Vibrational optical activity: From discovery and development to future challenges

Vibrational optical activity (VOA) consisting of infrared vibrational circular dichroism (VCD) and vibrational Raman optical activity (ROA) was predicted, discovered, and confirmed between 1971 and 1975. My path to VOA was mentored by three pioneers of chirality and vibrational spectroscopy: Professors Albert Moscowitz, Warner L. Peticolas, and Philip J. Stephens, and while they are no longer alive today, the Chirality Medal, my award address, and this paper are dedicated to each of them. Since the discovery of VOA, a number of key advances have made possible the current era of widespread applications. The principal instrumental advances were Fourier-transform VCD (FT-VCD) and multichannel charge coupled detector (CCD) ROA. Computational advances include the first complete quantum chemistry formulation of VCD leading to the magnetic field perturbation (MFP) and the nuclear velocity perturbation (NVP) theories. The strength of VOA is the comparison between measured and calculated spectra that enables the determination of absolute configuration and solution-state conformations. More recently, VCD has uncovered supramolecular chirality in amyloid fibrils and ROA to high-order protein structure. Future challenges for VOA include describing the effects of weak intermolecular interactions, transfer of chirality, solvent effects, supramolecular chirality, and the generation of nuclear velocity electron current density.     

Isotopic difference spectra as an aide in determining absolute configuration using vibrational optical activity: vibrational circular dichroism of 13C- and 2H-labelled nonamethoxy cyclotriveratrylene

The use of isotopic difference spectra in vibrational optical activity is demonstrated as a supplemental aide in determining the absolute configuration of chiral molecules. It is shown that IR and VCD difference spectra associated with isotopic substitution observed in experimental spectra can be accurately reproduced by density functional theory calculations when the IR and VCD spectra of the original isotopomer are calculated to reasonable accuracy. Results for isotopically substituted nonamethoxy cyclotriveratrylene are presented to illustrate the degree of agreement between measured and calculated IR and VCD difference spectra for several isotopomers of this molecule. These findings highlight the utility of isotoptic substitution as an aide to verifying the determination of absolute configuration using vibrational optical activity.     

Pressure dependence of vibrational optical activity of model biomolecules. A computational study

Change of molecular properties with pressure is an attracting means to regulate molecular reactivity or biological activity. However, the effect is usually small and so far explored rather scarcely. To obtain a deeper insight and estimate the sensitivity of vibrational optical activity spectra to pressure-induced conformational changes, we investigate small model molecules. The Ala-Ala dipeptide, isomaltose disaccharide and adenine-uracil dinucleotide were chosen to represent three different biomolecular classes. The pressure effects were modeled by molecular dynamics and density functional theory simulations. The dinucleotide was found to be the most sensitive to the pressure, whereas for the disaccharide the smallest changes are predicted. Pressure-induced relative intensity changes in vibrational circular dichroism and Raman optical activity spectra are predicted to be 2–3-times larger than for non-polarized IR and Raman techniques.     

CDSpecTech: A single software suite for multiple chiroptical spectroscopic analyses

The program CDSpecTech was developed to facilitate the analysis of chiroptical spectra, which include the following: vibrational circular dichroism (VCD) and corresponding vibrational absorption (VA) spectra; vibrational Raman optical activity (VROA) and corresponding vibrational Raman spectra; electronic circular dichroism (ECD) and corresponding electronic absorption (EA) spectra. In addition, the program allows for generating optical rotatory dispersion (ORD) as the Kramers–Kronig transform of ECD spectra. The simulation of theoretical spectra from transition strengths can be achieved using different bandshape profiles. The experimental and simulated theoretical spectra can be visually compared by displaying them together. A unique feature of CDSpecTech is performing spectral analysis using the ratio spectra; i.e., the dimensionless dissymmetry factor (DF) spectrum, which is the ratio of CD to absorption spectra, and the dimensionless circular intensity difference (CID) spectrum, which is the ratio of VROA to vibrational Raman spectra. The quantitative agreement between experimental and simulated theoretical spectra can also be assessed from the numerical similarity overlap between them. Two different similarity overlap methods are available. The program uses a graphical user interface which allows for ease of use and facilitates the analysis. All these features make CDSpecTech a valuable tool for the analysis of chiroptical spectra. The program is freely available on the World Wide Web.     

