Cl at 2.30 Å Normalized error

0.567 F/(No. pts)

Best Fit: 4 Pt-Cl at 2.30 Å and 1 Pt-N at 2.02 Å

Comparison of K2PtCl4 and C8H18N6O7Cl4Pt

-2.00E-01

0.00E+00

2.00E-01

4.00E-01

6.00E-01

8.00E-01

1.00E+00

1.20E+00

1.40E+00

1.60E+00

11540 11545 11550 11555 11560 11565 11570 11575 11580

Energy(eV)

Ab

sorb

ance

Pt

Cl Cl

Cl ClN

Normalized error: 0.547F/(No. pts)

-10

-8

-6

-4

-2

0

2

4

6

8

10

0 2 4 6 8 10 12 14 16

k(A^-1)

EX

AF

S*k

^3

EXAFS

FIT

0.00E+00

5.00E-01

1.00E+00

1.50E+00

2.00E+00

2.50E+00

3.00E+00

0.00E+00 5.00E-01 1.00E+00 1.50E+00 2.00E+00 2.50E+00 3.00E+00 3.50E+00 4.00E+00

R(A)

FT

Mag

nit

ud

e

FT

FT FIT

0.00E+00

5.00E-01

1.00E+00

1.50E+00

2.00E+00

2.50E+00

3.00E+00

0.00E+00 5.00E-01 1.00E+00 1.50E+00 2.00E+00 2.50E+00 3.00E+00 3.50E+00 4.00E+00

R(A)

FT

Ma

gn

itu

de

FT

FT FIT

Solution Pt-Std

-6

-4

-2

0

2

4

6

8

0 2 4 6 8 10 12 14

k(A^-1))

EX

AF

S*k

^3

EXAFS

FIT

4 Pt-Cl/S at 2.30 Å 1 Pt-N at 2.03 Å

Normalized error: 0.359F/(No. pts)

0.00E+00

2.00E-01

4.00E-01

6.00E-01

8.00E-01

1.00E+00

1.20E+00

1.40E+00

1.60E+00

1.80E+00

0.00E+00 5.00E-01 1.00E+00 1.50E+00 2.00E+00 2.50E+00 3.00E+00 3.50E+00 4.00E+00

R(A)

FT

Mag

nit

ud

e

FT

FT FIT

-6

-4

-2

0

2

4

6

8

0 2 4 6 8 10 12 14

k(A^-1)

EX

AF

S*k

^3

EXAFS

FIT

Solution Pt-Std Best Fit: 3 Pt-Cl/S at 2.30 Å and 2 Pt-N at 2.08 Å

Pt

Cl/S Cl/S

NN

Normalized error: 0.363 F/(No. pts)

0.00E+00

2.00E-01

4.00E-01

6.00E-01

8.00E-01

1.00E+00

1.20E+00

1.40E+00

1.60E+00

1.80E+00

0.00E+00 5.00E-01 1.00E+00 1.50E+00 2.00E+00 2.50E+00 3.00E+00 3.50E+00 4.00E+00

R(A)

FT

Mag

nit

ud

e

FT

FT FIT

Cl/S

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)