Explaining the Broadband

Absorbance of Melanins

Jenny Riesz

Condensed Matter Physics at UQ

People:– 3 Academic staff– 6 Postdocs– 8 PhD students– 3 Honours

students

Soft Condensed Matter– Establishing Structure ↔

Property relationships for:• Melanins• Organic photovoltaics and

optoelectronics• Novel organic electronic

materials

Condensed Matter Theory– Big Question focused, work

closely with experimentalists• Melanins• Organic superconductors• Frustrated quantum

antiferromagnets• Photosynthetic systems• Spintronics

Contour plot of the electron density in the highest occupied molecular orbital of an organic superconductor, calculated using density functional theory (NRLMOL).

Theme: The Structure-Property Jigsaw Puzzle

• Establishment of the Structure-Property Relationships for Bio-macromolecules (proteins, etc.)

Structure:

Properties:

Primary (Å) Secondary (nm) Tertiary (10’s nm) Aggregates (m)

QM QM/Classical Classical Classical

Molecular:-relaxed geometries-electronic structure-energies-phonon structure

Mesoscopics:-electron-phonon interactions-free spin dynamics-electron delocalisation-weak dispersive interactions

Macroscopic Observables:-optical-electrical-photochemical-chemical

I use several of these techniques:

– Quantitative optical spectroscopy• Absorbance and Emission spectra• Time resolved emission spectra• Infrared spectroscopy• Quantum Yields• Optical Scattering

– Quantum Chemistry• Density Functional Theory

– Physical structure– Energy structure– Vibrational structure

• Mesoscale theory

– Structural techniques• Inelastic Neutron Scattering

• Our approach is very multidisciplinary, with people specializing in:– Synthetic chemistry– Quantum chemistry– Many body theory– Mesoscopics– Electrical and optical measurements– Device physics– Molecular biophysics

What is Melanin?• Biological pigment found in a huge range of

species, including humans.• Responsible for photoprotection in our skin,

hair and eyes– Paradoxically, melanins are also implicated in

melanoma formation.– Malignant melanoma has a fatality rate

unparalleled by any other skin cancer type • Two types found in human skin:

– Eumelanin (black)– Pheomelanin (red-brown),

• Pheomelanin is the most closely linked with melanoma skin cancer

Albino giraffe (cannot produce melanin)Human melanoma

Not just a photoprotectant?

• Melanin is also found in the inner ear, and brain stem

• It’s role here is unknown, although

– White cats with blue eyes are often deaf

– Melanin deficiencies have been linked to Parkinson’s Disease

The exotic properties of melanin:• Broad band monotonic absorption in the UV & visible (black)• Condensed phase electrical conductivity and photoconductivity• Efficient non-radiative relaxation of photoexcitations

• An excellent material to study

Structure ↔ Property ↔ Function

relationships in a disordered organic system.

The big questions:• What is the physical and chemical structure of melanin?• How does this structure dictate its properties?

Melanins are macromolecules• Eumelanin has two different

monomers:• - Dihydroxyindole (DHI)

• - Dihydroxyindole-carboxylic acid (DHICA).

• Pheomelanin has one monomer:– - 3,4-dihydro-1,4-benzothiazine-3-

carboxylic acid (DHBCA)

The monomers can bind through several different positions.

DHICA

Eumelanin(black) R = -H or -COOH

DHI

Pheomelanin(red-brown)

DHBCA

N

S

O

NH2O

OH

O

OH

N

S

O

NH2O

OH

O

OH

Do monomers link to form long polymer chains, or instead terminate at smaller oligomers (2 - 10 monomers)?

OPTION 1: Hypothetical melanin polymer

OPTION 2: Oligomers, which can form aggregates through π stacking.

• The current favoured model of melanin secondary structure:– DHI and DHICA connect to form small, flat oligomers (2-10 monomers) – Oligomers stack to form aggregates (‘protomolecules’).

• This model has been widely accepted

• We believe that this model is unfounded– Melanin could have a different secondary structure that would be consistent with all the

experimental data.• The ‘protomolecule’ model was suggested on the basis of wide angle X-ray scattering

data• Other experiments such as:

can be interpreted as being consistent with the protomolecule model, but do not prove it

- None of these experiments reveals stacking of layers

Clancy and Simon (2001) Biochemistry 40 (44)

- Atomic force microscopy- Scanning tunnelling microscopy- Scanning electron microscopy

- Optical light scattering- NMR- Small angle X-ray scattering

Wide Angle X-ray Scattering

• The protomolecule model was proposed based on WAXS measurements by Cheng et al.

– The authors of this careful study agreed that the results were not conclusive!

