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