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ANALYTICAL SPOTLIGHT As published in LPI-October 2004

Advances in circular dichroism


spectroscopy and its applications
by Dr. Peter King

Circular Dichroism (CD) is a spectroscopic technique which reveals information about a mol-
ecule's chirality or "handedness". This technique has been used for many years, along with
the complementary techniques of polarimetry and optical rotatory dispersion (ORD), for
studying and quantifying optically active compounds and their interactions. The information
content of steady state CD spectra can be used to uniquely identify chiral compounds and
their configurations, predict the secondary structure of proteins and other biological macro-
molecules, and in kinetic mode as a probe to monitor the structural changes accompanying
protein folding or unfolding. CD can be used to monitor and quantify ligand binding process-
es and is an increasingly important tool in chiral drug development. The quality of CD instru-
mentation today is better than ever and the latest technological advances will be discussed
in this article.

Chirality arises in compounds which contain at least one chiral centre com- compounds. Such differential absorption at a chiral centre arises because of a
prising a central carbon atom that is bound to four different atomic or preferential interaction of the electrons in one optical isomer with the cir-
molecular groups in a tetrahedral configuration. This means such a com- cularly polarised fields of the monitoring light in either the left or right cir-
pound can exist in two distinct geometric configurations, or stereo-isomers, cularly polarised states. The difference in the relative absorptions of the two
which are non-superimposable mirror images of each other. Members of states as a function of wavelength yields a characteristic CD spectrum. It
such a pair of stereo-isomers are called enantiomers. If a larger molecule has must be emphasised that the CD signal of each pair of enantiomers is of
several (n) chiral centres, the compound will have 2n stereo-isomers, and opposite sign and so the amplitude of a measurement is not proportional
pairs of non-superimposable stereo-isomers in this population are also to concentration where a mixture of enantiomers exists; rather it provides
termed enantiomers. an indication of enantiomeric excess. A racemic mixture (50:50) of enan-
tiomers will show no CD signal at all.

Optical activity On a larger scale, the differential interaction of circularly polarised light
Chirality is the underlying basis for optical activity. In the simplest case an with asymmetric or helical electron distributions in macromolecular sec-
optically active sample causes rotation of the plane of a linearly polarised ondary structures yields the distinctive spectral features used to identify
beam of light transmitted through it. Each member of a pair of enantiomers those structures, their integrity and their relative concentration.
will cause a rotation of opposite sign and the terms dextro-rotatory and levo-
rotatory were first used to distinguish the two chemical forms responsible. Polarimetry, though a routine analytical tool, is not generally as sensitive
This gave rise to the corresponding name prefixes 'd' and 'l', although the and versatile as CD in terms of ultimate limits of detection and delivery of
alternative prefixes '+' and '-' are now preferred so as not to conflict with information content in either steady state or kinetic modes. The remainder
absolute configuration terminology. (The convention for assigning absolute of this article will therefore focus on the latest applications and develop-
configuration, based on the molecular groups around a chiral centre, uses the ments in CD technology.
prefix L and R to distinguish enantiomers. The L and R in this case does not
imply a corresponding rotational direction.) The mirrored geometry in
enantiomers is directly analogous to a pair of hands and it is from the Greek CD spectrometer design
for hand that the word "chiral" is derived. The basic CD spectrometer design most widely adopted by the leading
manufacturers comprises a high intensity broadband light source (usually
a high pressure Xenon arc lamp), a double prism monochromator which
Circular dichroism and not only serves to disperse the wavelength but is also used to linearly
optical rotation polarise the light beam, an electro-optic modulator which transforms the
Optical rotation arises from the differences in refractive index exhibited by linearly polarised light into circularly polarised states, a sample chamber
the two chiral forms of a molecule, and results in a rotation of the plane of and photomultiplier detector. Sophisticated control and signal processing
incident linearly polarised light when it passes though a sample of the mole- electronics, all under PC control, complete a typical instrument [Figure 1].
cule. Circular dichroism, on the other hand, arises as a result of the differen-
tial absorption of left and right circularly polarised light by chiral
ANALYTICAL SPOTLIGHT As published in LPI-October 2004

