Advances of Vibrational Circular Dichroism (VCD) in bioanalytical chemistry. A review

Abstract

Vibrational Circular Dichroism (VCD) is a unique and relatively new spectroscopic technique that is capable of determining an absolute configuration of chiral molecules. VCD can be also used to determine structure of large macromolecules. This review highlights the most recent advances of VCD in bioanalytical chemistry. It shows that VCD is capable of unraveling supramolecular organization of peptides, proteins, saccharides, glycerophospholipids, polypeptide microcrystals, as well as amyloid fibrils and DNA. This review also demonstrates how VCD can be utilized to explore molecule-molecule interactions that determine mechanisms of chiral separations in chromatography. It aims to attract attention of scientists from all different research areas demonstrating the strength and capability of this very powerful spectroscopic technique.

Graphical Abstract

1. Introduction

Chirality is one of the most interesting and fundamental properties of various objects in our universe ranging from molecules through living organisms to galaxies. There are two levels of molecular chiral essence: absolute configuration and supramolecular chirality [1]. A phenomenon of absolute configuration was first discovered in 1848 by French chemist Louis Pasteur. He observed that two different types of crystals were formed by crystallization of sodium ammonium salt of tartaric acid. Pasteur separated these morphologically different crystals with the help of tweezers and dissolved them in aqueous solvents. It has been found that solutions of the morphologically different crystals rotated the plane of polarized light in the opposite directions. Pasteur concluded that the rotation of polarized light caused by the solutions of different tartaric acid crystals was due to chiral property of molecules that were forming them [2]. These molecules lack a plane of symmetry and share the same connectivity but differ in their spatial arrangement. Such structurally identical but spatially distinct molecules are named enantiomers (one chiral center) or diastereomers (two chiral centers). Like a pair of hands, two enantiomers of a chiral compound are mirror images of each other that cannot be superimposed. The enantiomers often have substantially different physiological response. For example, one of enantiomers of limonene ((+)-limonene) has a smell of oranges, while another one ((−)-limonene) possesses a lemon like aroma [2].

Chirality of macroscopic objects, such as shells, can be easily visualized and characterized in the terms of handedness. Determination of a chiral organization of microscopic objects is often a challenging task. Microscopy, including bright-field and electron microscopy, is one of the most commonly used tools for the direct morphological characterization microscopic objects [3-6]. It is commonly used to investigate supramolecular chirality of microorganisms, protein aggregates and polymers. However, microscopy is often not capable of probing deeper levels of chiral organization of these specimens [7,8]. This limitation can be overcome by several chiroptical techniques, such as optical rotation (OR), electronic circular dichroism (ECD) and vibrational optical activity (VOA).

VOA is a spectroscopic determination of the differential response of a chiral molecule or a chiral supramolecular aggregate to left versus right circularly polarized light during a vibrational transition [9,10]. VOA includes infrared vibrational circular dichroism (VCD) and vibrational Raman optical activity (ROA). In the current review, only VCD will be discussed.

VCD spectra of small chiral molecules are equal in intensity but opposite in sign (mirror images of each other), while their infra red (IR) spectra appear the same. The VCD spectrum of the chiral molecule can be calculated using an ab initio density functional theory (DFT) method [11-16]. This makes VCD highly desirable technique for a determination of the absolute configuration of chiral molecules, elucidation of an influence of chiral molecules on surrounding solvents, and unraveling interactions between a simple chiral molecule with organized assembly like surfactant micelles [17,18]. Additionally, VCD can be used to monitor reactions, such as cyclobutane-1,2-d₂ thermolysis, that involve chiral compounds [19]. VCD is also capable of probing supramolecular chirality of macromolecules, such as collagen, amyloid fibrils and DNA [7,20-24], as well as unraveling supramolecular organization of self-assembled monolayer (SAM) films [25]. Finally, VCD can be used to unravel mechanisms of interactions of chiral molecules with polymers that are used as stationary phases in chromatography. These and many other applications of VCD will be discussed in the current review.

2. VCD instrumentation

A typical VCD spectrometer consists of three major components: an IR source, a photoelastic modulator (PEM) and a MCT camera. Electromagnetic radiation from the IR source is directed to an interferometer, which assigns a Fourier frequency to each point of the electromagnetic spectrum, and a polarizer to obtain a linearly polarized IR light. Next, the IR beam propagates to a PEM equipped by a zinc-selenide crystal (Zn_nSe). PEM sine-wave modulates the polarization between left- and right-circularly polarized states in the frequency range of tens of kilohertz. Both left- and right-circularly polarized light then passes through the sample, whereas the transmitted electromagnetic radiation is collected by a mercury cadmium telluride (MCT) camera (Fig. 1). On MCT chip, IR beam intensity is converted to an electrical signal that is processed by the subsequent electronics. Installation of the second PEM right after the sample cell drastically reduces possible birefringence artifacts in the acquired VCD spectra [26].