– Regardless, this model has been accepted as dogma

• Results from Cheng et al. (1994), Pigment Cell Res. (7):

– Figures: Experimentally measured structure factor of melanin (+) with theoretically predicted fits (solid line) for a) DHI and b) melanin protomolecule

• The fit for a four layer protomolecule is only marginally better than the fit for a single DHI monomer, with the exception of the peak at q = 1.74Å-1.

• This peak indicates the presence of a length scale of 3.45Å

– This corresponds to the interlayer spacing of defective graphite, hence the authors predicted a layered structure

– We believe that this 3.45Å spacing could be reproduced simply by considering multiple isolated DHI monomers spaced by their van der Waals radius

Fig a)

Fig b)

The Melanin Mystery• The most unique and distinguishing feature of melanin is its broadband

absorbance spectrum• It is highly unusual for a biological molecule

– Most molecules show distinct peaks– The broadband spectrum is likely related to its role as a photoprotectant

Submitted to J. Phys Chem. B. (2005) Riesz, Sarna and Meredith.

800700600500400300Wavelength (nm)

Ab

sorb

an

ce (

arb

. u

nits

)

Pheomelanin Eumelanin

Melanin Absorbance Spectra:

30

25

20

15

10

5

0P

erce

ntag

e sc

atte

red

(%)

500450400350300250200Wavelength (nm)

Percentage of total loss (measured as absorption) that is scattered

Is the broadband absorption spectrum

just due to scattering?

No!

5

4

3

2

1

0Abs

orpt

ion/

scat

terin

g co

effic

ient

(cm

-1)

800700600500400300200Wavelength (nm)

Wavelength (nm)

Percentage of loss (measured as absorption) due to scattering

Eumelanin Total Optical Density

Eumelanin Scattering

Biophysical Journal, in press (2006).

• It has been proposed that melanin is an amorphous semiconductor– This would explain the observed broad band absorption spectrum

– However, there are significant discrepancies in reported band gap values• Possibly due to the critical dependance of melanin solid state properties upon

humidity/hydration of the sample.

– At this time, the semiconducting properties of melanin have not been rigorously determined

EF

EV

EC

E

N(E)

1.40eV

0.78eV

0.2eV

{From DC conductivity Measurements}

Chemical Disorder Model

Soft Matter, 2, pp37-44 (2006).

• Another way that melanin might produce the broadband absorbance spectrum:

– Perhaps by the summation of the narrow absorption bands of many different oligomers• Chemical disorder – macroscopic properties are the ensemble average of a number of

chemically distinct species• Consistent with emission and excitation evidence• Only 11 inhomogeneously broadened transitions with typical polar solvent line widths are

required to produce a broadband spectrum.– The secondary structure is a central question!

Perhaps this is a “low cost” strategy for achieving robust functionality?

• Melanin functions biologically as an absorber• Does this mean that it has exceptionally strong absorbance?

– No, it has a similar oscillator strength / dipole strength to other biological molecules– Strong absorbance in skin is instead produced by high concentrations

14x103

12

10

8

6

4

2

0

Ext

inct

ion

Co

effi

cie

nt

(L m

ol-1

cm-1

)

1.2x1015

1.11.00.90.80.70.60.5Frequency (Hz)

Peak value: 13x104

L mol-1

cm-1

250nm600nm

Fluorescein

DHICA

Eumelanin

Tyrosine

 Dipole Strength

(debye2)

Eumelanin 37

DHICA 31

Tyrosine 1.6

Fluorescein 140

Soon to be submitted to Physical Review E. (2006) Riesz, Gilmore, McKenzie, Powell, Pederson, Meredith

• How does melanin harmlessly dissipate the energy that it absorbs?– Fluorescence?– Non-radiative relaxation?

• Melanin does emit fluorescence– It is not strong, and highly distorted by re-absorption effects, if one is not careful

• The emission is very broad – Suggests a disordered system

Ph

oto

lum

ine

sce

nce

(a

rb. u

nits

)

700650600550500450400Wavelength (nm)

Pheomelanin Eumelanin

Melanin Photoluminescence Spectra

Submitted to J. Phys Chem. B. (2005) Riesz, Sarna and Meredith.

1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4

0.0

2.0x105

4.0x105

6.0x105

8.0x105

1.0x106

1.2x106

Inte

nsi

ty (

cps)

Energy (eV)

• The emission peak shifts with excitation wavelength– Atypical

• This is consistent with the chemical disorder model– Selectively exciting sub-populations of oligomers

• We need a structural model for further analysis

Decreasing Eex

Eumelanin emission

Photochem. Photobiol. 79(2) pp211-216 (2003)

• The melanin emission spectrum violates the mirror image rule– It does not mirror the absorbance spectrum

30x10-3

20

10

0

Ab

sorb

an

ce (

bla

ck)

600550500450400Wavelength (nm)

2.0x106

1.5

1.0

0.5

0.0

Em

ission

(red

)

Fluorescein obeys the mirror image rule

300x103

250

200

150

100

50

Em

issio

n (re

d)

800700600500400300Wavelength (nm)

0.8

0.6

0.4

0.2

Ab

so

rba

nce

(b

lack)

• How much of the absorbed energy is dissipated radiatively?