Electronic and vibrational optical activity of several peptides related to neurohypophyseal hormones: disulfide group conformation

Electronic and vibrational optical activity of the set of neurohypophyseal hormones and their analogs was investigated to clarify the S-S bond solution conformation. The selected compounds include oxytocin (I), lysine vasopressin (II), arginine vasopressin (III), and their analogs (IV-IX), differing widely in their pharmacological properties. We have extended the already known electronic circular dichroism data by new information provided by vibrational circular dichroism (VCD) and Raman optical activity (ROA). The use of VCD brought additional details on three-dimensional structure of the chain reversal in the ring moiety and on its left handedness. Furthermore, Raman scattering and ROA allowed us to deduce the sense of the disulfide bond torsion.     

The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells

In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was rather due to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild type and MDR tumor cells. In fact, under treatment condition causing about 50% of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro- or anti-apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.     

Why is it important to simultaneously use more than one chiroptical spectroscopic method for determining the structures of chiral molecules?

In recent years, four different chiroptical spectroscopic methods, namely vibrational circular dichroism, vibrational Raman optical activity, electronic circular dichroism, and optical rotatory dispersion, have become popular for establishing the absolute configuration and predominant conformations of chiral molecules in solution state. Many individual laboratories normally utilize only one of these methods to derive the molecular structural information. Although that approach may be satisfactory for most of the molecules studied, it is to be noted that in some instances a single method can give ambiguous conclusions or may not give complete structural information. This article summarizes the situations where simultaneous use of more than one chiroptical spectroscopic method is required to obtain molecular structural information and recommends the routine application of more than one chiroptical spectroscopic method for any given molecule.     

Electronic and vibrational signatures of peptide helical structures: A tribute to Anton Mario Tamburro

Our efforts on the synthesis of peptides with well-characterized secondary structures, combined with detailed spectroscopic investigations, most of them performed in collaboration with internationally recognized experts, have allowed us to publish the electronic (electronic circular dichroism) and vibrational (FTIR absorption, vibrational circular dichroism, Raman, and Raman optical activity) signatures of the poorly studied peptide 3(10)-helix (and the related β-bend ribbon spiral conformation as well) in comparison with those already known for the classical α-helix.     

Spectroscopic properties of the nonplanar amide group: a computational study

Experimental studies suggest that amide bond may significantly deviate from planar arrangement even in linear peptides and proteins. In order to find out the extent to which such deviation may influence principal amide spectroscopic properties, we conducted a computational study of nonplanar N-methylacetamide (NMA) conformers. Vibrational absorption, Raman, and electronic spectra including optical activity were simulated with ab initio and density functional theory (DFT) methods. According to the results, small nonplanarity deviations may be detectable by nonpolarized spectroscopic techniques, albeit as subtle spectral changes. The optical activity methods, such as the vibrational circular dichroism (VCD), Raman optical activity (ROA), and electronic circular dichroism (CD, ECD), provide enhanced information about the amide nonplanarity, because planar amide is not optically active (chiral). For VCD, however, the inherently chiral contribution in most peptides and proteins most probably provides very weak signal in comparison with other contributions, such as the dipolar coupling. For the electronic CD, the nonplanarity contribution is relatively big and causes a strong CD couplet in the n-pi* absorption region accompanied by a red frequency shift. The pi-pi* CD region is relatively unaffected. The ROA spectroscopy appears most promising for the nonplanarity detection and the inherent chiral signal may dominate entire spectral parts. The amide I and III vibrational ROA bands are most challenging experimentally because of their relatively weak coupling to other peptide vibrations.     