Fig. 1.

Schematic illustration of a Dual-PEM VCD spectrometer.

A sample cell is typically made of calcium or barium fluoride (CaF₂ or BaF₂), since these materials have very low absorption in the IR part of the spectrum. Both solids (films) and liquid samples are suitable for VCD measurements. It should be mentioned that solid films should be rotated during the spectral acquisition to minimize possible spectral artifacts, such as linear CD, arising from the polarizable crystallinities. Spectral acquisition time directly depends on the concentration of the analyzed material and may range from several minutes to hours.

Since VCD is an absorption-based technique, samples have to be transparent or translucent for the IR light. This typically limits an application of VCD for studies of opaque samples. Rüther et al. demonstrated that utilization of a quantum cascade laser (QCL) as a light source, allowed to measure VCD spectra of highly absorbing samples. Using QCL-VCD setup Rüther et al. showed that VCD could be utilized to monitor a hydrolysis of a chiral complex nickel-(−)-sparteine chloride to free (−)-sparteine base in a biphasic system of sodium hydroxide solution and chloroform [27].

3. Analysis of protein and peptide structure

A typical VCD spectrum of a protein specimen is composed of contributions from two major types of vibrational modes that originate from the polypeptide backbone (amide bands) [7,28,29]. Amide modes include the amide I vibration (1600-1700 cm⁻¹), which primary represents C=O stretching and a small amount of out-of-phase C-N stretching, amide II vibration (~1550 cm⁻¹) that consists of out-of-phase combination of C-N stretching and N-H bending motions [30,31]. From the vibrational bands of amide chromophore, amide I band is the most commonly used to interpret structural organization and changes in the protein secondary structure. In early 90s, Keiderling group demonstrated that different protein secondary structures, such as α-helix and β-sheet, had distinct characteristic signatures of the amide I band (Fig. 2) [32-34].

Fig. 2.

VCD (top) and IR absorption (bottom) spectra in the amide I region for poly-L-lysine that is mostly α-helical, left (D2O at pH ~11), β-sheet, center (after heating to 65 °C for ~20 min followed by cooling), and PPII-like, right (at neutral pH). For comparison, all spectra were normalized to A = 1 for the amide I peak absorbance.

For instance, it has been shown that β-sheet exhibited two distinct minima at ~1610 and 1680 cm⁻¹, whereas α-helix showed a positive-negative (±) couplet at 1640/1660 cm⁻¹. Keiderling group has also found 3₁₀-helix and α-helix had different VCD spectra [32]. Using VCD, Dukor and co-workers discovered that poly-l-glutamic acid (Glu_n) did not have a random conformation, as it was expected. It rather consisted of short left-handed helical regions, at least 3–4 residues long, similar in conformation to the local structure of the 3₁₀-helix of poly-l-proline (Pro_n) [35].

It should be noted that a direct application of VCD for the structural characterization of proteins is a challenging task [36-38]. Most of proteins contain nearly all different types of secondary structure, including α-helix, β-sheet turns, reverse turns, β turns, β bends, hairpin bends, 3₁₀ bends, kinks, and widgets. At the same time, spectroscopic signatures of many of these secondary structure elements remain poorly understood. Also, not entire protein sequence is solvated, like in the case of peptides, while some parts of the sequence are located in hydrophobic pockets. Consequently, solvation effects of different types of secondary structure have to be taken into consideration. VCD does not have atomic resolution, which is often necessary for determination of the structural organization of proteins [39-43]. It also requires a high concentration of peptide or protein for the analysis (usually around 50–100 mg/ml). Nevertheless, VCD is much less labor-consuming than NMR or X-Ray, classical tools of structural biology that are capable of providing atomic resolution of protein molecules. Also, VCD probes solvated state of proteins that may be substantially different from their crystal structures [30,31]. For example, using VCD it has been found that bovine apo-α-lactol-bumin and lysozyme had substantially different secondary and tertiary structures in solution, while their crystals structures were found to be nearly identical [44].

It should also be mentioned that electronic CD (ECD) is a good alternative to VCD in determination of a protein secondary structure. ECD typically requires much less protein concentration for the analysis and is commonly used for determination of basic secondary structural features such as α-helix, β-sheet and disordered protein structure.