ing region of the spectrum in which the instru-


ment must operate. At this wavelength the optical
path must be purged with nitrogen to prevent light
absorption by atmospheric oxygen, and the speed
of all spectral scans is carefully controlled in order
to derive measurements with an acceptable signal
to noise ratio. From an experimental point of view
it must be remembered that whilst CD is a meas-
ure of the absorbance difference for CPL and CPR,
the conventional absorbance of the sample must
also be considered as it can severely impair the
transmission of the probe beam, particularly in the
far UV, to the point where no meaningful meas-
urement can be made. This not only applies to the
sample, but to the solvent as well, and oversights in
Figure 1. Schematic layout of the principle components of a modern CD spectrometer. this regard probably account for many unsuccessful
or erroneous CD measurements. The best protec-
tion from this mistake is to record a true absorption spectrum simultane-
Polarisation modulation ously, a feature offered on most modern CD spectrometers. Typically a
The measurement of CD requires the generation of a monochromatic background absorbance rising higher than 2 A.U. is likely to coincide with
probe light beam in each of the left circularly polarised (CPL) and right cir- serious CD signal degradation.
cularly polarised (CPR) states and a means of detecting the difference in
absorbance of the two states caused by the introduction of a sample into Modern CD applications
the light beam. The differential absorbance of a typical sample with respect Whilst small molecule chirality is of interest in its own right, the evolu-
to the two circularly polarised states is typically in the order of 1 part in 104 tionary selection of "handed" molecular building blocks (L-amino acids
or less. Therefore the differences to be measured in the CPL and CPR pho- and D-sugars for example) to synthesise larger biomolecules has lead to the
tometric signals are also of this order in relation to the transmission back- distinct chirality inherent in major biological macromolecules such as pro-
ground of the sample. The most reliable mechanism developed to date for teins and nucleic acids. The ubiquitous right-handed protein alpha helix
measuring such fine differences in sample absorbance utilises a modulation and right-handed DNA double helix are prime examples and yield distinc-
technique whereby the light beam from the monochromator is alternately tive CD spectra. This fundamental biochemical chirality also leads to many
switched at a high frequency (typically 50 kHz) between the CPL and CPR biomolecular interactions possessing chiral selectivity. It is no coincidence
states using a photo-elastic modulator (PEM). Following transmission that the basis for much commercial interest in small molecule chirality is a
through the sample, the beam strikes a photomultiplier detector which reflection of its relevance to that molecule's potential interaction with nat-
converts the incident photon flux to a photometric current.Any sample CD urally handed biological targets. Clearly, specific asymmetric binding sites
results in a small 50 kHz AC component superimposed on a much larger will, in general, preferentially interact with one enantiomer over its partner.
DC background resulting from the standard transmission of the sample. As a consequence, chirality and enantiomeric purity have become critical
This AC amplitude is detected using a tuned amplifier and demodulation issues during the drug development and formulation process.
circuit and the CD is derived from the ratio of the AC and DC amplitudes Probably the most common application of CD spectroscopy today is for
[1]. This optical modulation approach has the benefit of allowing sensitive protein secondary structure determination and, when applied in a kinetic
detection in a single channel and also inherent rejection of noise and drift mode, as a probe to monitor structural events during protein folding or
away from the modulation frequency. unfolding. Although the aromatic amino acid residues possess distinct CD
side chain contributions, the basis of much of the structural CD signal
information is derived from the amide groups in the polypeptide back-
Light throughput and bone. The various established protein secondary structures (alpha helix,
sensitivity beta sheet, beta turn, etc.) all yield distinctive spectral features in a CD spec-
The need to measure very small signals in the far-UV has been the biggest trum, which can be analysed to quantify each component's contribution.
influence driving improvements in instrument specification over the years
and significant increases to the limit of detection and far-UV performance Table 1 presents the results of a structural analysis of the lysozyme CD spec-
have been achieved. Specifically, the most critical requirement for all CD trum shown in figure 2. This table also serves to illustrate the sensitivity of
instruments is to maximise the intensity and quality of circularly polarised the result which is dependent on the wavelength range. The structural
light available, particularly in the far-UV, down to what is the experimen- information available from CD spectra can be a little imprecise and inter-
tally practical limit of 170-175 nm. 190 nm and below is the most demand- pretations do vary depending on the exact method of analysis. However,
ANALYTICAL SPOTLIGHT As published in LPI-October 2004

for why it is seldom taught at undergraduate level, a factor in itself unlike-


ly to promote its further uptake and utilisation.

