Chirality in Molecular Vibrations: VCD and ROA · 2014-06-19 · Chirality in Molecular Vibrations:...
Transcript of Chirality in Molecular Vibrations: VCD and ROA · 2014-06-19 · Chirality in Molecular Vibrations:...
Chirality in Molecular Vibrations:
VCD and ROA
Laurence A. Nafie
Department of Chemistry, Syracuse University
Syracuse, NY 13244 USA
Parma
June 2014
Outline
• Definitions of VOA
• Measurement of VCD and ROA
• Levels of Resonance Raman Scattering
• Resonance ROA
• Velocity Formulation of VCD
• Vibrational Current Density
• Determination of Absolute Configuration
• Enhanced VCD – Amyloid Fibrils and Low-
Lying Electronic States
• Conclusions
Definitions of VOA
3
( )L Rk k
Optical Rotation (Optical Rotatory Dispersion, ORD, CB)
( )L Rn n
Complex Refractive Index
n = n+ ik = ¢n + i ¢¢n
Ellipticity (Circular Dichroism, CD)
Classical Forms of Optical Activity
VIBRATIONAL OPTICAL ACTIVITY
Differential Interaction of a Chiral Molecule with Left and Right Circularly
Polarized Radiation During Vibrational Excitation
VIBRATIONAL CIRCULAR DICHROISM RAMAN OPTICAL ACTIVITY
Differential Absorption of Left and Right Differential Raman Scattering of Left
Circularly Polarized Infrared Radiation and Right Incident and/or Scattered
Radiation
Forms of Circular Polarization
Vibrational Optical Activity
( ) ( ) ( )L RA A A
ICP-ROA
(Incident CP)
SCP-ROA (Scattered CP)
DCPI-ROA
(In-Phase Dual CP )
DCPII-ROA (Out-of-Phase DCP)
( ) ( ) ( )R LI I I
( ) ( ) ( )R LI I I
( ) ( ) ( )R L
I R LI I I
( ) ( ) ( )R L
II L RI I I
VCD
VCD & ROA Short History
• VCD
• Discovered in 1974 by Holzwarth
• Confirmed by Nafie, Cheng, Keiderling & Stephens in 1975, 1976
• FT-VCD discovered by Nafie in 1978
• Commercialized by BioTools and ABB Bomem in 1997
• 2nd generation spectrometers w/time sampling - 2010
ROA
• Discovered in 1973 by Barron & Buckingham, ICP-ROA
• Confirmed by Hug in 1975
• SCP/DCP-ROA discovered by Nafie 1987
• New ROA Design, Hug 1999
• Commercialized by BioTools in 2003
• 2nd generation spectrometer w/ microscope - 2009/10
7
Classes of Molecules and Techniques
VCD & ROA
• VCD
• Small Organic Molecules, Pharmaceuticals and Natural Products
• Proteins, Peptides, Amino Acids, Sugars,Nucleic Acids, DNA, RNA Glycoproteins
• Transition Metal Complexes with Enhance VCD for Low-Lying States
• Chiral Polymers
• Supramolecular Structures including Protein Fibrils
• Solutions, Films, Solid Microcrystals, Spray-Dried Films
• Accurate Quantum Calculations
ROA
• Proteins, Peptides, Amino Acids, Sugars, Nucleic Acids, DNA, RNA, Glycoproteins
• Small Organic Molecules, mostly neat liquids
• Viruses
• Surface-Enhanced ROA (SEROA) of Adsorbed Molecules on Metal Surfaces
• Resonance ROA (RROA)
• Accurate Quantum Calculations
8
FT-VCD Measurements
9
FT-VCD Instrumental Layout
Photoelastic
Modulator(PEM)
Detector
Polarizer
Lock-in
Amplifier (LIA)
Optical
Filter
Process & Display
Sam ple
Cell
R L
VCD
IR
IA C
I D C
(1R,4R)-(+)-camphor
(1S,4S)-(-)-camphor
Enantiomers: IR spectra are identical, VCD spectra are opposite in sign
CCD-ROA Measurements
12
Path of Laser Activity in ChiralRAMAN 3000u
Aperture
Prism
3000u
Aperture
Beam
Compressor Lens
Beam Compressor Lens
Incident Shutter