4. Unraveling supramolecular organization of protein aggregates and amyloid fibrils

Amyloid fibrils are β-sheet rich protein aggregates that have been detected upon post mortem microscopic examination of brain tissues of patients who were diagnosed with neurodegenerative diseases [45,46]. These fibrils exhibited different morphologies, a phenomenon known as fibril polymorphism [6]. At the same time, morphologically different fibrils have very similar secondary structure, known as cross-β-sheet [47]. From the perspective of their morphology amyloid fibrils can be twisted and flat [7,48-51]. Twisted fibrils are built by several filaments that coiled along their longitude axis. At the same time, flat tape-like fibrils are composed of several side-by-side associated filaments [52,53]. One can imagine that such morphological and structural differences may have different toxicity [54,55]. Consequently, a clear understanding of fibril supramolecular organization may help to unravel the origin of fibril toxicity.

One could expect that such twisted fibrils would have strong VCD due to long-range coupling of carbonyl groups in their cross-β-sheet. In 1997, Ma et al. reported that VCD signal obtained from insulin and lysozyme fibrils was indeed many times stronger than the VCD signal of these proteins. Whereas insulin and lysozyme exhibited different VCD spectra, their fibrils had very similar VCD with bands at 1554, 1593, 1627, 1647, 1670 cm⁻¹ (sign pattern + + − + +) [22].

In 2010 Kurouski et al. discovered that supramolecular chirality of insulin fibrils could be controlled by pH [20]. It has been demonstrated that insulin fibrils grown at pH above 2 had a left-handed twist. These left-twisted fibrils exhibited a distinct VCD signal with + + − + + sign patter that was named ‘normal VCD’. At the same time, insulin fibril that were grown at pH below 2 showed nearly mirror-image VCD spectrum with − − + − − sign pattern (named ‘reversed VCD’) [24]. However, microscopic examination of reversed VCD fibrils did not reveal any right-handed twist on their surface. On the opposite, these fibrils were found to have flat tape-like morphology. Therefore, it has been proposed that these tape-like fibrils were composed of right-twisted filaments. Using VCD, AFM and SEM, Kurouski and co-workers followed insulin aggregation aiming to determine how fibril polymorphs with the opposite supramolecular chirality were formed. It has been found that initially, partially-denatured insulin aggregated into a thin twisted filament. If the filament adopted a left-handed twist, it was able to braid and coil forming left-twisted fibrils. In comparison, if the filament had a right-handed twist it could only associate side-by-side with other right-twisted filaments forming tape-like fibrils [24]. Based on this observation Kurouski and co-workers concluded that supramolecular chirality of the filament could be the underlying cause of the major morphology differences in all amyloid (twisted versus flat) fibrils.

Recently, Kurouski and co-workers demonstrated that pH controlled fibril polymorphism was very likely to be a general phenomenon [7]. It has been shown that pH directly controls formation of fibril polymorphs with opposite supramolecular chirality of lysozyme, apo-α-lactalbumin, HET-s (218–289) prion, and a short polypeptide fragment of transthyretin, TTR (105–115). Similar to insulin, left-twisted fibril polymorphs exhibited normal VCD, whereas fibrils with reversed VCD signal had tape-like topology (Fig. 3). It has been also found that insulin and lysozyme formed tape-like fibrils that exhibited reversed VCD at pH below 2, whereas twisted fibrils were grown at pH above this point. HET-s (218–289) prion and TTR (105–115), on the opposite, formed twisted fibrils that showed normal VCD at low pH (below 2) and tape-like fibrils at high pH. Authors did not reveal any correlation of the polypeptide amino acid sequence pI and tendency to form a particular fibril polymorph at high or low pH. Kurouski and co-workers also demonstrated that ionic strength has no impact on pH driven fibril polymorphism.

Fig. 3.

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 °C. Morphology of lysozyme fibrils (C–K) grown at pH 1.5 (C–E), pH 2.3 (F–H), and pH 2.7 (I–K) for 3 days at 65C. SEM (C, F, I) and AFM (D, E, G, H, J, K) images. Twists of fibrils (all left handed) are indicated by red arrows [7].

In addition to the full-length proteins and short peptides, Nieto-Ortega et al. also observed pH controlled fibrils polymorphism for aggregates formed from l-valine peptidomimetic compounds [56]. It has been reported that at different pH a compound with the molecular structure shown in Fig. 4A aggregated forming morphologically different fibrils. VCD revealed that these fibril polymorphs had opposite supramolecular chirality (Fig. 4). No clear evidence of the origin of fibril supramolecular chirality was evident from the obtained AFM and SEM images. This suggests that chiral essence of these fibrils lay below the detection limit of these microscopic techniques.

Fig. 4.

Molecular structure of the l-Valine peptidomimetic compound (A). VCD spectra (B), images (C) of the compound aggregates at pD 6.0 (left), 2.3 (middle) and 2.0 (right) obtained by SEM and AFM. From Nieto-Ortega et al. [56].

