Chemometric Methods to Quantify 1D and 2D NMR Spectral Differences Among Similar Protein Therapeutics

NMR spectroscopy is an emerging analytical tool for measuring complex drug product qualities, e.g., protein higher order structure (HOS) or heparin chemical composition. Most drug NMR spectra have been visually analyzed; however, NMR spectra are inherently quantitative and multivariate and thus suitable for chemometric analysis. Therefore, quantitative measurements derived from chemometric comparisons between spectra could be a key step in establishing acceptance criteria for a new generic drug or a new batch after manufacture change. To measure the capability of chemometric methods to differentiate comparator NMR spectra, we calculated inter-spectra difference metrics on 1D/2D spectra of two insulin drugs, Humulin R® and Novolin R®, from different manufacturers. Both insulin drugs have an identical drug substance but differ in formulation. Chemometric methods (i.e., principal component analysis (PCA), 3-way Tucker3 or graph invariant (GI)) were performed to calculate Mahalanobis distance (D _M) between the two brands (inter-brand) and distance ratio (D _R) among the different lots (intra-brand). The PCA on 1D inter-brand spectral comparison yielded a D _M value of 213. In comparing 2D spectra, the Tucker3 analysis yielded the highest differentiability value (D _M = 305) in the comparisons made followed by PCA (D _M = 255) then the GI method (D _M = 40). In conclusion, drug quality comparisons among different lots might benefit from PCA on 1D spectra for rapidly comparing many samples, while higher resolution but more time-consuming 2D-NMR-data-based comparisons using Tucker3 analysis or PCA provide a greater level of assurance for drug structural similarity evaluation between drug brands.     

Calculated vibrational properties of semiquinones in the A1 binding site in photosystem I

Time-resolved (P700⁺A₁^? – P700A₁) FTIR difference spectra have been obtained using photosystem I (PSI) particles with several different quinones incorporated into the A₁ protein binding site. Difference spectra were obtained for PSI with unlabeled and ¹⁸O labeled phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone) and 2-methyl-1,4-naphthaquinone (2MNQ) incorporated, and for PSI with unlabeled 2,3-dimethyl-1,4-naphthoquinone (DMNQ) incorporated. (¹⁸O – ¹⁶O), (2MNQ – PhQ) and (DMNQ – PhQ) FTIR double difference spectra were constructed from the difference spectra. These double difference spectra allow one to more easily distinguish protein and pigment bands in convoluted difference spectra. To further aid in the interpretation of the difference spectra, particularly the spectra associated with the semiquinones, we have used two-layer ONIOM methods to calculate corresponding difference and double difference spectra. In all cases, the experimental and calculated double difference spectra are in excellent agreement. In previous two and three-layer ONIOM calculations it was not possible to adequately simulate multiple difference and double difference spectra. So, the computational approach outlined here is an improvement over previous calculations. It is shown that the calculated spectra can vary depending on the details of the molecular model that is used. Specifically, a molecular model that includes several water molecules that are near the incorporated semiquinones is required.     

Characterizing aqueous solution conformations of a peptide backbone using Raman optical activity computations

Mounting spectroscopic evidence indicates that alanine predominantly adopts extended polyproline II (PPII) conformations in short polypeptides. Here we analyze Raman optical activity (ROA) spectra of N-acetylalanine-N′-methylamide (Ala dipeptide) in H₂O and D₂O using density functional theory on Monte Carlo (MC) sampled geometries to examine the propensity of Ala dipeptide to adopt compact right-handed (α_R) and left-handed (α_L) helical conformations. The computed ROA spectra based on MC-sampled α_R and PPII peptide conformations contain all the key spectral features found in the measured spectra. However, there is no significant similarity between the measured and computed ROA spectra based on the α_L- and β-conformations sampled by the MC methods. This analysis suggests that Ala dipeptide populates the α_R and PPII conformations but no substantial population of α_L- or β-structures, despite sampling α_L- and β-structures in our MC simulations. Thus, ROA spectra combined with the theoretical analysis allow us to determine the dominant populated structures. Including explicit solute-solvent interactions in the theoretical analysis is essential for the success of this approach.     