Polarizer
Degree of Circularity Converter
Fast Rotator
(Fast)
Fast Rotator
(Slow)
Circularity Converter
Prism
1000u
Aperture
Gradium Lens
Sample Holder
Beam Dump Slow
Rotator
LargeCircularity
Converter
S Branch
P Branch
Molch Filter
Beam Splitter
Launching Lenses
Fiber Mount
Fiber Mount
Incident Light
Raman Scattered
Light
(a) ROA of
S-(-)-α-pinene
(b) ROA of
R-(+)-α-pinene
(c) Raman spectrum of
α-pinene
Measured SCP-ROA Spectra
Degrees of RR and RROA
Degrees of RR and RROA
GU – General Unrestricted Theory
FFR – Far-From-Resonance Theory
NR – Near-Resonance Theory
SES – Single-Electronic-State Theory
MES – Multiple-Electronic-State Theory
General Unrestricted Theory (GU Level)
Exact Excited-State Vibronic Detail
•Raman tensor is not symmetric
•Raman tensor is time-reversal invariant
•3 Raman Invariants, 10 ROA Invariants
•ICP-ROA does not equal SCP-ROA
•DCPII-ROA is non-zero
•Software routines not available
Far-From-Resonance (FFR) Theory
No Excited State Vibronic Detail
•Raman tensor is symmetric
•Raman tensor is not time-reversal invariant
•Incident and scattered radiation have the
same degree of pre-resonance
•2 Raman Invariants, 3 ROA Invariants
•ICP-ROA, SCP-ROA and DCPI-ROA equal
•DCPII-ROA is equal to zero
•Software routines available commercially
from Gaussian, Inc.
Near-Resonance (NR) Theory (GU Invariant Level)
Simple Excited State Vibronic Detail
•Raman Tensor is not symmetric
•Raman tensor is time-reversal invariant
•Incident and scattered radiation have
different degrees of pre-resonance
•3 Raman Invariants, 10 ROA Invariants
•ICP-ROA, SCP-ROA, and DCPII-ROA
differ from each other
•DCPII-ROA is non-zero
•Software routines not available
Near Resonance (NR) Theory of
Vibrational Raman and ROA
SES Theory of natural vibrational RROA
First observation of natural RROA
Confirmation of SES-RROA Theory
First observation of natural RROA
Confirmation of SES-RROA Theory
Calculated ROA show monosignate spectra with the same form as the RR spectrum and with the same ratio as the electronic CD to the absorption spectrum, with the opposite sign, of the resonant electronic state
RR and ROA of Methyloxirane at 202 and 185 nm Excitation
Theoretical Background
of VCD
30
Dipole and Rotational Strength for Vibrational Transition of gv to gv’
m m m
. m > 0 . m < 0
IR
VCD
Vibrational Current Density
Continuity Equation - Conservation of Charge Density
BO Beyond BO
Vibrational Current Density
Anti-symmetric CH Stretch in Formaldehyde
H H
C
Vibrational Current Density
View along electric dipole transition moment of CH stretch
Determination of Absolute
Configuration using VOA
35
AC Determination of Small
Organic Molecules
39
W. Hug et al. Helv. Chim. Acta, 84, 1, 2001
Paper describing application of VCD to the Determination of Absolute Configuration of Chiral Pharmaceutical Molecules
VOA in
Pharma and Biopharma
43
VCD / ROA in Pharma
• Amgen, Astra-Zeneca, BMS, GSK, Eli Lilly, Wyeth/Pfizer, J&J,
Roche, Novartis, Boehringer-Ingelheim, Organon (Akzo Nobel, now
Merck), Merck, Pfizer, Abbott/AbbVie, Cell Therapeutics, Solvay,
Neurocrine, Sanofi-Aventis, Sepracor / Sunovion, Gilead, and many
more use VCD for AC by outsourcing measurements and calculations.