Aparicio et al. investigated self-assembly of oligo-p-phenylene-based organogelators using VCD, AFM and SEM [57]. It has been found that molecular structure of the investigated organogelators directly determined the aggregation propensity. Aparicio et al. discovered that aggregation of one of the investigated oligo-p-phenylene-based organogelators (OPPO) was highly sensitive to the initial conditions, which defined either a kinetic or thermodynamic pathway to produce helical structures of opposite handedness. It has been shown that aggregation of one of the OPPO at 3 × 10⁻³ M and 1.2 × 10⁻² led to the formation of different polymorphs with opposite supramolecular chirality. It has been proposed that structurally different nuclei were formed at low and high concentrations of the analyzed compound, which determined supramolecular organization of mature fibril aggregates. Aparicio and co-workers also found that aggregation of the same OPPO at 25 °C (8 × 10⁻³ M) produced aggregates that exhibited amide I with (±) pattern. However, if the sample was kelp at this temperature for 24 h, the inversion of the amide I band pattern was observed.

Kurouski et al. also investigated aggregation of polyglutamine (PolyQ) aggregates [58]. PolyQ deposits are a hallmark of Huntington disease. Specifically, expanded CAG-repeat diseases in which inheritance of an expanded polyQ sequence above a pathological threshold is associated with a high risk of disease. Application of VCD revealed that these PolyQ fibril aggregates exhibited a chiral supramolecular organization that was distinct from the supramolecular organization of all previously observed amyloid fibrils (Fig. 5). Moreover, PolyQ fibrils grown from monomers with Q repeats 35 and above (Q≥35) exhibited approximately 10-fold enhancement of the same VCD spectrum compared to the already enhanced VCD of fibrils formed from Q repeats 30 and below (Q ≤ 30). Using deep UV resonance Raman (DUVRR) spectroscopy coupled with hydrogen-deuterium (H/D) exchange, Kurouski and co-workers also demonstrated that the structure of Q≥35 PolyQ aggregates was much less compact comparing to the structure of the Q ≤ 30 fibrils.

Fig. 5.

VCD (A) and IR (B) spectra of Q 41 aggregates (blue), Q 26 aggregates (red), Q 25 aggregates (green) and Q 18 aggregates (black) and their morphologies (C) obtained using TEM [58].

This series of work demonstrated unique sensitivity of VCD to supramolecular organization of protein and peptide aggregates. It should be noted that due to the aggregation state of fibrils, which are insoluble and cannot be crystallized, such information may not be elucidated using NMR or X-Ray. Moreover, in many cases supramolecular chirality cannot be accessed using most advance microscopic tools, such as fluid-cell AFM or cryo-SEM.

A growing body of literature indicated that VCD has become a useful tool for the chiral characterization of amyloid aggregates. For example, Measey and Schweitzer-Stenner reported a large enhancement of VCD upon aggregation of short polypeptides [59]. They also demonstrated that mature fibrils formed from the N-terminal peptide fragment of the yeast prion protein, Sup35, and the amyloidogenic alanine-rich peptide AKY8 have opposite signed VCD. Fulara et al. demonstrated that opposite signed VCD spectra could be obtained for mature fibrils formed from poly- L or -D glutamic acid [60]. Polyglutamic acid forms spirally twisted aggregates with handedness determined by the amino acid chirality (left-handed for L and right-handed for D).

Using VCD, Kurouski et al. discovered that polypeptide microcrystals also exhibited supramolecular chirality similar to the one that was previously reported for amyloid fibrils. Authors pointed out that such a small twist of polypeptide units in the crystals is invisible for X-ray and could be observed only by VCD [8]. This work also demonstrated that supramolecular chirality and consequently arrangement of polypeptide monomers could be different in crystal and fibril form. It also demonstrated that VCD can be broadly used to elucidate supramolecular chirality of polypeptide microcrystals.

5. Elucidation of a structural organization of saccharides and glycerophospholipids

Sugars play a very important role in a cell metabolism. Monosaccharides, such as glucose and fructose, have direct physiological activity, whereas oligosaccharides determine antigen recognition in the immune system. More complex carbohydrate polymers define pathogenicity of microorganisms, serve as energy storage (glucagon) and build walls of plant cells (cellulose). Sugars often found as conjugates in proteins and lipids, modifying physiological properties of these molecules. Sugars possess enantiopurity and present exclusively in the D-form in living organisms. NMR is the commonly used tool for determination of the absolute configuration of sugars [61]. Tanigushi et al. demonstrated that VCD could unravel stereochemical organization of sugar anomers as well. It has been found that ⁴C₁ axial glycosides exhibited a negative, while ¹C₄ exhibited a positive band at around 1230 cm⁻¹. Interestingly, equatorial glycosides had no VCD band at ~1230 cm⁻¹ [62]. VCD was also extensively used to reveal supramolecular chemistry of carbohydrate polymers (will be discussed in the following sub-section), which commonly used as chiral stationary phases (CSPs) in chromatography.