Measurement of the fractional oxygenation of leghemoglobin in intact detached pea nodules by reflectance spectroscopy

A method is presented for the rapid measurement of the spectral properties of detached nodules of pea (Pisum sativum L. cv “Sparkle”) by diffuse reflectance spectroscopy. After correcting the spectra for surface light scattering, the spectrum of leghemoglobin is obtained. From this, the fractional oxygenation of leghemoglobin and the internal O₂ concentration can be calculated. With this method, we determined internal O₂ while measuring nitrogenase activity (C₂H₂) in detached pea nodules over a range of external O₂ concentrations. Nitrogenase activity was maximum when leghemoglobin was 25% oxygenated, corresponding to a calculated free O₂ concentration of 45 nanomolar in infected cells. Advantages of this method over previous methods which employed transmitted light are: (a) many nodules can be assayed simultaneously, (b) nitrogenase activity (C₂H₂) can be determined at the same time as spectra are recorded, and (c) spectra can be obtained from nodules submerged in buffer containing metabolic effectors.     

Experimental and calculated circular dichroism spectra of monoaza[5]helicenes

Circular dichroism (CD) spectra have been measured in the range of 400-200 nm on CH₃OH solutions of both enantiomers for the almost complete series of monoaza[5]helicenes, namely the molecules where the hetero N atom occupies positions 1, 3, 4, 5, 6, and 7, respectively (the 2 isomer is missing due to difficulties in the synthesis). CD spectra recorded at controlled room temperature allow one to define precise racemization rates, that are nicely interpreted on the basis of DFT molecular orbital calculations. Time-dependent DFT methods provide us with calculated CD and UV spectra, that are compared with the corresponding experimental data. We discuss the role of the N atom in determining the height of the racemization barrier and in shaping the appearance of the CD spectra.     

Characterisation of Authentic Lignin Biorefinery Samples by Fourier Transform Infrared Spectroscopy and Determination of the Chemical Formula for Lignin

Efficient methods for lignin characterisation are increasingly important as the field of lignin valorisation is growing with the increasing use of lignocellulosic feedstocks, such as wheat straw and corn stover, in biorefineries. In this study, we characterised a set of authentic lignin biorefinery samples in situ with no prior purification and minimal sample preparation. Lignin chemical formulas and lignin Fourier transform infrared (FTIR) spectra were extracted from mixed spectra by filtering out signals from residual carbohydrates and minerals. From estimations of C, H and O and adjustment for cellulose and hemicelluloses contents, the average chemical formula of lignin was found to be C₉H_10.2O_3.4 with slight variations depending on the biomass feedstock and processing conditions (between C₉H_9.5O_2.8 and C₉H_11.1O_3.6). Extracted FTIR lignin spectra showed many of the same characteristic peaks as organosolv and kraft lignin used as benchmark samples. Some variations in the lignin spectra of biorefinery lignin residue samples were found depending on biomass feedstock (wheat straw, corn stover or poplar) and on pretreatment severity, especially in the absorbance of bands at 1267 and 1032 cm^?1 relative to the strong band at ~1120 cm^?1. The suggested method of FTIR spectral analysis with adjustment for cellulose and hemicellulose is proposed to provide a fast and efficient way of analysing lignin in genuine lignin samples resulting from biorefineries.     

Molecules‐in‐molecules fragment‐based method for the calculation of chiroptical spectra of large molecules: Vibrational circular dichroism and Raman optical activity spectra of alanine polypeptides

The molecules‐in‐molecules (MIM) fragment‐based method has recently been adapted to evaluate the chiroptical (vibrational circular dichroism [VCD] and Raman optical activity [ROA]) spectra of large molecules such as peptides. In the MIM‐VCD and MIM‐ROA methods, the relevant higher energy derivatives of the parent molecule are assembled from the corresponding derivatives of smaller fragment subsystems. In addition, the missing long‐range interfragment interactions are accounted at a computationally less expensive level of theory (MIM2). In this work we employed the MIM‐VCD and MIM‐ROA fragment‐based methods to explore the evolution of the chiroptical spectroscopic characteristics of 3₁₀‐helix, α‐helix, β‐hairpin, γ‐turn, and β‐extended conformers of gas phase polyalanine (chain length n = 6–14). The different conformers of polyalanine show distinctive features in the MIM chiroptical spectra and the associated spectral intensities increase with evolution of system size. For a better understanding the site‐specific effects on the vibrational spectra, isotopic substitutions were also performed employing the MIM method. An increasing redshift with the number of isotopically labeled ¹³C=O functional groups in the peptide molecule was seen. For larger polypeptides, we implemented the two‐step‐MIM model to circumvent the high computational expense associated with the evaluation of chiroptical spectra at a high level of theory using large basis sets. The chiroptical spectra of α‐(alanine)₂₀ polypeptide obtained using the two‐step‐MIM model, including continuum solvation effects, show good agreement with the full calculations and experiment. This benchmark study suggests that the MIM‐fragment approach can assist in predicting and interpreting chiroptical spectra of large polypeptides.     