A point-process model for variance-occupancy-abundance relationships

The relationship between species abundance, the variance of the number of individuals, and species occupancy is a fundamental ecological characteristic of a community. Moreover, this relationship varies across scales, and any model for the variance-occupancy-abundance (VOA) relationship has to address its scale dependency in a consistent way. In this study, point-process theory was used to define a multiscale model that jointly predicts the VOA relationship across scales in a consistent way. This provides a tool to jointly analyze data sets collected at different scales and to give insights into the biological processes underlying the VOA relationship. This model can also account for different types of individual spatial pattern (clustered, random, or regular). Three stand-mapping data sets of tree species in tropical rain forests were used to assess the relevance of this model. When compared with four existing models, the model based on point-process theory provided the best fit to the data and was the most often ranked as the model with the best predictive performance.     

Correction: Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth

The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4–2.6) between time point 1 and 2; and median of 31 days (IQR: 28–36) between time point 2 and 3. Patients were median of 6 years (IQR: 3–12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2–8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R² = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log₁₀, and 2.23 log₁₀ between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the replication-competent virus in ART suppressed patients.     

The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells

In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in a competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was due instead to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild-type and MDR tumor cells. In fact, under treatment condition causing about 50 percent of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression, and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro- or anti- apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.     

Optical Rotatory Dispersion of Polypeptides in the Near-Infrared Region

For the first time ORD measurements in the near-infrared region from 0.7 to 2.0 μ for well-known polypeptides, namely, poly(γ-benzyl L -glutamate), poly(L -glutamic acid), poly-L -lysine·HCl, poly-S-carbobenzoxymethyl-L -cysteine, and Bombyx mori silk fibroin, were carried out. It was found that the value of the optical activity infrared term, which is proportional to the sum of rotational strengths of vibrational transitions, depends on polypeptide conformation. The optical activity infrared term value is equal to zero for the random-coil conformation, it is small but exceeds the measurement error for the α-helical state, and finally, for the β conformation it is an order of magnitude higher than for the α-helical state. The obtained results permit one to hope that on the basis of ORD measurement in the near-infrared region it will be possible to suggest a method of determining the β-form content in polypeptides and proteins     

Optical and vibrational properties of 1,2-benzenedicarboxylic anhydride

The solvent-induced changes in the optical and spectroscopic properties of 1,2-benzenedicarboxylic anhydride are studied using time dependent Hartree-Fock and density functional theory calculations within the framework of two reaction field procedures. To investigate the influence of the cavity shape, the Onsager reaction field is compared with the polarized continuum model (PCM). It is observed that solvent polarity has noticeable effects on the vibrational properties as well as the linear and nonlinear optical characteristics of the molecule. Furthermore, the Onsager and PCM procedures may lead to contradicted harmonic vibrational frequencies; in the case of the studied molecule the Onsager model predicts the blue-shifted CH stretching band while PCM leads to red-shifted CH stretching mode.     

Beta-sheet and associated turn signatures in vibrational Raman optical activity spectra of proteins.

We have measured the aqueous solution vibrational Raman optical activity (ROA) spectra of concanavalin A, alpha-chymotrypsin, and beta-lactoglobulin, all of which are rich in beta-sheet, together with that of the model beta-turn peptide L-pro-L-leu-gly-NH2. Possible ROA signatures of antiparallel beta-sheet include a strong sharp positive band at approximately 1,313 cm-1 associated with backbone amide III C alpha H and NH deformations, and an amide I couplet, negative at low wavenumber and positive at high, centered at approximately 1,658 cm-1. Negative ROA bands in the range approximately 1,340-1,380 cm-1, which might originate in glycine CH2 deformations, appear to be characteristic of beta-turns. Our results provide further evidence that ROA is a more incisive probe of protein conformation than conventional vibrational spectroscopy, infrared, or Raman, because only those few vibrational coordinates within a given normal mode that sample the skeletal chirality directly contribute to the corresponding ROA band intensity.     

