Induced Chirality in Canthaxanthin Aggregates Reveals Multiple Levels of Supramolecular Organization.

Carotenoids tend to form supramolecular aggregates via non-covalent interactions where the chirality of individual molecules is amplified to the macroscopic level. We show that this can also be achieved for non-chiral carotenoid monomers interacting with polysaccharides. The chirality induction in canthaxanthin (CAX), caused by heparin (HP) and hyaluronic acid (HA), was monitored by chiroptical spectroscopy. Electronic circular dichroism (ECD) and Raman optical activity (ROA) spectra indicated the presence of multiple carotenoid formations, such as H- and J-type aggregates. This is consistent with molecular dynamics (MD) and density functional theory (DFT) simulations of the supramolecular structures and their spectroscopic response.

The Stability of Hydrogen-Bonded Ion-Pair Complex Unexpectedly Increases with Increasing Solvent Polarity.

The generally observed decrease of the electrostatic energy in the complex with increasing solvent polarity has led to the assumption that the stability of the complexes with ion-pair hydrogen bonds decreases with increasing solvent polarity. Besides, the smaller solvent-accessible surface area (SASA) of the complex in comparison with the isolated subsystems results in a smaller solvation energy of the latter, leading to a destabilization of the complex in the solvent compared to the gas phase. In our study, which combines Nuclear Magnetic Resonance, Infrared Spectroscopy experiments, quantum chemical calculations, and molecular dynamics (MD) simulations, we question the general validity of this statement. We demonstrate that the binding free energy of the ion-pair hydrogen-bonded complex between 2-fluoropropionic acid and n-butylamine (CH3CHFCOO-…NH3But+) increases with increased solvent polarity. This phenomenon is rationalized by a substantial charge transfer between the subsystems that constitute the ion-pair hydrogen-bonded complex. This unexpected finding introduces a new perspective to our understanding of solvation dynamics, emphasizing the interplay between solvent polarity and molecular stability within hydrogen-bonded systems.

Glucagon-like peptide 1 aggregates into low-molecular-weight oligomers off-pathway to fibrillation.

The physical stability of peptide-based drugs is of great interest to the pharmaceutical industry. Glucagon-like peptide 1 (GLP-1) is a 31-amino acid peptide hormone, the analogs of which are frequently used in the treatment of type 2 diabetes. We investigated the physical stability of GLP-1 and its C-terminal amide derivative, GLP-1-Am, both of which aggregate into amyloid fibrils. While off-pathway oligomers have been proposed to explain the unusual aggregation kinetics observed previously for GLP-1 under specific conditions, these oligomers have not been studied in any detail. Such states are important as they may represent potential sources of cytotoxicity and immunogenicity. Here, we identified and isolated stable, low-molecular-weight oligomers of GLP-1 and GLP-1-Am, using size-exclusion chromatography. Under the conditions studied, isolated oligomers were shown to be resistant to fibrillation or dissociation. These oligomers contain between two and five polypeptide chains and they have a highly disordered structure as indicated by a variety of spectroscopic techniques. They are highly stable with respect to time, temperature, or agitation despite their noncovalent character, which was established using liquid chromatography-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results provide evidence of stable, low-molecular-weight oligomers that are formed by an off-pathway mechanism which competes with amyloid fibril formation.

Gold nanoshells with magnetic cores and a urea-based receptor for SERS sensing of fluoride anions: experimental and computational study.

The study demonstrates that a combination of plasmonic nanostructures and artificial receptors can be applied for sensing small molecular species. Gold nanoshells containing magnetic cores are used as the SERS-active substrates, which opens the way for the development of multimodal contrast agents with applicability extended to sensing or for the separation of analytes by magnetic solid-phase extraction. Disubstituted ureas forming hydrogen-bonded complexes with certain anions can be employed as molecular sensors. In this case study, gold nanoshells with silica-coated Mn-Zn ferrite cores were prepared by a multistep procedure. The nanoshells were co-functionalized with an N-(4-mercaptophenyl)-N'-(4-nitrophenyl)urea sensor synthesized directly on the gold surface, and with 4-nitrothiophenol, which is adopted as an internal standard. SERS measurements were carried out with acetonitrile solutions of tetrabutylammonium fluoride (Bu4NF) over a concentration range of 10-10-10-1 mol L-1. The spectral response of the sensor is dependent on the fluoride concentration in the range of 10-5-10-1 mol L-1. To investigate further the SERS mechanism, a model sensor, N-(4-bromophenyl)-N'-(4-nitrophenyl)urea, was synthesized and used in Raman spectroscopy with solutions of Bu4NF, up to a molar ratio of 1 : 20. The spectra and the interactions between the sensors and fluoride anions were also studied by extensive DFT computations.

Molecular dynamics and Raman optical activity spectra reveal nucleotide conformation ratios in solution.

Nucleotide conformational flexibility affects their biological functions. Although the spectroscopy of Raman optical activity (ROA) is well suited to structural analyses in aqueous solutions, the link between the spectral shape and the nucleotide geometry is not fully understood. We recorded the Raman and ROA spectra of model nucleotides (rAMP, rGMP, rCMP, and dTMP) and interpreted them on the basis of molecular dynamics (MD) combined with density functional theory (DFT). The relation between the sugar puckering, base conformation and spectral intensities is discussed. Hydrogen bonds between the sugar's C3' hydroxyl and the phosphate groups were found to be important for the sugar puckering. The simulated spectra correlated well with the experimental data and provided an understanding of the dependence of the spectral shapes on conformational dynamics. Most of the strongest spectral bands could be assigned to vibrational molecular motions. Decomposition of the experimental spectra into calculated subspectra based on arbitrary maps of free energies provided experimental conformer populations, which could be used to verify and improve the MD predictions. The analyses indicate some flaws of common MD force fields, such as being unable to describe the fine conformer distribution. Also the accuracy of conformer populations obtained from the spectroscopic data depends on the simulations, improvement of which is desirable for gaining a more detailed insight in the future. Improvement of the spectroscopic and computational methodology for nucleotides also provides opportunities for its application to larger nucleic acids.

Rotational circular dichroism of diamagnetic and paramagnetic molecules. A computational study.

Rotational circular dichroism (RCD) has not been observed yet, but it is expected to deliver information about chiral molecules useful in many branches of chemistry. In the past, rather weak RCD intensities were predicted for model diamagnetic molecules and a limited number of rotational transitions. Here, we review quantum-mechanical foundations and simulate entire spectral profiles, including larger molecules, open-shell molecular radicals, and high-momentum rotational bands. Contribution of the electric quadrupolar moment was considered, but it turned out that it does not contribute to field-free RCD. Two conformers of a model dipeptide provided clearly distinct spectra. The dissymmetry Kuhn parameter gK predicted for the diamagnetic molecules even for high-J transitions rarely exceeded 10-5, and the simulated RCD spectra were often biased to one sign. In the radicals, the coupling of the rotational angular momentum with the spin for some transitions raised gK to about 10-2, and the RCD pattern was more conservative. In the resultant spectra, many transitions had negligible intensities due to small populations of the involved states, and a convolution with a spectral function made the typical RCD/absorption ratios about 100-times smaller (gK ∼ 10-4). This is still comparable with values typical for electronic or vibrational circular dichroism, and paramagnetic RCD is thus likely to be measurable relatively easily.

Combination of Resonance and Non-Resonance Chiral Raman Scattering in a Cobalt(III) Complex.

Resonance Raman optical activity (RROA) spectra with high sensitivity reveal details on molecular structure, chirality, and excited electronic properties. Despite the difficulty of the measurements, the recorded data for the Co(III) complex with S,S-N,N-ethylenediaminedisuccinic acid are of exceptional quality and, coupled with the theory, spectacularly document the molecular behavior in resonance. This includes a huge enhancement of the chiral scattering, contribution of the antisymmetric polarizabilities to the signal, and the Herzberg-Teller effect significantly shaping the spectra. The chiral component is by about one order of magnitude bigger than for an analogous aluminum complex. The band assignment and intensity profile were confirmed by simulations based on density functional and vibronic theories. The resonance was attributed to the S0 →S3 transition, with the strongest signal enhancement of Raman and ROA spectral bands below about 800 cm-1 . For higher wavenumbers, other excited electronic states contribute to the scattering in a less resonant way. RROA spectroscopy thus appears as a unique tool to study the structure and electronic states of absorbing molecules in analytical chemistry, biology, and material science.

Structure of Zinc and Nickel Histidine Complexes in Solution Revealed by Molecular Dynamics and Raman Optical Activity.

The histidine residue has an exceptional affinity for metals, but solution structure of its complexes are difficult to study. For zinc and nickel complexes, Raman and Raman optical activity (ROA) spectroscopy methods to investigate the link between spectral shapes and the geometry were used. The spectra were recorded and interpreted on the basis of ionic equilibria, molecular dynamics, ab initio molecular dynamics, and density functional theory. For zwitterionic histidine the dominant tautomer was determined by the decomposition of experimental spectra into calculated subspectra. An octahedral structure was found to prevail for the ZnHis2 complex in solution, in contrast to a tetrahedral arrangement in the crystal phase. The solution geometry of NiHis2 is more similar to the octahedral structure found by X-ray. The Raman and ROA structural determinations of metal complexes are dependent on extensive computations, but reveal unique information about the studied systems.

Electronic Circular Dichroism-Circularly Polarized Raman (eCP-Raman): A New Form of Chiral Raman Spectroscopy.

This Concept article summarizes recent work on the development of a new form of chiral Raman spectroscopy, eCP-Raman, which combines two spectroscopies: electronic circular dichroism (ECD) and circularly polarized Raman (CP-Raman). First, some puzzling observations while carrying out Raman optical activity (ROA) measurements of several transition metal complexes under resonance are described, as well as the search for the mechanisms responsible. Then an equation for quantifying the eCP-Raman contribution is presented, followed by several examples of how eCP-Raman influences the IR -IL spectra of achiral and chiral solvent molecules and of a number of chiral solutes under resonance. The conditions to extract resonance ROA, when the eCP-Raman contribution is minimized, are also discussed. Finally, we comment on the potential applications of eCP-Raman.

Photochemical synthesis of pink silver and its use for monitoring peptide nitration via surface enhanced Raman spectroscopy (SERS).

Oxidative stress may cause extended tyrosine posttranslational modifications of peptides and proteins. The 3-nitro-L-tyrosine (Nit), which is typically formed, affects protein behavior during neurodegenerative processes, such as Alzheimer's and Parkinson's diseases. Such metabolic products may be conveniently detected at very low concentrations by surface enhanced Raman spectroscopy (SERS). Previously, we have explored the SERS detection of the Nit NO2 bending vibrational bands in a presence of hydrogen chloride (Niederhafner et al., Amino Acids 53:517-532, 2021, ibid). In this article, we describe performance of a new SERS substrate, "pink silver", synthesized photochemically. It provides SERS even without the HCl induction, and the acid further decreases the detection limit about 9 times. Strong SERS bands were observed in the asymmetric (1550-1475 cm-1) and symmetric (1360-1290 cm-1) NO stretching in the NO2 group. The bending vibration was relatively weak, but appeared stronger when HCl was added. The band assignments were supported by density functional theory modeling.

Low-frequency Raman optical activity provides insight into the structure of chiral liquids.

Vibrational frequencies of modes involving intermolecular motions in liquids are relatively small, in the Raman scattering close to the excitation frequency, and the bands may merge into a diverging uninterpretable signal. Raman optical activity (ROA) spectral shapes in this region, however, are structured more and may better reflect the nature of the studied systems. To understand the origin of the signal and its relation to the molecules, ROA spectra of six chiral neat liquids are recorded and analyzed on the basis of molecular dynamics and density functional theory computations. The theory of Raman scattering of liquids is discussed and adapted for modeling based on clusters and periodic boundary conditions. A plain cluster approach is compared to a crystal-like model. The results show that the low-frequency optical activity can be reliably modeled and related to the structure. However, momentary arrangement of molecules leads to large variations of optical activity, and a relatively large number of geometries need to be averaged for accurate simulations. The intermolecular modes are intertwined with intramolecular ones and start to dominate as the frequency goes down. The low-frequency ROA signal thus reflects the chemical composition and coupled with the modeling it provides a welcome means to study the structure and interactions of chiral liquids.

Unexpected Strengthening of the H-Bond Complexes in a Polar Solvent Due to a More Efficient Solvation of the Complex Compared to Isolated Monomers.

It is generally assumed that hydrogen-bonded complexes are less stable in solvents than in the gas phase and that their stability decreases with increasing solvent polarity. This assumption is based on the size of the area available to the solvent, which is always smaller in the complex compared to the subsystems, thereby reducing the solvation energy. This reduction prevails over the amplification of the electrostatic hydrogen bond by the polar solvent. In this work, we show, using experimental IR spectroscopy and DFT calculations, that there are hydrogen-bonded complexes whose stability becomes greater with increasing solvent polarity. The explanation for this surprising stabilization is based on the analysis of the charge redistribution in the complex leading to increase of its dipole moment and solvation energy. Constrained DFT calculations have shown a dominant role of charge transfer over polarization effects for dipole moment and solvation energy.

Insight into the Mechanism of Action and Peptide-Membrane Interactions of Aib-Rich Peptides: Multitechnique Experimental and Theoretical Analysis.

The increase in resistant bacterial strains necessitates the identification of new antimicrobial molecules. Antimicrobial peptides (AMPs) are an attractive option because of evidence that bacteria cannot easily develop resistance to AMPs. The peptaibols, a class of naturally occurring AMPs, have shown particular promise as antimicrobial drugs, but their development has been hindered by their mechanism of action not being clearly understood. To explore how peptaibols might interact with membranes, circular dichroism, vibrational circular dichroism, linear dichroism, Raman spectroscopy, Raman optical activity, neutron reflectivity and molecular dynamics simulations have been used to study a small library of peptaibol mimics, the Aib-rich peptides. All the peptides studied quickly partitioned and oriented in membranes, and we found evidence of chiral interactions between the phospholipids and membrane-embedded peptides. The protocols presented in this paper open new ground by showing how chiro-optical spectroscopies can throw light on the mechanism of action of AMPs.

Polymorphism of Amyloid Fibrils Induced by Catalytic Seeding: A Vibrational Circular Dichroism Study.

Amyloidal protein fibrils occur in many biological events, but their formation and structural variability are understood rather poorly. We systematically explore fibril polymorphism for polyglutamic acid (PGA), insulin and hen egg white lysozyme. The fibrils were grown in the presence of "seeds", that is fibrils of the same or different protein. The seeds in concentrations higher than about 5 % of the total protein amount fully determined the structure of the final fibrils. Fibril structure was monitored by vibrational circular dichroism (VCD) spectroscopy and other techniques. The VCD shapes significantly differ for different fibril samples. Infrared (IR) and VCD spectra of PGA were also simulated using density functional theory (DFT) and a periodic model. The simulation provides excellent basis for data interpretation and reveals that the spectral shapes and signs depend both on fibril length and twist. The understanding of fibril formation and interactions may facilitate medical treatment of protein misfolding diseases in the future.

Monitoring peptide tyrosine nitration by spectroscopic methods.

Oxidative stress can lead to various derivatives of the tyrosine residue in peptides and proteins. A typical product is 3-nitro-L-tyrosine residue (Nit), which can affect protein behavior during neurodegenerative processes, such as those associated with Alzheimer's and Parkinson's diseases. Surface enhanced Raman spectroscopy (SERS) is a technique with potential for detecting peptides and their metabolic products at very low concentrations. To explore the applicability to Nit, we use SERS to monitor tyrosine nitration in Met-Enkephalin, rev-Prion protein, and α-synuclein models. Useful nitration indicators were the intensity ratio of two tyrosine marker bands at 825 and 870 cm-1 and a bending vibration of the nitro group. During the SERS measurement, a conversion of nitrotyrosine to azobenzene containing peptides was observed. The interpretation of the spectra has been based on density functional theory (DFT) simulations. The CAM-B3LYP and ωB97XD functionals were found to be most suitable for modeling the measured data. The secondary structure of the α-synuclein models was monitored by electronic and vibrational circular dichroism (ECD and VCD) spectroscopies and modeled by molecular dynamics (MD) simulations. The results suggest that the nitration in these peptides has a limited effect on the secondary structure, but may trigger their aggregation.

Intense chiral signal from α-helical poly-L-alanine observed in low-frequency Raman optical activity.

Raman optical activity (ROA) spectral features reliably indicate the structure of peptides and proteins, but the signal is often weak. However, we observed significantly enhanced low-frequency bands for α-helical poly-L-alanine (PLA) in solution. The biggest ROA signal at ∼100 cm-1 is about 10 times stronger than higher-frequency bands described previously, which facilitates the detection. The low-frequency bands of PLA were compared to those of α-helical proteins. For PLA, density functional simulations well reproduced the experimental spectra and revealed that about 12 alanine residues within two turns of the α-helix generate the strong ROA band. Averaging based on molecular dynamics (MD) provided an even more realistic spectrum compared to the static model. The low-frequency bands could be largely related to a collective motion of the α-helical backbone, partially modulated by the solvent. Helical and intermolecular vibrational coordinates have been introduced and the helical unwinding modes were assigned to the strongest ROA signal at 101-128 cm-1. Further analysis indicated that the helically arranged amide and methyl groups are important for the strong chiral signal of PLA, while the local chiral centers CαH contribute in a minor way only. The strong low-frequency ROA can thus provide precious information about the motions of the peptide backbone and facilitate future protein studies.

α-Synuclein conformations followed by vibrational optical activity. Simulation and understanding of the spectra.

α-Synuclein is a neuronal protein which adopts multiple conformations. These can be conveniently studied by the spectroscopy of vibrational optical activity (VOA). However, the interpretation of VOA spectra based on quantum-chemical simulations is difficult. To overcome the hampering of the computations by the protein size, we used the Cartesian tensor transfer technique to investigate links between the spectral shapes and protein structure. Vibrational circular dichroism (VCD) and Raman optical activity (ROA) spectra of α-synuclein in disordered, α-helical and ß-sheet (fibril) forms were measured and analyzed on the basis of molecular dynamics and density functional theory computations. For the disordered and α-helical conformers, a high fidelity of the simulated spectra with a reasonable computational cost was achieved. Most experimental spectral features could be assigned to the structure. So far unreported ROA marker bands of the secondary structure were found for the lower-frequency and CH stretching vibrations. Fibril VCD spectra were simulated with a rigid periodic model of the geometry and the results are consistent with previous studies based on cryogenic electron microscopy. The fibrils also give a specific ROA signal, but unlike VCD it is currently not fully explicable by the simulations. In connection with the computational modeling the VOA spectroscopy thus appears as an extremely useful tool for monitoring α-synuclein and other proteins in solutions.

Chiral recognition via a stereodynamic vanadium probe using the electronic circular dichroism effect in differential Raman scattering.

Intermolecular interactions sensitive to chirality occur in many biological events. We report a complex formation between a versatile vanadium-based probe and a chiral co-ligand monitored via the combination of electronic circular dichroism (ECD) and Raman scattering. This "ECD-Raman" effect was discovered relatively recently and can be measured using a Raman optical activity (ROA) spectrometer. Simulated spectra based on experimental ECD and degree of circularity (DOC) values agree with the observed ones. Sensitive recognition of the chiral enantiopure co-ligand is thus enabled by a combination of resonance of the excitation light with the diastereoisomeric complex, co-ligand complexation, circular dichroism, and polarized Raman scattering from the achiral solvent. Relatively dilute solutions could be detected (10-4 mol dm-3), about 1000× less than is necessary for conventional ROA detection of the pure co-ligand and comparable to concentrations needed for conventional ECD spectroscopy. The results thus show that differential ECD-Raman measurements can be conveniently used to monitor molecular interactions and molecular spectroscopic properties.

Can One Measure Resonance Raman Optical Activity?

Resonance Raman optical activity (RROA) is commonly measured as the difference in intensity of Raman scattered right and left circularly polarized light, IR -IL , when a randomly polarized light is in resonance with a chiral molecule. Strong and sometimes mono-signate experimental RROA spectra of several chiral solutes were reported previously, although their signs and relative intensities could not be reproduced theoretically. By examining multiple light-matter interaction events which can occur simultaneously under resonance, we show that a new form of chiral Raman spectroscopy, eCP-Raman, a combination of electronic circular dichroism and circularly polarized Raman, prevails. By incorporating the finite-lifetime approach for resonance, the experimental patterns of the model chiral solutes are captured theoretically by eCP-Raman, without any RROA contribution. The results open opportunity for applications of eCP-Raman spectroscopy and for extracting true RROA experimentally.

Recognition of the True and False Resonance Raman Optical Activity.

Resonance Raman optical activity (RROA) possesses all aspects of a sensitive tool for molecular detection, but its measurement remains challenging. We demonstrate that reliable recording of RROA of chiral colorful compounds is possible, but only after considering the effect of the electronic circular dichroism (ECD) on the ROA spectra induced by the dissolved chiral compound. We show RROA for a number of model vitamin B12 derivatives that are chemically similar but exhibit distinctively different spectroscopic behavior. The ECD/ROA effect is proportional to the concentration and dependent on the optical pathlength of the light propagating through the sample. It can severely alter relative band intensities and signs in the natural RROA spectra. The spectra analyses are supported by computational modeling based on density functional theory. Neglecting the ECD effect during ROA measurement can lead to misinterpretation of the recorded spectra and erroneous conclusions about the molecular structure.