Development of a method for reconstruction of crowded NMR spectra from undersampled time-domain data

NMR is a unique methodology for obtaining information about the conformational dynamics of proteins in heterogeneous biomolecular systems. In various NMR methods, such as transferred cross-saturation, relaxation dispersion, and paramagnetic relaxation enhancement experiments, fast determination of the signal intensity ratios in the NMR spectra with high accuracy is required for analyses of targets with low yields and stabilities. However, conventional methods for the reconstruction of spectra from undersampled time-domain data, such as linear prediction, spectroscopy with integration of frequency and time domain, and analysis of Fourier, and compressed sensing were not effective for the accurate determination of the signal intensity ratios of the crowded two-dimensional spectra of proteins. Here, we developed an NMR spectra reconstruction method, “conservation of experimental data in analysis of Fourier” (Co-ANAFOR), to reconstruct the crowded spectra from the undersampled time-domain data. The number of sampling points required for the transferred cross-saturation experiments between membrane proteins, photosystem I and cytochrome b ₆ f, and their ligand, plastocyanin, with Co-ANAFOR was half of that needed for linear prediction, and the peak height reduction ratios of the spectra reconstructed from truncated time-domain data by Co-ANAFOR were more accurate than those reconstructed from non-uniformly sampled data by compressed sensing.     

At-line monitoring of key parameters of nisin fermentation by near infrared spectroscopy,chemometric modeling and model improvement

An analytical procedure has been developed for at-line (fast off-line) monitoring of 4 key parameters including nisin titer (NT), the concentration of reducing sugars, cell concentration and pH during a nisin fermentation process. This procedure is based on near infrared (NIR) spectroscopy and Partial Least Squares (PLS). Samples without any preprocessing were collected at intervals of 1 h during fifteen batch of fermentations. These fermentation processes were implemented in 3 different 5 l fermentors at various conditions. NIR spectra of the samples were collected in 10 min. And then, PLS was used for modeling the relationship between NIR spectra and the key parameters which were determined by reference methods. Monte Carlo Partial Least Squares (MCPLS) was applied to identify the outliers and select the most efficacious methods for preprocessing spectra, wavelengths and the suitable number of latent variables (n _LV). Then, the optimum models for determining NT, concentration of reducing sugars, cell concentration and pH were established. The correlation coefficients of calibration set (R _c) were 0.8255, 0.9000, 0.9883 and 0.9581, respectively. These results demonstrated that this method can be successfully applied to at-line monitor of NT, concentration of reducing sugars, cell concentration and pH during nisin fermentation processes.     

NMR studies of the conformation of the natural sweetener rebaudioside A

Rebaudioside A is a natural sweetener from Stevia rebaudiana in which four β-d-glucopyranose units are attached to the aglycone steviol. Its ¹H and ¹³C NMR spectra in pyridine-d₅ were assigned using 1D and 2D methods. Constrained molecular dynamics of solvated rebaudioside using NMR constraints derived from ROESY cross peaks yielded the orientation of the β-d-glucopyranose units. Hydrogen bonding was examined using the temperature coefficients of the hydroxyl chemical shifts, ROESY and long-range COSY spectra, and proton-proton coupling constants.     

Conformational analysis of peracetylated hexononitriles

The conformations of six peracetylated hexononitriles in solution have been investigated by Fourier-transform, proton n.m.r. spectroscopy at 90 MHz, with iterative analysis and simulation of many of the spectra. The conformation of tetra-O-acetyl-L-arabinononitrile has been re-examined by the same methods. A shift reagent [Eu(fod)₃-d₃₀] and spectra at 220 MHz were used to improve spectral dispersion, where necessary. For practically all of the derivatives studied, the vicinal, proton-proton coupling-constants are consistent with a zigzag conformation in which the cyano group lies in the plane of the other carbon atoms of the chain, unless this conformation contains a parallel 1,3-interaction of substituents. Other conformers that are also consistent with the coupling constants observed are discussed, including rotamers about chain-terminal, carbon-carbon bonds.     