Using VCD, Monde's group explored chiral organization of glycerophospholipids (GPLs) that had bacterial, eukaryotic, and mitochondrial origin. GPLs are the most abundant lipids in all mammalian membranes. They are composed of a polar phosphoester headgroup, a prochiral glycerol, and acylchains that typically range from C₁₄ to C₂₀ with various levels of unsaturation. Tanigushi et al. shown that supramolecular chirality of GPLs isolated from bacteria, eukaryotes, and mitochondria could be identified by the sign of a VCD exciton couplet. Specifically, bacterial and eukaryotic sn-3 GPLs showed a positive–negative couplet in the carbonyl stretching region (~1750 cm⁻¹). Archaeal GPLs and mammalian bis(monoacylglycero)phosphates (BMPs) that had sn-1 configuration, on the opposite, exhibited a negative–positive couplet in that spectral region (Fig. 6) [63].

Fig. 6.

(A) VCD and IR spectra of phosphatidylcholines. Each spectrum was measured in CDCl₃ at a concentration of 0.1 M (sn-3-PC-C8a and sn-3-PC-C8b) or 0.08 M (sn-3-PC-C8 and sn-1-PC-C8). (B) Schematic orientation of the two carbonyl groups of sn-3-PC (left) and the relationship between the arrangement of the electric transition moments (red arrows parallel to the C=O bonds) and the sign of the VCD couplet (right). The carbonyl orientation is depicted on the basis of the predicted stable conformers of sn-3-PCC4, where the C=O group at C1 prefers a syn orientation with regard to C1–HS. From Taniguchi et al. [63].

6. VCD in chromatographic chiral separations

Chiral purity of drug substances is a major concern in the modern pharmaceutical industry. In 1960s, n-phthalyl-glutamic acid imide was marketed as sedative Thalidomide. Its therapeutic activity resided exclusively in the R-enantiomer, while its enantiomer, S-enantiomer had teratogenic effect. Since the drug substrate was not enantiomerically pure, Thalidomide caused several hundred births of malformed infants [2]. After this horrifying incident, pharmaceutical companies enforced control of chiral purity of developed drugs. Since most of small molecule drugs are chiral, control of a drug chiral purity becomes vitally important [64,65].

Current methods of chiral analysis include such non-chromatographic techniques as polarimetry, NMR, isotopic dilution, calorimetry, and enzyme techniques [66,67]. Nevertheless, the vast majority of chiral analyses, which includes enantiomeric and diasteriomeric purity, are done by gas chromatography (GC), supercritical fluid chromatography (SFC) and HPLC [68]. If diasteriomers can be separated on achiral columns (such as C8 or C18 (HPLC and SFC) and HP-5 (GC)), separation of enantiomers requires utilization of chiral stationary phases (CSPs) [69,70]. CSPs that are based on cyclodextrins, macrocyclic glycopeptides, as well as the cellulose and amylose are commonly used in chiral separations [71-73]. It has been proposed that non-chiral interactions, such as hydrogen bonding in normal phase HPLC and π-π interactions. Consequently, CSPs are typically modified by electron rich or electron poor aromatic systems, such as tris-(3,5-dimethylphenylcarbamate, to enhance their selectivity to a specific molecule. In reversed phase HPLC, inclusion complexation rather than interactions described above, determine the mechanisms of chiral separations [68,74].

Ma and co-workers used VCD to investigate mechanisms of chiral separations on cellulose and amylose tris-(3,5-dimethylphenylcarbamate (CDMPC and ADMPC) [75]. First, supramolecular chirality of these CSPs was characterized. It appeared that VCD spectra of CDMPC and ADMPC exhibited significant differences (Fig. 7). The VCD spectrum of ADMPC polymer had a −/+ couplet in the amide I (1701 cm⁻¹) band, whereas CDMPC exhibited a converse (±) couplet in this spectral region. Based on these spectral differences, as well as on the opposite signs of the VCD band that was assigned to phenylalanine (1612 cm⁻¹), Ma and co-workers concluded that CDMPC and ADMPC had opposite handedness of the backbone structure.

Fig. 7.

VCD and IR spectra of the ADMPC polymer film (black curve) and the CDMPC polymer film (red curve) (noise level is offset for clarity). From Ma et al. [75].