Solution structure and dynamics of biomolecules from Raman optical activity

Raman optical activity (ROA) measures vibrational optical activity by means of a small difference in the intensity of Raman scattering from chiral molecules in right and left circularly polarized incident laser light. The ROA spectra of a wide range of biomolecules in aqueous solution can now be measured routinely. Because of its sensitivity to the chiral elements of biomolecular structure, ROA provides new information about solution structure and dynamics complementary to that supplied by conventional spectroscopic techniques. This article provides a brief introduction to the theory and practice of ROA spectroscopy followed by a review of recent ROA results on polypeptides, proteins, carbohydrates, nucleic acids and viruses which illustrate how new insight into current problems of structure, folding and function may be obtained from ROA studies.     

Influence of methane enrichment by aeration of recirculated supernatant on microbial activities during anaerobic digestion

A methane enrichment process (MEP) was evaluated that involved air purging of recycled digester contents to strip CO₂ and increase biogas methane content. The objective of this work was to determine if the aeration resulted in oxygen inhibition of microbial activities involved in anaerobic digestion of municipal solid waste. To assess the degree of biological perturbation associated with the MEP, the reactor effluent was sampled twice while the MEP was operating and twice while it was not operating. Analyses were run on composite samples (representing several different sample ports of the non-mixed reactor) and samples of digester effluent entering and leaving the MEP process. The analyses included volatile organic acids (VOA), effluent solids content, dehydrogenase activity, specific methanogenic activities (SMA), and enzyme-linked immunosorbent assay (ELISA) for methanogenic, sulfate-reducing, and cellulolytic bacterial species. Methane enrichment in these experiments occurred with the methane content of the biogas exceeding 90%. There were no effects of the MEP on effluent VOA concentration. The MEP had no effect on volatile solids (VS) levels of composite samples representing the non-mixed digester contents, however, VS was reduced in effluent passing through the MEP. Although MEP had an inhibitory effect on anaerobic populations leaving the MEP process, no inhibitory effects were observed in measurements of microbial activities in digester samples, including specific methane production rate, dehydrogenase, and numbers of specific organisms estimated using ELISA techniques.     

Raman,FT-IR spectroscopic analysis and first-order hyperpolarisability of 3-benzoyl-5-chlorouracil by first principles

3-Benzoyl-5-chlorouracil (3B5CU), a biologically active synthetic molecule, has been analysed at DFT/6-311+ + G(d,p) level and reported for the first time as a potential candidate for nonlinear optical (NLO) applications. The optimised skeleton of 3B5CU molecule is non-planar. The frontier orbital energy gap, dipole moment, polarisability and first static hyperpolarisability have been calculated. The first static hyperpolarisability is found to be almost 15 times higher than that of urea. The high value of first static hyperpolarisability (2.930 × 10^? 30 e.s.u.) due to the intra-molecular charge transfer in 3B5CU has been discussed using first principles. A complete vibrational analysis of the molecule has been performed by combining the experimental Raman, FT-IR spectral data and the quantum chemical calculations. In general, a good agreement of calculated modes with the experimental ones has been obtained. The strong vibrational modes contributing towards NLO activity, involving the whole charge transfer path, have been identified and analysed.     

Determination of absolute configuration--an overview related to this special issue

Rapid progress in asymmetric synthesis stimulated a further development of methods and techniques for the determination of absolute configuration of chiral molecules. In recent years the direct methods, i.e. X-ray diffraction analysis, circular dichroism (vibrational and electronic), Raman optical activity, optical rotation measurements, as well as indirect methods for relative configuration assignment with the use of NMR spectroscopy or enzymatic transformations, are receiving increasing attention not only by specialists in the field but also by synthetic and structural chemists alike. This paper provides a short overview of the methods currently used, as well as references to contributions collected in this Thematic Issue of Chirality.     

Super-multiplexed vibrational probes: Being colorful makes a difference

Biological systems with intrinsic complexity require multiplexing techniques to comprehensively describe the phenotype, interaction, and heterogeneity. Recent years have witnessed the development of super-multiplexed vibrational microscopy, overcoming the ‘color barrier’ of fluorescence-based optical techniques. Here, we will review the recent progress in the design and applications of super-multiplexed vibrational probes. We hope to illustrate how rainbow-like vibrational colors can be generated from systematic studies on structure–spectroscopy relationships and how being colorful makes a difference to various biomedical applications.