Circular dichroism studies of α-aminoisobutyric acid-containing peptides: Chain length and solvent effects in alternating Aib-L-Ala and Aib-L-Val sequences

The CD spectra of the peptides Boc-X-(Aib-X)_n-OMe (n = 1, 2, 3) and Boc-(Aib-X)₅-OMe, where X = L -Ala or L -Val have been examined in several solvents. The X = Ala and Val peptides behave similarly in all solvents, suggesting that the Aib residues dominate the folding preferences of these peptides. The decapeptides adopt helical conformations in methanol and trifluoroethanol, with characteristic negative CD bands at 222 and 205 nm. In the heptapeptides, similar spectra with reduced intensities are observed. Comparison with nmr studies suggest that estimates of helical content in oligopeptides by CD methods may lead to erroneous conclusions. The pentapeptides yield solvent-dependent spectra indicative of conformational perturbations. Peptide association in dioxane results in an unusual spectrum with a single negative band at 210 nm for the decapeptides. Disaggregation is induced by the addition of methanol or water to dioxane solutions. Aggregation of the heptapeptides is less pronounced in dioxane, suggesting that a critical helix length may be necessary to promote association stabilized by helix dipole–dipole interactions.     

Potential bias in NMR relaxation data introduced by peak intensity analysis and curve fitting methods

We present an evaluation of the accuracy and precision of relaxation rates calculated using a variety of methods, applied to data sets obtained for several very different protein systems. We show that common methods of data evaluation, such as the determination of peak heights and peak volumes, may be subject to bias, giving incorrect values for quantities such as R₁ and R₂. For example, one common method of peak-height determination, using a search routine to obtain the peak-height maximum in successive spectra, may be a source of significant systematic error in the relaxation rate. The alternative use of peak volumes or of a fixed coordinate position for the peak height in successive spectra gives more accurate results, particularly in cases where the signal/noise is low, but these methods have inherent problems of their own. For example, volumes are difficult to quantitate for overlapped peaks. We show that with any method of sampling the peak intensity, the choice of a 2- or 3-parameter equation to fit the exponential relaxation decay curves can dramatically affect both the accuracy and precision of the calculated relaxation rates. In general, a 2-parameter fit of relaxation decay curves is preferable. However, for very low intensity peaks a 3 parameter fit may be more appropriate.     

Studies of the induction of mitotic gene conversion by ultraviolet irradiation: II. Action spectra

Action spectra for the induction of intragenic mitotic recombination (gene conversion) at the trp5 locus by UV are presented for three cell stages (T₀, T₉ and T₁₆) taken from synchronously growing cultures of Saccharomyces cerevisiae. The spectra over the range from 230 to 300 nm were taken mostly in 5-nm steps. The peak of action spectra was significantly shifted, regardless of the stage, toward the longer wavelengths as compared with that of the absorption spectrum of DNA (258 nm) or even that of thymine (265 nm). In one extreme case (T₁₆), the peak was shifted 17 nm from the absorption peak of DNA. Further, the spectrum changed its shape at the cell stage advanced from non-dividing (unbudded) (T₀) to a dividing phase (T₁₆). Furthermore, the induction cross section decreased by a large factor (about 40), regardless of the wavelength, in going from T₀ to T₁₆. From observations of the high photoreversibility of induced conversions, the major primary damage was thought to be pyrimidine dimers in the DNA.One plausible explanation, though not quite satisfactory from the quantitative viewpoint for these findings was that the increasing RNA during growth would screen the incident UV differentially with respect to the stage. If this explanation is correct, thymine dimers may still be considered, in spite of the shifts and deformations in the action spectra, as the major primary damage that triggers the long series of processes leading to gene conversion. Conventional methods for obtaining action spectra are discussed in comparison with the present method, which was based on sensitivity parameter a in the proposed dose (t)-frequency (f) relation, f = (at)^α (α is the multiplicity parameter).     