• Not much!– Melanin has a very low quantum yield (0.1% - 0.2%)

– Strong phonon coupling (photoprotectant)

• The pheomelanin quantum yield is approximately twice that of eumelanin– Related to its higher photoreactivity?

J. Phys. Chem. B., 109(43), pp20629-20635 (2005).

2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.75.5x10-4

6.0x10-4

6.5x10-4

7.0x10-4

7.5x10-4

Qua

ntum

Yie

ld

Excitation Energy (eV)

350nm

380nm

410nm

Photochem. Photobiol.(Rapid Comm), 79(2), pp211-216 (2004).

Radiative Quantum Yield of Eumelanin

• The quantum yield is dependant upon excitation energy– This is atypical, and is consistent with emission from a collection of

oligomers (chemical disorder model)

Mapping the energy dissipation pathways:• What percentage of the energy absorbed at each wavelength is emitted at

each wavelength?– The “Specific” quantum yield

J. Chem. Phys., 123, 194901:pp01-06 (2005)

• Melanin is an extremely challenging system to work with– Highly insoluble– Strong, broadband absorbance– Highly disordered!

• These factors all make elucidating the structure very difficult

• To shed light on this problem, we have begun to study the melanin formation process– Quantitative spectroscopy to monitor the polymerisation process– Combined with DFT applied to melanin:

• Monomers• Dimers• Small oligomers

• A synthetic “bottom-up” approach.

DHICA: A key melanin monomer• Different redox states show substantially different energy gaps

Oxidation state

Predicted Excitation Energy (DFT)

1 3.04eV (407nm)

2a 2.67eV (465nm)

2b 2.64eV (470nm)

3a 1.96eV (634nm)

3b 1.10eV (1129nm)

3c 1.25eV (994nm)

Powell (2005), Chem. Phys. Lett. 402, 111-115.

For example:

HOMO-LUMO Gap

Monomer 3.04eV (407nm)

Dimer 2.16eV (580nm)

Possible DHICA dimerBiophysical Journal (2005), Tran, Powell and Meredith.

• The gap also changes significantly upon dimerization

• For this particular dimer, the gap of the dimer is red-shifted by 170nm relative to the monomer!

• This is consistent with the disordered oligomer model– Large polymers are not necessary to produce broadband absorbance

8

6

4

2

0

-2

Exp

erim

enta

lly m

easu

red

% T

rans

mitt

ance

4000 3000 2000 1000 0Wavenumber (cm

-1)

40

30

20

10

0

DF

T C

alculated Intensity

IR Spectrum• NRLMOL accurately predicts the positions of significant IR peaks

Experimental

Theoretical

Spectra are mirroredIR Spectrum for DHICA

CO2

Water band

• Further analysis of these spectra is currently underway

800x106

600

400

200

0

Em

issi

on I

nten

sity

(cp

s)

380360340320300280260Excitation Wavelength (nm)

25x103

20

15

10

5

0

Extinction C

oefficient (L mol -1cm

-1)

Excitation spectrum Absorbance spectrum

3.8eV

4.2eV

DHICA Spectroscopy

• Emission:– Single peak– Insensitive to excitation

wavelength– Suggest internal conversion

occurs for higher energy excitations

1.0

0.8

0.6

0.4

0.2

0.0

Em

issi

on (

arb.

uni

ts)

3.63.43.23.02.82.62.42.2Emission Energy (eV)

Excitation Wavelength: 323nm 350nm 380nm

3.1eV

• Excitation:– Double peak– Corresponds to absorbance

spectrum as expected

• Calculations are currently underway to compare the energy structure derived from these spectra to that from density functional theory

Scaled for comparison

20x103

15

10

5

0Ext

inct

ion

Co

effi

cie

nt (

L m

ol-1

cm-1

)

600500400300Wavelength (nm)

t = 0

NaOH added

t = 250min

• We can then observe how these properties change as DHICA evolves into melanin

– UV illumination and/or alkaline pH stimulate melanin formation

• Emission decreases as eumelanin forms– But most significantly, the spectrum does not change shape until the final

timepoints– DHICA is the only fluorescent species with a significant quantum yield!– Larger oligomers dissipate energy non-radiatively!

