NOE Measurements Research Articles

Oxidative stress-induced degradation of Brinzolamide: Isolation and in-depth characterization of unique hydroxylamine and oxime degradation products

Since the safety and efficacy of therapeutic products are strongly related to their stability and purity, impurities including the unavoidable degradation products may affect the pharmacological effect. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines Q3A requires the identification of process impurities and as well as degradation products in any drug substance to assess the inherent stability of the drug. The present work involves an ICH-guided degradation study for the Brinzolamide (BRZ), a topical ophthalmic drug which is generally used to lower the intraocular pressure (IOP) during glaucoma. Under oxidative stress at room temperature for 20 h, four degradation products (namely BRZ-Pk1, BRZ-PK2, BRZ-Pk3, and BRZ-Pk4) are isolated using advanced chromatographic techniques. Upon confirming the masses of the compounds using High-resolution mass spectrometry (HRMS), functional groups are identified with the help of Fourier-transform infrared spectroscopy (FT-IR). Extensive 1-dimensional (1D) and 2-dimensional (2D) Nuclear Magnetic Resonance spectroscopic (NMR) experiments especially 1D nOe, 1H-13C-HSQC and 1H-13C-HMBC unequivocally confirm the structures. Among the four compounds analyzed, three (BRZ-Pk1, BRZ-Pk2, and BRZ-Pk4) are novel, while BRZ-Pk3 was previously reported solely with mass spectrometric data. Nitrogen-based 2D NMR experiments are crucial for determining the oxidation state of hydroxylamine and oxime products within the molecules, and 1D nOe measurements help confirming E/Z isomerism (geometrical isomerism) for BRZ-Pk2 and BRZ-Pk4. All the proposed structures are justified with appropriate analytical data. The proposed mechanisms are expected to help in identifying the possible degradation pathways for similar pharmaceutical candidates.

Insights into mechanisms of MALT1 allostery from NMR and AlphaFold dynamic analyses

Mucosa-associated lymphoid tissue lymphoma-translocation protein 1 (MALT1) is an attractive target for the development of modulatory compounds in the treatment of lymphoma and other cancers. While the three-dimensional structure of MALT1 has been previously determined through X-ray analysis, its dynamic behaviour in solution has remained unexplored. We present here dynamic analyses of the apo MALT1 form along with the E549A mutation. This investigation used NMR 15N relaxation and NOE measurements between side-chain methyl groups. Our findings confirm that MALT1 exists as a monomer in solution, and demonstrate that the domains display semi-independent movements in relation to each other. Our dynamic study, covering multiple time scales, along with the assessment of conformational populations by Molecular Dynamic simulations, Alpha Fold modelling and PCA analysis, put the side chain of residue W580 in an inward position, shedding light at potential mechanisms underlying the allosteric regulation of this enzyme.

Conformations of 4‐tert‐Butyloxy‐, 4‐(Trimethylsilyl)oxy‐ and 4‐(Trimethylstannyl)oxy‐6‐methyl‐2H‐pyran‐2‐ones in the Crystalline State and in Solution

AbstractThe conformational studies in solution and in the solid state of substituted 6‐methyl‐4‐hydroxy‐2‐pyrones indicated that the substituents on the 4‐hydroxy group clearly favored the s‐cis arrangement towards the formal double bond. Steady state kinetic NOE measurements of the tert‐butyl, trimethylsilyl and the trimethylstannyl derivatives showed a marked preference for this conformation in solution. The structures determined by X‐ray diffraction demonstrated the exclusive presence of the same conformation in the solid state. In these three derivatives, the pertinent dihedral angle deviates only slightly from the value for coplanarity with the formal double bond. The predictions of the τ‐bond model are in accordance with the observations. The X‐ray structure of the trimethylstannyl derivative reveals pentacoordinated tin with short contacts to the oxygens at the 2 and 4 position of neighboring molecules. The tin center plays both the role of a protecting group for the 4‐hydroxy moiety and of a Lewis acid towards the 2‐carbonyl oxygen. The X‐ray structure of the stannyl derivative shows unique features which can be compared to the extended transition state of aldol reactions.

Molecular Dynamics-Assisted Optimization of Protein NMR Relaxation Analysis.

NMR relaxation analysis of the mobile residues in globular proteins is sensitive to the form of the experimentally fitted internal autocorrelation function, which is used to represent that motion. Different order parameter representations can precisely fit the same set of 15N R1, R2, and heteronuclear NOE measurements while yielding significantly divergent predictions of the underlying autocorrelation functions, indicating the insufficiency of these experimental relaxation data for assessing which order parameter representation provides the most physically realistic predictions. Molecular dynamics simulations offer an unparalleled capability for discriminating among different order parameter representations to assess which representation can most accurately model a wide range of physically realistic autocorrelation functions. Six currently utilized AMBER and CHARMM force fields were applied to calculate autocorrelation functions for the backbone H–N bond vectors of ubiquitin as an operational test set. An optimized time constant-constrained triexponential (TCCT) representation was shown to markedly outperform the widely used (Sf2,τs,S2) extended Lipari–Szabo representation and the more closely related (Sf2,SH2, SN2) Larmor frequency-selective representation. Optimization of the TCCT representation at both 600 and 900 MHz 1H converged to the same parameterization. The higher magnetic field yielded systematically larger deviations in the back-prediction of the autocorrelation functions for the mobile amides, indicating little added benefit from multiple field measurements in analyzing amides that lack slower (∼ms) exchange line-broadening effects. Experimental 15N relaxation data efficiently distinguished among the different force fields with regard to their prediction of ubiquitin backbone conformational dynamics in the ps–ns time frame. While the earlier AMBER 99SB and CHARMM27 force fields underestimate the scale of backbone dynamics, which occur in this time frame, AMBER 14SB provided the most consistent predictions for the well-averaged highly mobile C-terminal residues of ubiquitin.