Recent improvements in CD instrumentation


However, CD is alive and well and is now a far more accessible and user-
friendly technique than it has ever been. The emergence of exciting new
applications has driven marked improvements in the reliability, ease-of-use
and cost of the latest generation of CD instruments. Probably the best known
manufacturers of equilibrium CD instruments over the last 30 years are
Jasco, Aviv, Jobin-Yvon (who no longer manufacture CD instruments) and
more recently Applied Photophysics Ltd (APL) and OLIS, Inc. (The OLIS
Figure 2. A CD spectrum of lysozyme (1 mg/mL). The measured RSM CD spectrometer is of a fundamentally different design from those of
differential absorbance (∆Α) has been converted to Molar CD other manufacturers and will not be described here.) APL's involvement in
(∆ε) by dividing by concentration (M) and pathlength (cm). CD began 10 years ago with the development of the first CD stopped flow
instrument enabling the measurement of kinetic CD on fast timescales at sin-
gle wavelengths. Chirascan is the first CD instrument from APL designed
unlike alternative tools such as X-ray crystallography (which requires sam- specifically for equilibrium CD measurements, but includes data acquisition
ple crystallisation) or NMR (which needs high concentrations and has lim- technology derived from kinetic CD systems and novel optical design fea-
itations in terms of protein size), sample conditions are benign in the sense tures aimed at raising the existing performance ceiling significantly.
that measurements can be carried out on native structural conformations
in aqueous environments. Modern mechanical, optical and software engineering methods coupled with
Although CD is most commonly used for structural studies on proteins, it the latest manufacturing technologies underpin the improvements. For
has also been extensively applied to other biological macromolecules such Chirascan, they have been applied to improve the efficiency of light collection
as nucleic acids and carbohydrates. The latter are less straightforward to and transmission, resulting in greatly improved light throughput, particular-
probe without chemical substitution to enhance the native CD signature. ly in the far-UV. Additionally, its unique dual-polarising prism monochro-
CD is also potentially useful when the chirality of a molecule is relevant to mator doubles the available wavelength bandwidth, which is especially useful
its functional interactions, e.g. ligand binding and enzyme kinetics. In cer- in the far-UV, and can be used to further increase the light intensity at the
tain cases a non-chiral ligand will become chiral, and therefore observable sample. The driving force behind augmenting light intensity at the sample is
by CD, only on binding to a chiral host. This phenomenon is called to reduce the time taken to measure CD spectra and also to extend the range
induced-CD (ICD). Here the detection of a CD signal is evidence for the of samples that are accessible to the technique. The unique throughput-
binding event. Other commercially important applications for CD detec- enhancing features of this instrument offer great potential in both areas.
tion are emerging, including the online discrimination of enantiomers in Furthermore, the introduction of digital adaptive sampling allows scan
automated chiral HPLC separations and as a quality control tool for assess- rates to be varied continuously during an experiment according to on-the-
ing structural and functional integrity of chiral drugs during manufacture fly light levels. This means the entire scan speed is no longer dictated by far-
and formulation. UV throughput, resulting in significantly shorter overall scan times and
increased sample throughput.
Attention to startup time, with respect to rapid nitrogen purging, has been
Challenges facing CD significantly improved by virtue of an hermetically sealed light path which
Although the applications of CD are diverse, it is nevertheless still a fairly retains the purge even when the supply of nitrogen is switched off for sev-
uncommon tool in the spectroscopy laboratory. This may be partly due to eral days. Instrument preparation time is thereby reduced to the time for
its esoteric reputation, but has also arisen from difficulties stemming from the lamp to stabilise (less than 1/2 an hour) and consumption of nitrogen
experimental technique and the limitations, not to mention cost, of avail- is significantly lowered.
able instrumentation. The typical sptral operating range (~170-800 nm) is All manufacturers offer a range of accessories for their instruments.
demanding in terms of instrument and sample preparation. To reach the Examples include peltier temperature controlled sample holders and
far-UV the instruments require nitrogen purging and samples must be multi-syringe titration systems. Modern instruments also offer simultane-
provided in very short pathlength cells (<0.1 mm) to allow measurable ous detection of more than one optical property with the CD signal. The
photometric signals to be transmitted. The light throughput limitation in simultaneous collection of an absorbance spectrum was mentioned earlier,
the UV is also the reason scan times are slow compared to other spectro- but the simultaneous detection of fluorescence is also of growing interest.
scopic methods. Other detection modes such as fluorescence detected CD (FDCD), where-
by only fluorescent chiral centres are observed, and magnetic CD (MCD),
The complexity of CD measurement, both technically and experimentally, which monitors CD induced by strong magnetic fields, are also available
compared to other UV-Vis spectrosopy methods probably also accounts through appropriate upgrades.
ANALYTICAL SPOTLIGHT As published in LPI-October 2004•