• VCD is now used as a routine tool for AC and not just a research
technique. Hundreds of AC determinations carried out in Pharma each
year. Over 100 US Patents cite VCD for AC determination of new
drugs
• VCD is ‘accepted’ by regulatory agencies as proof of Absolute
Configuration.
• VCD is in the initial stages of becoming a standard method for AC
determination in the US Pharmacopeia (Stimuli article published in
July)
AC Determination of
Pharmaceutical Molecules
46
50
L.A. Nafie, Nat. Prod. Comm. 3, 451-466 (2008)
VOA Analysis of Proteins
for Biopharma Applications
52
VCD and IR protein spectra
Alpha helical
Beta sheet
Nonlinear mapping algorithm for classification 80 protein ROA spectra
Barron et al. J Mol Bio 363 19-26 (2006)
Aggregation of Human IgG1
Work and slides courtesy of Dr. Tiansheng Li – Amgen, Inc.
CAUSES OF SOLUBLE & INSOLUBLE AGGREGATES: - temperature - shear force - freeze-thawing - pH - high concentration -- long term storage
Cynthia Li and Tiansheng Li Current Pharmaceutical Biotechnology, 2009, 10, 391-399
Higher-Order Pre-Aggregation seen by ROA but not by Raman
Enhanced VCD Intensity
57
Examples of Enhanced
VCD Intensity
• Protein Fibrillation and Development
• Molecules with Low-Lying Electronic States
•Negative Index Materials and Helicene and
Cyclocene Molecules
•Spray-Dried Films of Amino Acids and Peptides
•Heme Protein Ligands
Enhanced VCD Intensity
in Protein Fibrils
59
Initial Stages of Fibril Formation
More Advanced Fibril Formation and Development
J. Am. Chem. Soc. 129, 12364-12365 (2007)
1700 1600 1500
Wavenumber
-20
-10
0
10
A
X 1
05
Native Protein
VCD
1700 1600 1500
Wavenumber
-20
-10
0
10
Fibril
VCD
VCD of Fibrils of
Lysozyme and Insulin
A BA B
Lysozyme Insulin
Normal VCD and IR
for Lysozyme and
Insulin
-30
-20
-10
0
10
20
A X
10
5
1700 1600 1500
Wavenumber (cm-1)
0.0
0.1
0.2
0.3
Ab
so
rba
nce
Lysozyme
Insulin
1670
1666
1624
1639
1643
1515
15581596
1677
1627
1635
15151546
g ~ 10-2 to 10-3
-8
-4
0
4
A
X 1
05
Observed VCD
Noise
Native
Heat 1 hr
Heat 2 hrs
heat 2.5 hrs
1900 1700 1500 1300
Wavenumber (cm-1)
0.0
0.2
0.4
Absorb
ance
Observed IR
Insulin Fibril Formation
and Growith at pH 2
with heating at 60oC
Reversed Supramolecular Chirality
in Insulin Fibrils
Reversed Large
VCD of Insulin
Protein Fibrils
Implications of
Reversed
Supramolecular
Chirality
Comparison of Insulin
Fibrils in the Solution
and Film State for
Normal and Reversed
Supramolecular
Chirality
Proto-
Filaments
pH 2.4-3.2
Proto-fibrils
intertwining
nVC
D
LH
nVCDLH
Fibrils
Height: ~1.5 nm
Height: ~4 nm
Height ~8 nm
pH 1.1-2.1
nVCDLH?
rVCD
RH?
rVCD
RH?
intertwining
RH
?