120x106

100

80

60

40

20

0

Em

issi

on

Inte

nsi

ty (

cps)

550500450400Emission Wavelength (nm)

t = 0

t = 250min

Synthetic Chemistrycombined with

DFT analysis:

3.81eV 3.10eV 2.13eV

• We have successfully synthesized these model compounds• Methyl groups block potential polymerization sites and direct synthesis• DFT shows red-shifting of the excitation energy with increased coupling• Spectroscopic analysis for comparison is underway…

Conclusions• All of the available data is consistent with the chemical

disorder structural model for melanin– Broadband absorbance and other properties are caused by

summation of the properties of many chemically distinct species

• We have yet to find strong evidence for the stacked oligomer structural model– Further theoretical analysis requires a better knowledge of the

melanin structure to understand Structure ↔ Property relationships

Clancy and Simon (2001), Biochemistry, 40, 13353-13360

Next steps…• The question of secondary structure is critical to interpretation of almost

all our results.• Inelastic neutron scattering spectroscopy (INS) may prove useful

– Measures the phonon spectrum without the selection rules imposed by infrared and Raman spectra

– This has been a useful technique for secondary structure in other disordered systems

– DFT will form an essential part of the analysis

• I am here to do INS at ISIS at the Rutherford Appleton Laboratory, the world's brightest neutron source.

• Continuing directed chemical synthesis of oligomers with quantitative spectroscopy, and comparison to density functional theory

Publications:1. P. Meredith and J. Riesz (2004) “Radiative Relaxation Quantum Yields for Synthetic

Eumelanin” Photochemistry and Photobiology, 79(2) 211-216.

2. J. Riesz, J. Gilmore and P. Meredith (2005) “Quantitative photoluminescence of broad band absorbing melanins: a procedure to correct for inner filter and re-absorption effects”. Spectrochimica Acta A, Vol 61(9) 2153-2160

3. P. Meredith, B. Powell, J. Riesz, R. Vogel, D. Blake, S. Subianto, G. Will & I. Kartini “Broad Band Photon-harvesting Biomolecules for Photovoltaics”, in Artificial Photosynthesis: From Basic Biology to Industrial Application (ed: A.F. Collings & C. Critchley), ISBN: 3-527-31090-8, Ch3, p37 (2005).

4. S. Nighswander-Rempel, J. Riesz, J. Gilmore, P. Meredith (2005) “A quantum yield map for synthetic eumelanin”. The Journal of Chemical Physics 123, 194901. Also selected for the November 15 issue of Virtual Journal of Biological Physics Research (2005).

5. S. Nighswander-Rempel, J. Riesz, J. Gilmore, J. Bothma, P. Meredith (2005) “Quantitative Fluorescence Excitation Spectra of Synthetic Eumelanin”. J. Phys. Chem. B, 109(43) 20629-20635.

6. P. Meredith, B. Powell, J. Riesz, S. Nighswander-Rempel, M. Pederson, E. Moore (2006) “Towards Structure-Property-Function Relationships for Eumelanin”, Soft Matter, 2, 37 - 44.

7. J. Riesz, J. Gilmore, P. Meredith (2006) “Quantitative scattering of melanin solutions” Biophysical Journal, 90 (11).

8. J. Riesz, T. Sarna, P. Meredith (2006) “Radiative Relaxation in Synthetic Pheomelanin”, Journal of Physical Chemistry B.

9. J. Riesz, J. Gilmore, R. McKenzie, B. Powell, M. Pederson, P. Meredith (2006) “The Dipole Strength of Melanin”. Soon to be submitted to Phys. Rev. E.

10. J. Riesz, I. Mahadevan, A. Coutts, B. Powell, R. McKenzie, M. Pederson, P. Meredith (2006) “Quantitative spectroscopy of DHICA, a key melanin monomer”. In prep.

11. J. Riesz, A. Coutts, P. Meredith (2006) “Spectroscopic observation of melanin formation”. In prep.

AcknowledgementsSynthesisEvan Moore (Berkley)Kirsten LaurieRoss McGearySurya Subianto (QUT)Indu MadehavanPaul Burn (Oxford)

DevicesAdam Micolich (UNSW)David Blake

My PhD AdvisorsPaul MeredithRoss McKenzieBen Powell

Optical & StructuralJose Eduardo de Albuquerque (Viscosa)Stephen Nighswander-RempelTad Sarna (Jagiellonian)John Tomkinson (RAL)Aaron CouttsJoel Gilmore

TransportClare GiacomantonioAdam Micolich (UNSW)Andrew Watt (Oxford)Francis Pratt (RAL)

Quantum Chemistry, Computation & TheoryMark Pederson (NRL Washington)

Biological FunctionStephen Nighswander-RempelPeter Parsons (QIMR)

Funding: