Magnetic circular dichroism
Magnetic circular dichroism spectroscopy is based upon the measurement of the difference in absorption between left circularly polarized (LCP) light and RCP light, induced in a sample by a strong magnetic field oriented parallel to the direction of light propagation (124,125). In fact, the origin of CD and MCD is quite different. Electronic circular dichroism requires a molecular environment where molecular structure features distributed electric charge in a spatial array that has helical handedness. In contrast, MCD is due to electromagnetic interaction of the external field with electronic charge within the sample, no matter how it is distributed, and is a universal property of light absorption for all matter when placed in a magnetic field. Hence, a chiral molecular structure is not a requirement for MCD technique (124,125).
The most important use of MCD spectra in the UV–vis–near-IR region is to assist the interpretation of electronic spectra and provide experimentally based information about the electronic states involved in the observed transitions (124,126). This requires the companion use of absorption spectra, ideally obtained simultaneously and under the same experimental conditions as the MCD spectra. MCD analyses without the companion absorption spectrum can provide nothing more than a fingerprint measurement for the system under experiment (124,126).
In MCD, the sample is placed in a longitudinal magnetic field (i.e. coincident with light propagation axis). It is actually an application of the well-known Faraday effect, the magnetic field induces optical activity in any sort of samples. Magnetic CD is a consequence of the interaction of electronic energy levels with the magnetic field and shows up only when absorption of light occurs (124,125).
As the most extensive applications, electronic and magnetic properties of metalloproteins and inorganic complexes have been studied by MCD (127). There are various studies in the literature have been made on iron-sulphur proteins (128), porphyrins and haeme proteins (129,130), copper, rare earth, cobalt and non-haeme iron bioinorganic systems (131), and non-haeme ferrous enzymes (132) using this technique. Both excited- and ground-state information and a more powerful probe of the coordination geometry and structure of metal chromophores have been provided by employing MCD (127).
Magnetic CD has also been observed for vibrational or rotation–vibrational excitations in the infrared (MVCD) and for magnetic core to valence level absorption edges in the X-ray region (XMCD). It is obvious that the experimental setup for these measurements must be appropriate to the spectral region involved, and therefore, the necessary apparatus will depend upon the wavelength of the light used for the measurement. Infrared or X-ray polarizers and optics are quite different from than those used in the UV-Vis region (124,125).
For XMCD particularly, intense X-ray photon sources are required, which means proximity to synchrotron laboratories is necessary for measurements. Nevertheless, the measured differential absorption between LCP and RCP light by a sample in a strong magnetic field oriented along the propagation direction is entirely analogous to measurements in the UV-Vis, and the origin of MVCD and XMCD likewise results from the Zeeman interaction of the field with magnetic moments within the sample (124,125).
Magnetic vibrational circular dichroism has been developed largely by Keiderling et al., and it was applied initially to vibrational studies of molecules with high symmetries in the condensed phase (124,126). More recently, with improvements in instrumental methods for higher resolution, MVCD was used to study the molecular Zeeman effect (ZE) of rotationally resolved vibrations of small molecules in the gas phase. The infrared measurements necessitated the use of sources and detectors appropriate for the IR region and grid polarizers, lenses, and photo elastic modulator (PEM) elements that have high transmission in the IR (124,133). Since the range of measurement and resolution were greatly enhanced over dispersive spectrometers, the applications of Fourier transform IR (FTIR) methods to MVCD were also notable. However, the differential signals for LCP and RCP light are often very small and instrumental artefacts have been, and are still, problematic because they can be large compared with the signals of interest (124,133).
Magnetic CD measurements in the X-ray region (XMCD) involve excitation of core electrons to empty or partly filled valence orbitals. For example, the L-edge absorptions in transition metals involve excitation of the spin–orbit split 2p core electrons to the valence 3d or 4p levels. Interpretation of the MCD of these transitions requires experimental measurements that occur typically at energies of hundreds of electron volts (eV) to several thousand electron volts (keV). An interesting feature of XMCD is that it provides an element-specific spectrum that is a measure of orbital and spin angular momentum and the related magnetic properties which result from such momentum. Hence, XMCD has largely involved solid-state samples, often at low temperature (124,125).
Fluorescence detected circular dichroism
Fluorescence detected circular dichroism combines the structural sensitivity and chiral specificity of CD with the sensitivity and specificity of fluorescence (134,135). It consists in measuring the differential emission of light from a sample excited with LCP (left-circularly polarized) and RCP (right-circularly polarized) radiations, and is based on the assumption that the amount of light emitted depends exclusively on the amount absorbed; in other words, the excitation spectrum of the fluorophore parallels the absorption spectrum, and the molecular quantum yield is the same for both circularly polarized components (136). FDCD is then an indirect way of measuring a differential absorption (134,135). It is sensitive to both chiral and fluorescent molecules, and therefore is far more specific than standard transmission CD, because in principle the CD associated with a single fluorophoric molecule or moiety may be selectivity extracted in the presence of many non-fluorescent chromophores. Moreover, similar to the higher sensitivity of fluorescence compared with absorption spectroscopy, the direct measurement of emitted radiation against a zero background renders FDCD more sensitive than conventional CD (137).
In FDCD, the difference in fluorescence intensity for left and RCP excitation is measured. In practice, the technique is very selective, because only fluorophores are detected (even in a multichromophoric molecule). Additionally, exciton coupled FDCD seems a very promising approach, since selectivity and sensitivity are highly enhanced (138,139).
Fluorescence detected circular dichroism helps in separating contributions from multi-component systems, and the quenching detection of chiral molecules also through multidimensional FDCD experiments (140). It could also be employed in numerous examples of mixtures of a fluorophoric probe and a chromophoric species, (i.e., a fluorescent dye associated with a biopolymer), or of a fluorophore inserted into a non-fluorescent chiral biomolecules such as proteins or nucleic acids (140). Fluorescence detected circular dichroism may be applied as a detection tool in chromatography, electrophoresis, and determination of enantiomeric excesses, whereas only a few other reports have provided data regarding FDCD sensitivity enhancement (139,140).
Using a fluorescence cuvette and placing at 90° a second photomultiplier tube with transfer optics, it is possible to collect CD and fluorescence from the sample, simultaneously. Emission signal should be filtered by a long-pass filter, or, alternatively, by an emission monochromator. In fact, the approach is getting today rather popular, mainly in applications requiring long thermal melting experiments to monitor conformational changes, since double information can be obtained, with limited hardware investment (140).
Applications of circular dichroism in the near-infrared region (NIR) are quite rare, but not uncommon. Many metals linked with proteins and several chiral metal complexes may give CD active bands in the NIR region (NIR is starting from 700 nm up) (141,142). Conventional CD spectroscopies feature double prism monochromators for best efficiency in the far-UV (<250 nm). Double prism monochromators have very low dispersion toward the NIR field, so the actual limit of standard units are around 1100 nm. This wavelength is also the practical NIR limit of red extended (S1) photomultiplier tubes and of Si diodes (141,142). The capability of NIR-CD has been employed by Eglinton et al. to show that both the oxidized and reduced states of cytochrome c oxidase (as a metalloprotein) contain electronic states in the NIR region. The spectra have revealed all of the new bands could be assigned with reasonable confidence to one or more of the metal centres in the protein. Consequently, it was clear this spectral region allows observation of the two hems α and α3 separately (143).
To approach the NIR further a different hardware is therefore necessary. Jasco’s J-730 was designed for this purpose (144,145), it features a filament source (halogen), a plane grating single monochromator, a linear polarizer, a conventional PEM and a liquid nitrogen cooled in Sb detector. But the most important difference is that a further light modulator (a chopper) must be inserted in the beam, since typical IR detectors would not operate with CD signals (141,142).
Vibrational circular dichroism
Vibrational circular dichroism (VCD) is a spectroscopic technique, which uses circularly polarized light to provide information about a substance (146,147). An interesting feature of many substances is that they respond differently to incident light having different polarization, which they may absorb, reflect, and or transmit different amounts of differently polarized light. VCD technique is generally directed to determining the difference in absorption that a substance exhibits between right and LCP light (148). VCD measurements are particularly useful in the field of stereochemistry, i.e., the study of the shapes of molecules and the spatial arrangement of atoms therein (149). More particularly, VCD analyses are useful in the study of substances which contain chiral molecules (molecules having structures which cannot be superimposed on their mirror images; The concept of chirality is illustrated by a person’s right hand, which can be said to be chiral: it is a mirror image of their left hand, but the hands cannot be superimposed no matter how one orients them relative to each other) (150,151). For example, many substances, particularly biological substances, contains chiral molecules of opposite senses-that is, the molecules are mirror images of each other, in which case they are known as enantiomers or optical isomers. Each of the enantiomers may have different properties, in particular, different biological response (e.g. sugars are chiral molecules, and the human body can digest and use ‘right-handed’ sugars, but not their left-handed counterparts) (152). Since VCD spectral bands of enantiomers have opposite sign, VCD spectroscopy can allow one to differentiate between enantiomers, a result which is extremely useful in pharmaceutical and chemical fields. Similarly, one can determine how much of one enantiomer is present with respect to its twin, by looking at the spectrum of the mixture of enantiomers and comparing it to one of the pure enantiomers (since the difference will reflect how much the spectrum of one enantiomer attenuates the other) (148,149).
One of VCD-based technique has been improved using IR spectroscopy is FTIR (Fourier transform infrared)-VCD (146). As it was indicated, infrared absorption spectroscopy (IR) is the principal tool available for the study of molecular vibrations (147). Vibrational circular dichroism (VCD) is the differential absorption of left and RCP infrared light by vibrating molecules. The two techniques were combined to create the spectroscopic measurement technique that will be described here, FTIR-VCD (153). FTIR-VCD is used to study the subtle differences in vibrational spectra that result from molecules that differ only in their three dimensional geometry. The technique has been shown to be particularly useful for the study of the conformational characteristics of biological macromolecules such as proteins and nucleic acids and smaller molecules like chiral pharmaceuticals (153).
In summary, the technique can be used in conjunction with calculations (i.e., density functional theory (DFT) calculations) to determine the absolute configuration (AC) of newly synthesized molecules, and can be used to determine enantiomeric purity in molecules whose AC is already known (151). However, if the molecules are enormously large, DFT calculations are impractical. Consequently, biomolecules (e.g. proteins and nucleic acids) cannot be studied. If the molecule is enormously flexible, and the number of populated conformations is enormously large, the prediction of its VCD spectrum becomes very time-consuming and less reliable. Therefore, VCD is not applicable to the determination of ACs. VCD is a practical technique for the majority of medium-sized organic molecules (151).
HPLC- circular dichroism
Presently, pharmaceutical, food and biotechnology companies are facing more and more problems producing enantiomerically pure products. The companies have to verify and certify the purity of their products to meet the regulations that are getting more and more restrictive every day. A large investment is necessary for companies to build commercial-scale production processes for optically pure pharmaceuticals and intermediates. These processes are generally based on various methods such as catalytic asymmetric synthesis, biocatalytic resolution, diastereomeric crystallization, enantioselective absorption or combinations of these methods (154). These processes must include facilities to verify and certify the optical purity of the intermediate and end products or of their metabolites at the laboratory level. For three last decades, HPLC has been the most preferred separation method in these industries (155). Chiral HPLC, both for analytical and preparative scale, is probably the easiest answer to these requests (156). Chiral HPLC separations have been booming through continues development and a growing number of new chiral stationary phases, by the use of chiral modifiers in the mobile phase or by precolumn derivatization with chiral reagents. As well-known enantiomers have the same chemical and physical properties and the only way to discriminate and quantify is their interactions with polarized light, so chiroptical HPLC detectors are the logical complement to any chiral separation (157,158). The tandem combination of chiral and mass-sensitive detectors is very valuable when chromatographic separation can only be partially achieved. This is probably the main appeal of modern chiral HPLC detectors: the possibility to reliably quantify enantiomeric purity without separation is indeed a dramatic advantage. So while these detectors may well be used with chiral columns to verify elution order or to measure (in circular dichroism) the spectra of the compounds, the main interest is to quantify optical purity when using non-chiral chromatographic conditions (159). The capabilities have been applied in several studies (160,161). For instance, Gergely and colleagues have reported the application of HPLC-CD instrument could provide the selective detection of chiral compounds, and subsequently it allowed the employment of high detection wavelengths, presenting more selective detection with minimal background noise (161).
Many important progresses have been achieved in the last few years and various chiral detectors are now commercially available. Jasco has pioneered this field and is today offering chiral detectors based on circular dichroism (e.g. Jasco model OR-2090 Chiral detector; http://www.jasco.co.uk/chiral.asp), which has the unique advantage to provide two different signals (CD and UV) simultaneously, the first proportional to the optical activity and the second related to the mass or amount of the sample compound (162). In addition to dedicated detectors also a conventional CD, equipped with a proper flow cell, can be profitably used for the application, providing chiral and mass information, simultaneously (162).
Stopped-flow circular dichroism
Combination of CD spectroscopies with high mixing speed stopped-flow devices is a well-known technique for fast kinetic measurements (12,163,164). Apart from obvious applications in the organic chemistry field, much interest is now going toward protein folding analyses. For example, Clarcke et al. have shown that stopped-flow synchrotron CD can give outstanding information on the kinetics of secondary structure formation. They demonstrated α-helix initiation in peptides is at least 105 times slower than expected and long α-helices can fold with an overshoot in helix content (165).
These measurements may be tasking because of multiple shots accumulation may be necessary to extract a valuable result (166). In addition to CD, an instrument equipped with a suitable stopped-flow cell may become the proper optical bench to collect simultaneously absorption and fluorescence data (167).
Proper integration between cell and spectrometer is the first prerequisite, but it is also essential to rely on suitable software to control at the same time both the units. More advanced stopped-flow cells feature today syringes (2, 3 or 4) controlled by stepping-motors, and the possibility to exchange easily the observation cell for different experiments. However, main application of stopped-flow CD is protein folding (166,167).
Synchrotron radiation circular dichroism
Synchrotron radiation circular dichroism is an emerging technique which offers significant improvements to the well-established method of conventional circular dichroism spectroscopy (168). Particularly, developments in instrumentation of CD using vacuum-ultraviolet synchrotron radiation circular dichroism (VUV-SRCD) have made CD a potentially powerful tool in proteomics (169). Using VUV-SRCD, the spectra can be taken at lower range of wavelengths relative to that of conventional CD-spectroscopies. These types of measurements allow us to elucidate finer details of secondary structures of proteins and determine all the predicted folds and motifs in protein secondary structures (170). As a consequence, it takes advantage of the high light flux available from synchrotron sources over a wide range of wavelengths, which results in higher signal-to-noise ratios and enables the collection of lower wavelength data than possible using xenon arc lamps that are typically the illumination source in conventional CDs (171). Therefore considerably smaller amounts of protein are required to obtain a spectrum of comparable quality, and smaller differences can be distinguished (172).
Additionally, averaging times, defined as the times taken at each wavelength to acquire a suitable signal, can be greatly reduced, thereby speeding up the process of obtaining a spectrum. This is advantageous not only for increasing the rate of data collection (useful for structural genomics projects which seek to examine a large number of proteins), but ultimately also for increasing the time resolution of stopped-flow experiments (165).
With SRCD it is possible to collect data <205 nm in almost any solvent and buffer commonly used in biological studies. Thus, this method should provide especially good improvements for assessing secondary structure in protein folding and unfolding studies. Although the technological advancements enabling these measurements were first demonstrated in two last decades (173), growth of applications in structural and functional genomics has been performed more recently (172). For example, applications of SRCD in secondary structure analyses (169), monitoring protein folding and kinetics (165), and drug investigation (169) have been introduced (174).
The applications of SRCD will mainly be divided to investigations of membrane proteins and in the pharmaceutical industry. Integral membrane proteins constitute an important class of proteins that is greatly under-represented in the databases of crystal structures (175). It has been estimated that about one-third of the proteins in the human genome may be membrane-associated, and at present nearly 60% of all drug targets are against membrane proteins. To date, CD studies of membrane proteins have been limited in the accuracy of their secondary structure determinations because none of the reference databases currently available include any membrane proteins, even though it is obvious that such proteins have different spectral characteristics from their soluble ones (176–178). Particularly, with its increased sensitivity and the potential for automation of data collection, SRCD could provide a regular assay for monitoring drug–target interactions (179). Additionally, SRCD is currently being used as a method for examining integrity of protein folding, and may assume an even greater role as more proteins are expressed in high yields as inclusion bodies, requiring refolding to regain their native structures. Indeed, SRCD could find a use as a standard reference method for assays of fidelity of folded products (175).
In summary, SRCD extends the measurements possible with conventional CD instruments, providing much higher information content and the ability to examine biological samples under a richer variety of conditions. On the other hand, the availability of the three-dimensional crystal structure of a protein causally associated with a disease is very valuable in understanding the molecular bases of the disease and in aiding the process of rational drug design for development of pharmaceuticals to treat the disease (180). Knowledge of how the native protein differs from the mutant protein is especially important for this process, although it is not always possible to obtain structures for the mutant proteins, perhaps due to differences in solubility or stability or other characteristics necessary for producing crystals. In this case, SRCD can be used to compare the native and mutant proteins in solution, and in combination with the native protein crystal structure and other biophysics and bioinformatics studies, provide information on the changes associated with the mutant that produces the diseased state (181).