Native
insulin
Partially
unfolded
protein
aggregation
denaturation
Chirality
visible for
VCD
Chirality
visible for
microscopy
Unstable,
requires pairing
Height: ~1.5 nm
Height: ~2 nm
Parallel aggregation
Parallel aggregation
D. Kurouski, X. Lu, R. K. Dukor, L. A. Nafie and I. K. Lednev, Biophys. J., 2012, in press
69
0
.2
.4
.6
.8
1
1800 1750 1700 1650 1600 1550 1500 1450 1400
-1
-0.5
0
0.5
1
1.5
ΔA
x 1
02
Ab
sorb
ance
Wavenumber (cm-1)
a)
b)
1549
1680
1624
1647
1623
1544
1663
1610
1677
1556
VCD (a) and IR (b)
spectra of lysozyme
fibrils grown at pH 1.0
(blue), 1.5 (green), 2.3
(black) and 2.7 (red) for
3 days at 65 ⁰C.
0
.2
.4
.6
.8
1
1800 1750 1700 1650 1600 1550 1500 1450 1400
-2
0
2
4
a)
ΔA
x 1
04
b)A
bso
rba
nce
Wavenumber (cm-1)
1666 1639
1617
1624
1659
1577
1544
1722
1655
1626
1546
1532
0
1700 1650 1600 1550
ΔA
x 1
04
Wavenumber (cm-1)
1611
1616
1636
1658 1561-2
2
4
-1
-0.5
0
0.5
1800 1750 1700 1650 1600 1550 1500 1450 1400
ΔA
x 1
02
0
.2
.4
.6
.8
1
Ab
sorb
an
ce
Wavenumber (cm-1)
a)
b)
1671
1614
1557
15781666
1640
1611
1636
1725
1655
1624
1550
1620
VCD (a) and IR (b)
spectra of apo-alpha
lactalbumin fibrils
grown at pH 1.5 (blue),
2.5 (red), 3.0 (green)
and 4.0 (black) for 3
days at 37 ⁰C.
VCD (a) and IR (b)
spectra of HET-s
mouse prion protein
fibrils grown at pH 2.0
(red), 3.3 (green), 3.9
(black) room
temperature, 2 months
-2.0
0
2.0
4.0
6.0
1800 1750 1700 1650 1600 1550 1500 1450 1400
0.0
0.2
0.4
0.6
0.8
1.0
ΔA
x 1
04
Wavenumber (cm-1)
Abs
orba
nce
a)
b)
1647
1623
15461525
1624
VCD (a) and IR (b)
spectra of TRR 105-115
fragment of
transthyretin fibrils
grown at pH 1.0 (blue),
1.5 (green), 2.0 (black),
2.5 (red) and 3.0
(violet) for 2 days at 37
⁰C. For insulin, lysozyme, apo-alpha-lactalbumin, HET-s mouse prion protein and the TTR105-115 peptide fragment maintain correlation of normal VCD to left-twisted fibril morphology and reversed VCD to flat tape-like fibrils
VCD versus pH for 4 additional proteins/peptides
Enhanced VCD Intensity
in Molecules with Low-Lying
Electronic States
73
Sparteine Transition Metal
Complexes
Sparteine Transition Metal
Complexes
Sparteine Transition Metal
Complexes
Conclusions
• VCD and ROA are a sensitive spectroscopic
probes of absolute molecular stereochemistry of
all classes of chiral molecules and biomolecules
• Velocity formulation of VCD allows
visualization of vibrational current density
• Enhanced VCD can be seen in
– Protein fibril formation and development
– Molecules with low-lying excited electronic states
– Extended chiral cyclic π-electron systems including
chiral conducting polymers
Acknowledgments
– Prof. Tess Freedman, Dr. Xiaolin Cao, Shengli Ma,
Rosina Lombardi, Syracuse University
– Dr. Rina K. Dukor, BioTools, Inc.
– Professor Igor Lednev and Dmitry Kurouski,
University of Albany, SUNY
– Funding: NIH, NSF, NASA, AFOSR, Johnson
Pharmaceutical R&D, BioTools, Inc. and the CASE
Center, Syracuse University