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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 molecule'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 macromolecules, 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 processes and is an increasingly important tool in chiral drug development. The quality of CD instrumentation 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 comprising a central carbon atom that is bound to four different atomic or molecular groups in a tetrahedral configuration. This means such a compound can exist in two distinct geometric configurations, or stereo-isomers, which are non-superimposable mirror images of each other. Members of such a pair of stereo-isomers are called enantiomers. If a larger molecule has several (n) chiral centres, the compound will have 2n stereo-isomers, and pairs of non-superimposable stereo-isomers in this population are also termed enantiomers. compounds. Such differential absorption at a chiral centre arises because of a preferential interaction of the electrons in one optical isomer with the circularly polarised fields of the monitoring light in either the left or right circularly polarised states. The difference in the relative absorptions of the two states as a function of wavelength yields a characteristic CD spectrum. It must be emphasised that the CD signal of each pair of enantiomers is of opposite sign and so the amplitude of a measurement is not proportional to concentration where a mixture of enantiomers exists; rather it provides an indication of enantiomeric excess. A racemic mixture (50:50) of enantiomers will show no CD signal at all. On a larger scale, the differential interaction of circularly polarised light with asymmetric or helical electron distributions in macromolecular secondary structures yields the distinctive spectral features used to identify those structures, their integrity and their relative concentration. Polarimetry, though a routine analytical tool, is not generally as sensitive and versatile as CD in terms of ultimate limits of detection and delivery of information content in either steady state or kinetic modes. The remainder of this article will therefore focus on the latest applications and developments in CD technology.
Optical activity Chirality is the underlying basis for optical activity. In the simplest case an optically active sample causes rotation of the plane of a linearly polarised beam of light transmitted through it. Each member of a pair of enantiomers will cause a rotation of opposite sign and the terms dextro-rotatory and levorotatory were first used to distinguish the two chemical forms responsible. This gave rise to the corresponding name prefixes 'd' and 'l', although the alternative prefixes '+' and '-' are now preferred so as not to conflict with absolute configuration terminology. (The convention for assigning absolute configuration, based on the molecular groups around a chiral centre, uses the 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 for hand that the word "chiral" is derived.
Circular dichroism and optical rotation Optical rotation arises from the differences in refractive index exhibited by the two chiral forms of a molecule, and results in a rotation of the plane of incident linearly polarised light when it passes though a sample of the molecule. Circular dichroism, on the other hand, arises as a result of the differential absorption of left and right circularly polarised light by chiral
CD spectrometer design 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 not only serves to disperse the wavelength but is also used to linearly polarise the light beam, an electro-optic modulator which transforms the linearly polarised light into circularly polarised states, a sample chamber and photomultiplier detector. Sophisticated control and signal processing electronics, all under PC control, complete a typical instrument [Figure 1].
As published in LPI-October 2004
ing region of the spectrum in which the instrument 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 measure 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 measurement 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 protection from this mistake is to record a true absorption spectrum simultaneously, a feature offered on most modern CD spectrometers. Typically a Polarisation modulation 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 evoluto 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 proground 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 distinctechnique 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 natconverts 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 . 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 backbone. The various established protein secondary structures (alpha helix, Light throughput and beta sheet, beta turn, etc.) all yield distinctive spectral features in a CD specsensitivity 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 spechave 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 intertally 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,
As published in LPI-October 2004
for why it is seldom taught at undergraduate level, a factor in itself unlikely 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 userfriendly 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 RSM CD spectrometer is of a fundamentally different design from those of other manufacturers and will not be described here.) APL's involvement in 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 single wavelengths. Chirascan is the first CD instrument from APL designed specifically for equilibrium CD measurements, but includes data acquisition technology derived from kinetic CD systems and novel optical design features aimed at raising the existing performance ceiling significantly. Modern mechanical, optical and software engineering methods coupled with the latest manufacturing technologies underpin the improvements. For Chirascan, they have been applied to improve the efficiency of light collection and transmission, resulting in greatly improved light throughput, particularly in the far-UV. Additionally, its unique dual-polarising prism monochromator doubles the available wavelength bandwidth, which is especially useful in the far-UV, and can be used to further increase the light intensity at the sample. The driving force behind augmenting light intensity at the sample is to reduce the time taken to measure CD spectra and also to extend the range of samples that are accessible to the technique. The unique throughputenhancing features of this instrument offer great potential in both areas. Furthermore, the introduction of digital adaptive sampling allows scan rates to be varied continuously during an experiment according to on-thefly light levels. This means the entire scan speed is no longer dictated by farUV throughput, resulting in significantly shorter overall scan times and increased sample throughput. Attention to startup time, with respect to rapid nitrogen purging, has been significantly improved by virtue of an hermetically sealed light path which retains the purge even when the supply of nitrogen is switched off for several days. Instrument preparation time is thereby reduced to the time for the lamp to stabilise (less than 1/2 an hour) and consumption of nitrogen is significantly lowered. All manufacturers offer a range of accessories for their instruments. Examples include peltier temperature controlled sample holders and multi-syringe titration systems. Modern instruments also offer simultaneous detection of more than one optical property with the CD signal. The simultaneous collection of an absorbance spectrum was mentioned earlier, but the simultaneous detection of fluorescence is also of growing interest. Other detection modes such as fluorescence detected CD (FDCD), whereby only fluorescent chiral centres are observed, and magnetic CD (MCD), which monitors CD induced by strong magnetic fields, are also available through appropriate upgrades.
Figure 2. A CD spectrum of lysozyme (1 mg/mL). The measured differential absorbance (∆Α) has been converted to Molar CD (∆ε) by dividing by concentration (M) and pathlength (cm).
unlike alternative tools such as X-ray crystallography (which requires sample crystallisation) or NMR (which needs high concentrations and has limitations in terms of protein size), sample conditions are benign in the sense that measurements can be carried out on native structural conformations in aqueous environments. Although CD is most commonly used for structural studies on proteins, it has also been extensively applied to other biological macromolecules such as nucleic acids and carbohydrates. The latter are less straightforward to probe without chemical substitution to enhance the native CD signature. CD is also potentially useful when the chirality of a molecule is relevant to its functional interactions, e.g. ligand binding and enzyme kinetics. In certain cases a non-chiral ligand will become chiral, and therefore observable by CD, only on binding to a chiral host. This phenomenon is called induced-CD (ICD). Here the detection of a CD signal is evidence for the binding event. Other commercially important applications for CD detection are emerging, including the online discrimination of enantiomers in automated chiral HPLC separations and as a quality control tool for assessing structural and functional integrity of chiral drugs during manufacture and formulation.
Challenges facing CD Although the applications of CD are diverse, it is nevertheless still a fairly uncommon tool in the spectroscopy laboratory. This may be partly due to its esoteric reputation, but has also arisen from difficulties stemming from experimental technique and the limitations, not to mention cost, of available instrumentation. The typical sptral operating range (~170-800 nm) is demanding in terms of instrument and sample preparation. To reach the far-UV the instruments require nitrogen purging and samples must be provided in very short pathlength cells (<0.1 mm) to allow measurable photometric signals to be transmitted. The light throughput limitation in the UV is also the reason scan times are slow compared to other spectroscopic methods. The complexity of CD measurement, both technically and experimentally, compared to other UV-Vis spectrosopy methods probably also accounts
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 . 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 competion 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 improvements and, given the unique capability of CD as a probe for this fascinatSoftware 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-Luthertions 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 . 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, particularly 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 cannot 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
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