Next, Ma and co-workers utilized VCD to investigate how opposite enantiomers interacted with CDMPC [76]. It has been observed that VCD spectrum of a complex of CDMPC-R-enantiomer of 1,3-diphospha-allen had more intense bands at ~1720 and ~1693 cm⁻¹ (Fig. 8, A) comparing to CDMPC-S-enantiomer complex. Based on this observation, Ma and co-workers concluded that R-enantiomer had stronger interactions with CDMPC comparing to the S-enantiomer. It has been also found that a VCD peak corresponding to the glycosidic (C-O-C) region (~1060 cm⁻¹, Fig. 8, B) showed greater intensity and sharper peak shape for R-enantiomer compared to S-enantiomer. Based on this evidence, Ma and co-workers suggested that R-enantiomer would be included deeper in the cavities of the stationary phase comparing to the S-enantiomer (Fig. 8, C) [76].

Fig. 8.

High wavenumber (1760-1660 cm⁻¹) (A) and low wavenumber (1100-1000 cm⁻¹) (B) spectra region of the VCD spectra of R- and S-allene with CDMPC from Chiralcel OD-3 column (Chiral Technologies). Modelled interactions (C) between R- and S-allene with CDMPC. From Ma et al. [76].

Quinine and quinidine are also commonly used as stationary phases in chiral separations. In 2009, Lindner group showed that VCD could be used to shed light on the mechanism of interaction of (R) and (S)-3,5-dinitrobenzoylleucine (DNB-Leu) with these tert-butylcarbamoylquinine (t-BuCQN) and tert-butylcarbamoylquinidine (t-BuCQD) [77]. Authors observed significant changes in amides carbamate, and carboxyl groups upon interactions of DNB-Leu with quinine and quinidine (Fig. 9). Moreover, it has been shown that molecule-CSPs complex strongly depends on the nature of the solvent. Julinek et al. compared IR and VCD spectra of S- and R-DNB-Leu complexed with t-BuCQN in methanol and acetonitrile. It has been found that VCD bands for R-DNB-Leu-t-BuCQN complex in methanol is significantly lower and the spectrum exhibits broad peaks with intense noise which the authors could not assign. In contrast, VCD spectra of weakly bound complexes in acetonitrile exhibit sharp peaks with intensity comparable with those observed for strongly bound complexes, giving a hint on the effect of the solvents on the associative equilibria [77].

Fig. 9.

VCD (top) and IR absorption (bottom) spectra of weakly bound complexes of DNB-(R)-Leu + t-BuCQN (solid line) and DNB-(S)-Leu + t-BuCQD (dashed line) in (a) MeOH-d4 and (b) ACN-d3 [77].

Independently, Shen et al. investigated interactions of chiral aromatic amines with crown ethers-based CSPs (Fig. 10). These CSPs often used for chiral separations of compounds that contain primary and secondary amines [68].

Fig. 10.

VCD spectra of the complexes of the two enantiomers of 1-(4-bromophenyl)-ethylamine with (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid in DMSO solution. From Sherry et al. [78].

Shen and co-workers found that VCD spectrum of amine-ether complex (Fig. 10) exhibited two peaks around 1731 cm⁻¹ and 1670 cm⁻¹. The maximum at 1731 cm⁻¹ was assigned to the free carboxylic functional group of the crown ether. The maximum at 1669 cm⁻¹ was expected to be the carboxyl group either present in the form of an inter or intramolecular dimer or interact with the enantiomeric analytes. It has been found that the frequency of the VCD band of S-enantiomer-ether complex (1669 cm⁻¹) appeared to be red-shifted relative to the frequency of R-enantiomer-ether complex (1677 cm⁻¹). This indicated a stronger interactions between the S-enantiomer and crown ethers-based CSPs ((+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid), relative to the R-enantiomer [78].

Shen and co-workers demonstrated that VCD could be used to investigate changes in CSPs induced by organic solvents [79]. Specifically, Shen et al. used VCD to investigate conformational changes in crown ether stationary phases upon solvation by methanol and acetonitrile, the most commonly used solvents in chiral separations [78]. It has been found that as the concentration of methanol in the mobile phase increased, a VCD band around 1753 cm⁻¹ exhibited a red shift. It was concluded that such a shift was an indicative of possible hydrogen bonded dimerized carboxyl, as well as interactions between methanol and the crown ether tetracarboxylic acid. These interactions occurred at the expense of the analyte interaction with crown ether tetracarboxylic acid, leading to no change in the chromatographic enantioselectivity. The similar shift of the VCD band around 1753 cm⁻¹ was induced by acetonitrile. Comparing the two band shifts in this region (for methanol and acetonitrile), Shen et al. found that the band shift is larger in methanol compared to acetonitrile (17 vs. 10 wave-numbers, respectively). The larger shift in methanol could be attributed to the concerting interaction of the dimerized carboxyls, along with the H-bond interaction between the carboxylic groups of crown ether and methanol. Shen at al. concluded that such behavior correlated well with the chromatographic enantioselectivity for the two enantiomers. Thus, VCD can be used to predict conformational changes in crown ether stationary phase that would favor selection of one or another enantiomer.

