Interaction of nitrite with ferric protoglobin from Methanosarcina acetivorans - an interesting model for spectroscopic studies of the haem-ligand interaction.

Protoglobin from Methanosarcina acetivorans (MaPgb) is a dimeric globin belonging to the same lineage of the globin superfamily as globin-coupled sensors. A putative role in the scavenging of reactive nitrogen and oxygen species has been suggested as a possible adaptation mechanism of the host organism to different gaseous environments in the course of evolution. A combination of optical absorption, electronic circular dichroism (ECD), resonance Raman (rRaman), and electron paramagnetic resonance (EPR) reveal the unusual in vitro reaction of ferric MaPgb with nitrite. In contrast to other globins, a large excess of nitrite did not induce the formation of a nitriglobin form in MaPgb. Surprisingly, the addition of nitrite in mildly acidic pH led to the formation of a stable nitric-oxide ligated ferric form of the protein (MaPgb-NO). Furthermore, the 300-700 nm ECD spectrum of ferric MaPgb is for the first time reported and discussed, showing strong differences in the Soret and Q ellipticity compared to ferric myoglobin, in line with the unusually strongly ruffled haem group of MaPgb and the related quantum-mechanical admixture of the S = 5/2 and S = 3/2 state of its ferric form. The Soret and Q ellipticity change strongly upon formation of MaPgb-NO, revealing a significant effect of the nitric-oxide ligation on the haem group and pocket. The related changes in the asymmetric pyrrole half-ring stretching vibration modes observed in the rRaman spectra give experimental support to earlier theoretical models, in which an important role of the in-plane breathing modes of the haem was predicted for the stabilization of the binding of diatomic gases to MaPgb.

Improving the IR spectra alignment algorithm with spectra deconvolution and combination with Raman or VCD spectroscopy.

The relative stereochemistry of organic molecules can be determined by comparing theoretical and experimental infrared (IR) spectra of all isomers and assessing the best match. For this purpose, we have recently developed the IR spectra alignment (IRSA) algorithm for automated optimal alignment. IRSA provides a set of quantitative metrics to identify the candidate structure that agrees best with the experimental spectrum. While the correct diastereomer could be determined for the tested sets of rigid and flexible molecules, two issues were identified with more complex compounds that triggered further development. First, strongly overlapping peaks in the IR spectrum are not treated adequately in the current IRSA implementation. Second, the alignment of multiple spectra from different sources (e.g. IR and VCD or Raman) can be improved. In this study, we present an in-depth discussion of these points, followed by the description of modifications to the IRSA algorithm to address them. In particular, we introduce the concept of deconvolution of the experimental and theoretical spectra with a set of pseudo-Voigt bands. The pseudo-Voigt bands have a set of parameters, which can be employed in the alignment algorithm, leading to improved scoring functions. We test the modified algorithm on two data sets. The first set contains compounds with IR and Raman spectra measured in this study, and the second set contains compounds with IR and VCD spectra available in the literature. We show that the algorithm is able to determine the correct diastereomer in all cases. The results highlight that vibrational spectroscopy can be a valuable alternative or complementary method to inform about the stereochemistry of compounds, and the performance of the updated IRSA algorithm suggests that it is a powerful tool for quantitative-based spectral assignments in academia and industry.

GLB-3: A resilient, cysteine-rich, membrane-tethered globin expressed in the reproductive and nervous system of Caenorhabditis elegans.

The popular genetic model organism Caenorhabditis elegans (C. elegans) encodes 34 globins, whereby the few that are well-characterized show divergent properties besides the typical oxygen carrier function. Here, we present a biophysical characterization and expression analysis of C. elegans globin-3 (GLB-3). GLB-3 is predicted to exist in two isoforms and is expressed in the reproductive and nervous system. Knockout of this globin causes a 99% reduction in fertility and reduced motility. Spectroscopic analysis reveals that GLB-3 exists as a bis-histidyl-ligated low-spin form in both the ferrous and ferric heme form. A function in binding of diatomic gases is excluded on the basis of the slow CO-binding kinetics. Unlike other globins, GLB-3 is also not capable of reacting with H2O2, H2S, and nitrite. Intriguingly, not only does GLB-3 contain a high number of cysteine residues, it is also highly stable under harsh conditions (pH = 2 and high concentrations of H2O2). The resilience diminishes when the N- and C-terminal extensions are removed. Redox potentiometric measurements reveal a slightly positive redox potential (+8 ± 19 mV vs. SHE), suggesting that the heme iron may be able to oxidize cysteines. Electron paramagnetic resonance shows that formation of an intramolecular disulphide bridge, involving Cys70, affects the heme-pocket region. The results suggest an involvement of the globin in (cysteine) redox chemistry.

Vibrational Optical Activity Study of Four Antibiotic (Lipo)glycopeptides: Vancomycin, Oritavancin, Dalbavancin, and Teicoplanin.

The antibiotic glycopeptide class, of which vancomycin is the original compound, has received due attention over the past few decades in search of antibiotics to overcome resistances developed by bacteria. Crucial for the understanding and further development of glycopeptides that possess desired antibacterial effects is the determination of their conformational behavior, as this sheds light on the mechanism of action of the compound. Among others, vibrational optical activity (VOA) techniques (vibrational circular dichroism and Raman optical activity) can be deployed for this, but the question remains to what extent these spectroscopic techniques can provide information concerning the molecular class under investigation. This contribution takes the last hurdle in the search for the capabilities of the VOA techniques in the conformational analysis of the antibiotic glycopeptide class by extending research that was previously conducted for vancomycin toward its three derivatives: oritavancin, dalbavancin, and teicoplanin. The principal information that can be drawn from VOA spectra is the conformation of the rigid cyclic parts of the glycopeptides and the aromatic rings that are part hereof. The addition or removal of carbohydrates does not induce noticeable VOA spectral responses, preventing the determination of the conformation they adopt.

Insights in the vibrational optical activity spectra of the antibiotic vancomycin in DMSO.

Vibrational circular dichroism (VCD) and Raman optical activity (ROA) are two spectroscopic techniques that are sensitive towards the conformational behaviour of molecules, and are often complementary herein. In this work we pursue the determination of the conformational ensemble of the antibiotic glycopeptide vancomycin in DMSO through comparison of experimental and computational spectra, both for VCD and ROA. ROA is found to be highly suitable for the task, identifying an ensemble that strongly resembles the NMR conformation. In the case of VCD, however, a too high sensitivity of the intensities with respect to minor conformational changes hampers a reliable conformational analysis. Whence attempting to improve the match between the VCD experiment and calculations by any means - e.g., by inducing minor conformational changes or including solvent effects in the calculations - we show that there is the risk of going down the rabbit hole. In conclusion, this work contributes to the broader understanding of where, when and how VCD and ROA can be deployed as techniques for conformational analysis.

Epimeric Mixture Analysis and Absolute Configuration Determination Using an Integrated Spectroscopic and Computational Approach-A Case Study of Two Epimers of 6-Hydroxyhippeastidine.

Structural elucidation has always been challenging, and misassignment remains a stringent issue in the field of natural products. The growing interest in discovering unknown, complex natural structures accompanies the increasing awareness concerning misassignments in the community. The combination of various spectroscopic methods with molecular modeling has gained popularity in recent years. In this work, we demonstrated, for the first time, its power to fully elucidate the 2-dimensional and 3-dimensional structures of two epimers in an epimeric mixture of 6-hydroxyhippeastidine. DFT calculation of chemical shifts was first performed to assist the assignment of planar structures. Furthermore, relative and absolute configurations were established by three different ways of computer-assisted structure elucidation (CASE) coupled with ORD/ECD/VCD spectroscopies. In addition, the significant added value of OR/ORD computations to relative and absolute configuration determination was also revealed. Remarkably, the differentiation of two enantiomeric scaffolds (crinine and haemanthamine) was accomplished via OR/ORD calculations with cross-validation by ECD and VCD.

Tackling Stereochemistry in Drug Molecules with Vibrational Optical Activity.

Chirality plays a crucial role in drug discovery and development. As a result, a significant number of commercially available drugs are structurally dissymmetric and enantiomerically pure. The determination of the exact 3D structure of drug candidates is, consequently, of paramount importance for the pharmaceutical industry in different stages of the discovery pipeline. Traditionally the assignment of the absolute configuration of druggable molecules has been carried out by means of X-ray crystallography. Nevertheless, not all molecules are suitable for single-crystal growing. Additionally, valuable information about the conformational dynamics of drug candidates is lost in the solid state. As an alternative, vibrational optical activity (VOA) methods have emerged as powerful tools to assess the stereochemistry of drug molecules directly in solution. These methods include vibrational circular dichroism (VCD) and Raman optical activity (ROA). Despite their potential, VCD and ROA are still unheard of to many organic and medicinal chemists. Therefore, the present review aims at highlighting the recent use of VOA methods for the assignment of the absolute configuration of chiral small-molecule drugs, as well as for the structural analysis of biologics of pharmaceutical interest. A brief introduction on VCD and ROA theory and the best experimental practices for using these methods will be provided along with selected representative examples over the last five years. As VCD and ROA are commonly used in combination with quantum calculations, some guidelines will also be presented for the reliable simulation of chiroptical spectra. Special attention will be paid to the complementarity of VCD and ROA to unambiguously assess the stereochemical properties of pharmaceuticals.

Exploring machine learning methods for absolute configuration determination with vibrational circular dichroism.

The added value of supervised Machine Learning (ML) methods to determine the Absolute Configuration (AC) of compounds from their Vibrational Circular Dichroism (VCD) spectra was explored. Among all ML methods considered, Random Forest (RF) and Feedforward Neural Network (FNN) yield the best performance for identification of the AC. At its best, FNN allows near-perfect AC determination, with accuracy of prediction up to 0.995, while RF combines good predictive accuracy (up to 0.940) with the ability to identify the spectral areas important for the identification of the AC. No loss in performance of either model is observed as long as the spectral sampling interval used does not exceed the spectral bandwidth. Increasing the sampling interval proves to be the best method to lower the dimensionality of the input data, thereby decreasing the computational cost associated with the training of the models.

Employing complementary spectroscopies to study the conformations of an epimeric pair of side-chain stapled peptides in aqueous solution.

Understanding the conformational preferences of free ligands in solution is often necessary to rationalize structure-activity relationships in drug discovery. Herein, we examine the conformational behavior of an epimeric pair of side-chain stapled peptides that inhibit the FAD dependent amine oxidase lysine specific demethylase 1 (LSD1). The peptides differ only at a single stereocenter, but display a major difference in binding affinity. Their Raman optical activity (ROA) spectra are most likely dominated by the C-terminus, obscuring the analysis of the epimeric macrocycle. By employing NMR spectroscopy, we show a difference in conformational behavior between the two compounds and that the LSD1 bound conformation of the most potent compound is present to a measurable extent in aqueous solution. In addition, we illustrate that Molecular Dynamics (MD) simulations produce ensembles that include the most important solution conformations, but that it remains problematic to identify relevant conformations with no a priori knowledge from the large conformational pool. Furthermore, this work highlights the importance of understanding the scope and limitations of the available techniques for conducting conformational analyses. It also emphasizes the importance of conformational selection of a flexible ligand in molecular recognition.

Paving the way to conformationally unravel complex glycopeptide antibiotics by means of Raman optical activity.

It is crucial for fundamental physical chemistry techniques to find their application in tackling real-world challenges. Hitherto, Raman optical activity (ROA) spectroscopy is one of the examples where a promising future within the pharmaceutical sector is foreseen, but has not yet been established. Namely, the technique is believed to be able to contribute in investigating the conformational behaviour of drug candidates. We, herein, strive towards the alignment of the ROA analysis outcome and the pharmaceutical expectations by proposing a fresh strategy that ensures a more complete, reliable, and transferable ROA study. The strategy consists of the treatment of the conformational space by means of a principal component analysis (PCA) and a clustering algorithm, succeeded by a thorough ROA spectral analysis and a novel way of estimating the contributions of the different chemical fragments to the total ROA spectral intensities. Here, vancomycin, an antibiotic glycopeptide, has been treated; it is the first antibiotic glycopeptide studied by means of ROA and is a challenging compound in ROA terms. By applying our approach we discover that ROA is capable of independently identifying the correct conformation of vancomycin in aqueous solution. In addition, we have a clear idea of what ROA can and cannot tell us regarding glycopeptides. Finally, the glycopeptide class turns out to be a spectroscopically curious case, as its spectral responses are unlike the typical ROA spectral responses of peptides and carbohydrates. This preludes future ROA studies of this intriguing molecular class.

Synthesis, Structure and Photophysical Properties of a New Class of Inherently Chiral Boron(III) Chelates-The tert-Leucine Complexes.

A new family of boron(III) chelates is introduced whereby molecular chirality, confirmed by circular dichroism, is imported during synthesis such that isolation of the diastereoisomers does not require separation procedures. The photophysical properties of two members of the family have been examined: the N,O,O-salicylaldehyde-based derivative shows pronounced intramolecular charge-transfer character in fluid solution and is weakly fluorescent, with a large Stokes shift. The corresponding 2-methylamino-benzaldehyde-derived N,N,O-chelate absorbs and fluoresces in the visible region with a much smaller Stokes shift. Orange fluorescence is also observed for this compound as a cast film. Temperature-dependence studies show that decay of the fluorescent state is weakly activated but emission is less than quantitative at 77 K. Quite rare for boron(III)-based chelates, this derivative undergoes intersystem crossing to form a meta-stable triplet-excited state. X-ray crystal structures are reported for both compounds, along with simulated ECD spectra.

A combined Raman optical activity and vibrational circular dichroism study on artemisinin-type products.

Artemisinin and two of its derivatives, dihydroartemisinin and artesunate, which are front line drugs against malaria, were investigated using Raman optical activity (ROA) and vibrational circular dichroism (VCD) experiments, both supported by density functional theory (DFT) level calculations. The experimental techniques combined with DFT calculations could show that dihydroartemisinin was present as an epimeric mixture in solution. In addition, an approximation of the epimeric ratio could be extracted which was in agreement with the ratio obtained by 1H-NMR spectroscopy. The current study also demonstrates that both ROA and VCD are able to assign the correct absolute configuration (AC) of artemisinin and artesunate out of all their possible diastereomers without any explicit knowledge on their correct stereochemistry and accentuates the synergetic effect between ROA and VCD in AC determination.