Through the Eye of a Chromophore CD Spectroscopy

Some gemstones show different colours if one observes them from different angles. They are dichroic; the effect is called dichroism. In contrast to the optical rotation dispersion, circular dichroism (CD) spectroscopy does not describe the specific rotation [a] of an optically active substance as a function of the wavelength. CD spectroscopy measures the differential absorption of circularly polarized light by mirror image isomers. An explanation:

By definition, a chiral molecule does not show a plane of symmetry. Therefore, the excitation of electrons in a chiral molecule into an electronically excited state will not occur in a symmetric matter. The pushing of electron density from a starting state to a higher energy final state triggered by electromagnetic radiation proceeds in an asymmetric manner by some kind of helical electron movement. This helical

8 As a matter of fact, the optical rotation dispersion in polyglycine is entirely determined by helical chirality, since glycine-monomers are achiral. Polyglycine can occur in left- and right-helices (M- and P-helices). In contrast to this, proteins (enzymes) are right-helices (P-helices), determined by the l-configuration of the a-amino acid monomers. So is DNA a right-helix (P-helix), determined by its chiral monomers the d-ribofuranose units. As an exception, Z-DNA, a type of DNA structure that is characterized by a regularity in its sequence adopts a left-helix (M-helix).

excitation of electrons in chiral molecules is affected by chiral photons in a way that right-circularly polarized light (RCPL) is not absorbed by chiral molecules in the same manner as left-circularly polarized light (LCPL). The interaction is different. The above explication is to illustrate and visualize CPL absorption and is, of course, not based on physical reality.

CD spectroscopy determines the difference of the molar extinction coefficient Ae of a given substance between LCPL and RCPL as written in Eq. 2.9. This differential absorption depends on the wavelength, a dependence that is measured by CD spectropolarimeters and given in a CD spectrum.

Typically, CD spectra show an extremum (minimum or maximum) if the energy of the incoming circularly polarized light is closely matching the energy of absorption of a chromophore. The energy of this extremum coincides with the energy of the point of inflection described by the Cotton effect in abnormal ORD curves which itself ideally coincides with the maximum of an electronic absorption band (Barron 2004). Summarizing, a maximum in an ultraviolet and visible absorption spectrum results in an extremum in the CD spectrum and a Cotton effect with [a] = 0 in ORD. In this context, it is sometimes stated that ORD and CD techniques are used to look at the stereochemistry of a chiral molecule "through the eyes of the chromophore".

A positive ORD plain curve shows a positive CD signal, a negative ORD plain curve shows a negative CD signal. In Fig. 2.10, the CD spectra of (-)-^-a-alanine and (+)-S-a-alanine enantiomers are given, in which each curve shows a maximum and a minimum and the two spectra are "opposite" to each other.

For CD measurements of chiral compounds spectropolarimeters are used, composed of a. a light source, which is in most instruments a water-cooled xenon arc lamp for UV and visible CD measurements, b. an optical device that produces monochromatic electromagnetic radiation that is alternatively left- and right-circularly polarized. This is performed by a series of mirrors and prisms creating linearly-polarized light, connected to a photo-elastic modulator (PEM) or a Pockels cell, transforming linearly- into circularly polarized light, typically switching between right- and left-circular polarizations light with a frequency v = 50 kHz by applying an AC voltage.

c. A cuvette for the analyte in solution, and d. a photo-multiplier detection unit able to determine the energy-dependent differential absorption for the two circular polarizations.

For historical reasons, different instruments with different units are used in CD spec-troscopy. In order to transform values for dichroic absorption Ae into the old but common unit of molar ellipticity Mq in mol-1 dm3 cm-1, Eq. 2.10 can be applied. After absorption, the transmitted polarized light leaves a sample of a chiral molecule in solution as elliptically-polarized light. Formerly, such ellipticity was determined experimentally for recording circular dichroism spectra.

Fig. 2.10 Circular dichroism spectra of the amino acids (—)-R-a-alanine (blue line) and (+)-S-a-alanine (black line) recorded as zwitterions in hexafluoroisopropanol (HFIP) solution at the ISA synchrotron facility in Arhus, Denmark. The spectra of the two enantiomers are depicted between 150 and 300 nm and show - as expected - opposite signs

Fig. 2.10 Circular dichroism spectra of the amino acids (—)-R-a-alanine (blue line) and (+)-S-a-alanine (black line) recorded as zwitterions in hexafluoroisopropanol (HFIP) solution at the ISA synchrotron facility in Arhus, Denmark. The spectra of the two enantiomers are depicted between 150 and 300 nm and show - as expected - opposite signs

The molar ellipticity M0 is related with the specific ellipticity [0] in g-1 dm3 cm-1 by Eq. 2.11 where MW is the molecular mass in g mol-1.

The ellipticity 0 itself is linked with the specific ellipticity by Eq. 2.12.

The differential absorption AA, often encountered in chemistry literature, is obtained by Eq. 2.13, the circular dichroism version of the Beer-Lambert law (Rodger and Norden 1997).

Note that even a molecule with defined P-helicity may show positive or negative CD signals since rearrangement of electron density with different handedness not necessarily following our P/M-notation might be involved.

Comparing ORD and CD techniques, the experimentator should note that CD spectroscopy is often advantageous and now generally preferred, since a better resolution of signals can be obtained, particularly if chromophores absorb at wavelengths close to each other. If there are several adjacent absorption bands, the net Cotton effect will be a superposition of the individual Cotton effect curves and the CD lineshape function drops to zero much more rapidly than the ORD lineshape function (Barron 2004). However, CD signals are generally weaker than ORD signals and the ORD technique allows determining values even in a certain distance from the Cotton effect.

In order to convert data on optical rotation [m']% obtained from ORD measurements into the specific molar ellipticity [0]* from CD records and vice versa, the Kronig-Kramers theorem represented in Eq. 2.14 can be used.

ra 2

Besides the above outlined experimental approach, one may attempt to theoretically predict the interaction of electromagnetic radiation with chiral organic molecules. Quantum mechanical calculations are required, which are based on the Hamiltonian operator HTint of the energy of interaction between light and matter. According to Eq. 2.15, this is the difference of the vector products between the electric field vector E and the operator (indicated with for the electric dipole moment 1 of the molecule and the vector product between the magnetic field vector B and the operator for the magnetic dipole moment m of the molecule. The software Gaussian 03 offers a tool for quantum mechanical calculations of CD signals.

Resulting from experimental data and quantum mechanical calculations, CD spectra of peptides and proteins in solution typically show electronic transitions of the occupied «-orbital to the unoccupied n-orbital (denoted with *) at about 220 nm with negative values. A negative (n*,n)-electronic transition is at about 210 nm and a positive (n*,n)-transition is often found at about 190 nm.

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