Action Spectra for in vivo and in vitro Conversions of Phycochrome b, a Reversibly Photochromic Pigment in a Blue-Green Alga, and Its Separation from Other Pigments

Phycochrome b, one of the reversibly photochromic pigments found in Tolypothrix distorta seems to exist in only two forms: P^b₅₀₀ and P^b₅₇₀. The pigment has been spectroscopically demonstrated in vivo. It has also been separated from other pigments. Two different methods for separation have been used: isoelectric focusing and gel filtration. Preparations of purified phycobilisomes contain phycochrome b. The in vivo and in vitro absorption difference spectra were determined as well as action spectra for the conversions in vitro and in vivo of P^b₅₀₀ to P^b₅₇₀ and vice versa. Transformation kinetics of phycochrome b show that the conversions in both directions are initially first-order reactions.     

Oxoperoxofluoromolybdate(VI) complexes. The first synthesis of oxodiperoxofluoromolybdate(VI) complexes

A number of oxoperoxofluoromolybdate(VI) complexes, viz. M₂[MoO(O₂)F₄] and M₂[MoO(O₂)₂F₂] where M = K and NH₄ and K[MoO(O₂)₂F]·2H₂O have been synthesised by various methods. The infrared spectra suggest that the peroxide groups are bonded to the molybdenum centre in a triangular bidentate manner.     

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes

The β-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D₂O for days and is assigned to the transmembrane β-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D₂O are assigned to the extracellular and periplasmic loops. The two-dimensional ¹H/¹⁵N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5° tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to α-helical membrane proteins.     

Comparative study of two highly purified forms of liver microsomal cytochrome P-450: circular dichroism and other properties.

The two major forms of rabbit liver microsomal cytochrome P-450, P-450_LM2 and P-450_LM4, which were previously shown to differ in their absorption spectra, electrophoretic and immunochemical properties, and substrate specificities, have been further characterized by several methods, (a) The two cytochromes have different CD spectra in the ferric state but similar spectra when reduced. Upon conversion of P-450_LM2 to P-420 by treatment with sodium dodecyl sulfate, the CD spectrum is greatly diminished except in the far ultraviolet region, whereas the conversion of P-450_LM4 toP-420 with this detergent results in a spectrum with a new positive band in the visible region, (b) Although P-450_LM4 has a much higher tryptophan content than P-450_LM2, the fluorescence spectra of these proteins are similar in magnitude. Upon denaturation, the fluorescence of P-450_LM4 increases, thereby indicating a large quenching effect in the native protein, (c) Studies on the interaction of dilauroylglyceryl-3-phosphorylcholine with the cytochromes showed that P-450_LM2 gives a much stronger Type I difference spectrum than does P-450_LM4. This phospholipid has no significant effect on the state of aggregation of these cytochromes as judged by calibrated gel filtration. The CD spectra of P-450_LM2 and P-450_LM4 are unchanged in the visible region but are enhanced in the far ultraviolet region upon the addition of phosphatidylcholine. The results appear to indicate an increase in α-helical content, particularly with P-450_LM4, in the presence of the phospholipid.     

Exploration of interaction of canthaxanthin with human serum albumin by spectroscopic and molecular simulation methods

The interaction between the food colorant canthaxanthin (CA) and human serum albumin (HSA) in aqueous solution was explored by using fluorescence spectroscopy, three‐dimensional fluorescence spectra, synchronous fluorescence spectra, UV–vis absorbance spectroscopy, circular dichroism (CD) spectra and molecular docking methods. The thermodynamic parameters calculated from fluorescence spectra data showed that CA could result in the HSA fluorescence quenching. From the K_SV change with the temperature dependence, it was concluded that HSA fluorescence quenching triggered by CA is the static quenching and the number of binding sites is one. Furthermore, the secondary structure of HSA was changed with the addition of CA based on the results of synchronous fluorescence, three‐dimensional fluorescence and CD spectra. Hydrogen bonds and van der Waals forces played key roles in the binding process of CA with HSA, which can be obtained from negative standard enthalpy (ΔH) and negative standard entropy (ΔS). Furthermore, the conclusions were certified by molecular docking studies and the binding mode was further analyzed with Discovery Studio. These conclusions can highlight the potential of the interaction mechanism of food additives and HSA.