This series of works demonstrated how VCD could be used to explore a nature of molecule-molecule interactions that determine mechanisms of chiral separations. It also showed how VCD could be used to determine supramolecular organization of CSPs. Detailed understanding of supramolecular chirality of CSPs, as well as elucidation of mechanisms that are responsible for chiral separations, are vitally important to improve selectivity of currently used chiral columns. Nowadays, invention of new CSPs is often achieved via a decoration of organic polymers, such as cellulose or amylose, with various chemical groups. If such new CSP allows for a separation of some enantiomers that could not been previously separated on any of the commercially available columns, it can be used to make a new chiral column. However, such a “random” search for new CSPs indicates that our understanding of mechanisms that lie behind chiral separations is quite incomplete [80-82].

At the same time, there are several ground breaking discoveries in a development of new CSPs. For instance, Armstrong group developed new brush-type (CSPs) that allowed for ultrafast “chiral” separations in the 4–40 s range. Patel et al., demonstrated that these CSPs were used in all mobile phase modes and with high flow rates and pressures to separate over 60 pairs of enantiomers [83]. Additionally, Chankvetadze group reported high efficiency of polysaccharide-based (CSP) in chiral separations of asymmetric sulfoxides [84].

VCD is a promising tool that is capable of shedding light on such mechanisms. However, one can envision that unambiguous band assignment in IR and VCD spectra of CSPs is extremely challenging. In addition, the reported VCD spectra are the net of VCD of complex (molecule-CSPs) minus VCD of CSPs. Such subtraction was usually not normalized on a particular reference band. Therefore, unambiguous interpretation of small differences in the intensity of bands in two VCD spectra might be tentative. Finally, observed spectra changes allowed to determine the interaction of both enantiomers with CSPs, whereas only limited information about the mechanisms of such interactions could be revealed.

7. Supramolecular organization of DNA and collagen

Deoxyribonucleic acid (DNA) is probably the most well-known and extensively characterized molecule in a cell. Primarily, because DNA is the carrier of genetic information used for the development, functioning and reproduction of all known living organisms and many viruses. DNA exists in several possible conformations including A-DNA, B-DNA, and Z-DNA forms. A- and B-forms of DNA are right-handed double helixes. The major difference between A-form and B-form nucleic acid is the conformation of the deoxyribose sugar ring. B-form has C2′ endo-conformation, whereas A-form exhibits C3′ endo-conformation. In Z-DNA, two strands coil in left-handed helices pronouncing a zig-zag pattern in the phosphodiester backbone [85].

In 1994, Keiderling group utilized VCD to probe supramolecular chiral organization of DNA. It has been found that B- and Z-DNA exhibited significant spectral differences in phosphate (PO₂) stretching region (~1100 cm⁻¹) [86], Fig. 11.

Fig. 11.

Comparison of VCD (top panel) and IR (bottom panel) spectra of B-form (dash line) and Z-form (solid line) in the PO₂ stretching region. From Wang et al. [86].

Wang et al. also investigated whether changes in the DNA sequence could be probed by VCD [86]. It has been found that vibrational bands in C=O stretching region of VCD spectra (1550-1750 cm⁻¹) could be used to elucidate the nucleobase composition of the DNA sequence, while little to no changes were observed in PO₂ stretching part of the spectrum (Fig. 12).

Fig. 12.

Sequence-dependent VCD and absorption of B-form DNA in C=O stretching region (A). (−−−): poly(dG-dC) poly(dG-dC); (→ → → →):m. lysodeikticus, 72% GC; (….): calf thymus, 44% GC; (−. −. −): c. perfringens, 26% GC; (solid line): poly(dA-dT)-poly(dA-dT). Corresponding VCD spectra (1–5) in PO₂ stretching region (B). From Wang et al. [86].

VCD was also used to elucidate changes in DNA that could be caused by transitional metals such as Cu²⁺ [87]. Andrushchenko et al. investigated the interaction of Cu²⁺ ions with DNA using VCD. It has been found that metal ions bind to phosphate groups causing significant distortion of most guanine-cytosine (GC) base pairs, while a minor effect on adenine-thymine (AT) base pairs was observed [87].

Shanmugam and Polavarapu recently utilized VCD to unravel temperature-induced conformational changes of collagen type I [23]. It has been found that thermal denaturation of collagen resulted in a biphasic transition from poly-l-proline II (PPII) to unordered structure. The PPII structure was assigned to collagen based on negative VCD couplet in the amide I region, while the formation of unordered structure was evident from the disappearance of VCD signal in the amide I region.