domain on a PC it can be inspected along with a residual plot of the raw


data minus the smooth, thus yielding incontrovertible evidence that the
data has not been distorted. This facility is not available with traditional
pre-acquisition analogue filtering.

Summary
The combination of advances in technology described above has led to a
significant improvement in the speed and reliability of CD measurement.
Whilst sample preparation and an understanding of the limitations
caused by sample and solvent absorption and some knowledge of the
Table 1. The results of a protein secondary structure analysis of the theory of CD measurement is unavoidable, the latest CD instruments are
lysozyme data shown in Figure 2 using the CDNN protein analysis highly sophisticated, easy-to-use and highly reliable. The cost of the
application [2]. Note the sensitivity of the results is dependant on the required technology is dropping and so the entry cost for CD instruments
wavelength range included. The sum row provides a net contribu- has come down and there are a number of manufacturers keeping compe-
tion of all underlying structures and should ideally equal 100%. tition keen. The importance of chirality in so many areas of chemistry and
biochemistry is also likely to drive further developments and improve-
ments and, given the unique capability of CD as a probe for this fascinat-
Software and data ing phenomenon, the future of CD can only be described as looking bright.
processing References
All current CD instruments are accompanied by sophisticated PC software 1. Velluz L, Legrand M, Grosjean M. Optical circular dichroism, principles,
providing data acquisition control, data visualisation and management fea- measurements and applications. 1965, Academic, New York.
tures. These software packages also provide a range of data processing func- 2. CDNN: Gerald Böhm, Institut für Biotechnologie, Martin-Luther-
tions such as spectral baseline subtraction and basic math functions Universität, Halle-Wittenberg, Germany. SELCON: Prof. Robert Woody,
including averaging, curve fitting and data filtering. The protein analysis Colorado State University, USA. CONTIN: Stephen Provencher, 48
described earlier is frequently performed using programs available in the Chancery Lane East, Oakville, Ontario L6J 5P6, Canada.
public domain such as CDNN, SELCON and CONTIN [2]. Data files and
sample concentrations must be submitted in precise formats to these pro- The author
grams and so facilities to export correctly formatted data files are also pro- Peter King, Ph.D., Technical Director,
vided. For added convenience, client-server software architecture Applied Photophysics Ltd
developed for Chirascan allows experiments to be conducted remotely and 203-205 Kingston Road
monitored over a network. Leatherhead, Surrey KT22 7PB, UK
Tel.: +44 1372 386537
Fax: +44 1372 386 477
Data smoothing and filtering Email: Petek@photophysics.com
This topic is discussed in its own right as it is a matter of considerable
importance. CD measurements are often small and frequently noisy, par-
ticularly in the far-UV. To improve their signal-to-noise ratio filtering is
usually applied, either electronically to the raw analogue signals themselves
or, with the advent of fast digital signal processing, to the raw acquired data
in the digital domain. It can-
not be over-stressed that
electronic analogue filtering
is a risky process and can
lead to the distortion of
measured spectra. The
selection of time constants
to filter real time signals
during scans is highly
Figure 3. The Chirascan CD spectrom- dependent on both spectral
eter from Applied Photophysics Ltd, complexity and required
Leatherhead, UK. scan speed and should be
considered obsolete given the speed and reversibility of digital filtering
approaches. Most importantly, if a noisy trace is filtered in the digital