These works point out the unique sensitivity of VCD to the supramolecular organization of biopolymers. These studies demonstrate that VCD can be used to probe conformational change in DNA, as well as the changes in the nucleotide sequence and collagen structure.

8. Other applications of VCD

An interesting application of VCD was recently reported by Merten et al. It has been shown that VCD could be used to monitor transmission of stereochemical information from a chiral phosphate anion to a flexible manganese (III)–salen cation [88]. It was demonstrated how a chiral anion forces its accompanying catalytically relevant cation into an enantiomeric conformation. Merten et al. could also prove the origin of the enantio control of the catalyst system and correlate the degree of stereochemical transmission with the experimentally observed enantio selectivities. This study demonstrated that VCD could be utilized for unraveling the underlying induction mechanisms and experimentally achieved enantiomeric excesses obtained with chiral catalysts. Merten et al. pointed out that such determination of the catalytic mechanism could be possible if the intermediate reaction complex, in which the chirality transfer occurs, is stable without addition of a second reactant [88]. Otherwise, the reaction between the two will take place and the complex would change during the spectral acquisition. Also, the component of the system that becomes chiral ideally should possess a functional group and an associated vibrational mode that can be easily separated from modes of the chiral auxiliary/catalyst.

9. Conclusions and future directions

Over the last 40 years since the discovery and experimental confirmation, VCD emerged into the powerful analytic technique. This review highlighted the most recently reported applications of VCD in bioanalytical chemistry. It showed that VCD is extremely powerful in the elucidation of the structural organization of saccharides, peptides, proteins and glycerophospholipids. VCD exhibits unique sensitivity to the supramolecular organization of biological polymers, such as collagen, DNA and amyloid fibrils. It should be noted that electron and probe microscopy is often not capable of unraveling chiral organization of these macromolecules. This review also demonstrated how VCD can be utilized to unravel molecule-molecule interactions that determine chiral separations in chromatography. Moreover, VCD could be used to determine the absolute configuration of de novo synthesized or extracted compounds, which is extremely important for their practical utilization in pharmacy. Numerous examples of such determination of the absolute configuration are discussed in excellent reviews from research laboratories of Nafie [89,90] Keiderling [91], and Joseph-Nathan [92]. It should be also mentioned that conformational and configurational assignments of natural product molecules that were reported over the last 15 years are summarized in the excellent review by Batista Jr. and co-workers [93].

These works pawed a way for the development of new exciting applications of VCD in various areas ranging from pharmaceutics to solid state physics. BioTools Inc. demonstrated micro-VCD devise that used a pair of fast-focusing lenses, one before and one after the sample to focus IR light down to 1 mm [94]. Using this micro-VCD spectrometer, Lu et al. investigated supramolecular heterogeneity of insulin and lysozyme fibril films. While the VCD and IR spatial results for insulin fibril films showed variations primarily associated with local film thickness, there were in addition variations in the magnitude of the VCD relative to IR that showed different local degrees of supramolecular fibril development. Lu et al. found more significant differences in the VCD of lysozyme fibril films that showed sensitivity to both fibril development and the dominant local sense of fibril chirality.

Reiter at al. presented an implementation that can be used for calculation of VCD spectra at the density functional theory (DFT) level [95]. This allowed for the exploitation of symmetry and the usage of effective core potentials. Using this approach, one may account for scalar relativistic effects in the calculation of VCD spectra for molecules containing heavy elements. Finally, Taniguchi et al. et al. showed that VCD could be used for elucidation of the equibrated state of the furanose ring puckers, which is often difficult to study by other techniques [96]. These studies highlight the growth of VCD as a powerful analytical technique with far reaching practical implications.

HIGHLIGHTS.

• Vibrational Circular Dichroism (VCD) determines absolute configuration of chiral molecules.

• VCD is capable of probing supramolecular chirality of macromolecules, such as protein aggregates and DNA.

• Using VCD, mechanisms of molecule-molecule interactions and chiral separations can be investigated.

• Recent advances of VCD in bioanalytical chemistry are critically discussed.

Biography

Dmitry Kurouski earned his M. S. in Biochemistry from Belarusian State University, Belarus and Ph. D. (Distinguished Dissertation) in Analytical Chemistry from SUNY Albany, NY, USA. After a Postdoc in the laboratory of Professor Richard P. Van Duyne at Northwestern University, Dr. Kurouski worked at Boehringer-Ingelheim Pharmaceuticals, as Senior Research Scientist. In 2017, Dr. Kurouski joined Biochemistry and Biophysics Department at Texas A&M University as Assistant Professor.