LOW MOLECULAR METABOLITES OF FUNGI. 13, 3-DIMETOXYSTACHIBOTRINIS STACHYBOTRYS CHARTARUM

Stachybotrys chartarum produces a number of low molecular weight compounds. Column chromatography of the latter isolated the nitrogen-containing compound 3-methoxystachibotrin. The structure of 13,3-dimethoxystachibotrin was established on the basis of chemical transformations using the data of ЯМР1H, 13C, 2МЯМР1H-1H, 1H -13C chemical shift correlations (COSY), long-range 1H -13C interaction (НМВС) correlation, NOE measurements in a rotating coordinate system (ROESY), IR and mass- spectra of electron impact.

Cross-Linked Poly-4-Acrylomorpholine: A Flexible and Reversibly Compressible Aligning Gel for Anisotropic NMR Analysis of Peptides and Small Molecules in Water.

Determination of the solution conformation of both small organic molecules and peptides in water remains a substantial hurdle in using NMR solution conformations to guide drug design due to the lack of easy to use alignment media. Herein we report the design of a flexible compressible chemically cross-linked poly-4-acrylomorpholine gel that can be used for the alignment of both small molecules and cyclic peptides in water. To test the new gel, residual dipolar couplings (RDCs) and J-coupling constants were used in the configurational analysis of strychnine hydrochloride, a molecule that has been studied extensively in organic solvents as well as a small cyclic peptide that is known to form an α-helix in water. The conformational ensembles for each molecule with the best fit to the data are reported. Identification of minor conformers in water that cannot easily be determined by conventional NOE measurements will facilitate the use of RDC experiments in structure-based drug design.

Cross‐Linked Poly‐4‐Acrylomorpholine: A Flexible and Reversibly Compressible Aligning Gel for Anisotropic NMR Analysis of Peptides and Small Molecules in Water

AbstractDetermination of the solution conformation of both small organic molecules and peptides in water remains a substantial hurdle in using NMR solution conformations to guide drug design due to the lack of easy to use alignment media. Herein we report the design of a flexible compressible chemically cross‐linked poly‐4‐acrylomorpholine gel that can be used for the alignment of both small molecules and cyclic peptides in water. To test the new gel, residual dipolar couplings (RDCs) and J‐coupling constants were used in the configurational analysis of strychnine hydrochloride, a molecule that has been studied extensively in organic solvents as well as a small cyclic peptide that is known to form an α‐helix in water. The conformational ensembles for each molecule with the best fit to the data are reported. Identification of minor conformers in water that cannot easily be determined by conventional NOE measurements will facilitate the use of RDC experiments in structure‐based drug design.

Application of Relaxation Dispersion of Hyperpolarized 13C Spins to Protein–Ligand Binding

AbstractNuclear spin relaxation dispersion parameters are proposed as indicators of the binding mode of a ligand to a protein. Hyperpolarization by dissolution dynamic nuclear polarization (D‐DNP) provided a 13C signal enhancement between 3000–6000 for the ligand 4‐(trifluoromethyl) benzene‐1‐carboximidamide binding to trypsin. The measurement of 13C R2 relaxation dispersion was enabled without isotope enrichment, using a series of single‐scan Carr–Purcell–Meiboom–Gill experiments with variable refocusing delays. The magnitude in dispersion for the spins of the ligand is correlated to the position with respect to the salt bridge between protein and the amidine group of the ligand, indicating the ligand binding orientation. Hyperpolarized relaxation dispersion is an alternative to chemical shift or NOE measurements for determining ligand binding modes.

Application of Relaxation Dispersion of Hyperpolarized 13 C Spins to Protein-Ligand Binding.

Nuclear spin relaxation dispersion parameters are proposed as indicators of the binding mode of a ligand to a protein. Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) provided a 13 C signal enhancement between 3000-6000 for the ligand 4-(trifluoromethyl) benzene-1-carboximidamide binding to trypsin. The measurement of 13 C R2 relaxation dispersion was enabled without isotope enrichment, using a series of single-scan Carr-Purcell-Meiboom-Gill experiments with variable refocusing delays. The magnitude in dispersion for the spins of the ligand is correlated to the position with respect to the salt bridge between protein and the amidine group of the ligand, indicating the ligand binding orientation. Hyperpolarized relaxation dispersion is an alternative to chemical shift or NOE measurements for determining ligand binding modes.

Dynamic 15N{1H} NOE measurements: a tool for studying protein dynamics

Intramolecular motions in proteins are one of the important factors that determine their biological activity and interactions with molecules of biological importance. Magnetic relaxation of 15N amide nuclei allows one to monitor motions of protein backbone over a wide range of time scales. 15N{1H} nuclear Overhauser effect is essential for the identification of fast backbone motions in proteins. Therefore, exact measurements of NOE values and their accuracies are critical for determining the picosecond time scale of protein backbone. Measurement of dynamic NOE allows for the determination of NOE values and their probable errors defined by any sound criterion of nonlinear regression methods. The dynamic NOE measurements can be readily applied for non-deuterated or deuterated proteins in both HSQC and TROSY-type experiments. Comparison of the dynamic NOE method with commonly implied steady-state NOE is presented in measurements performed at three magnetic field strengths. It is also shown that improperly set NOE measurement cannot be restored with correction factors reported in the literature.

Regioselectivity of the Chan–Lam coupling of ambident nitropyrazoles with trans-styrylboronic acid

Regioselectivity of the Chan–Lam coupling of ambident nitropyrazoles with trans-styrylboronic acid depends on the base and catalyst nature and can vary the N(1)/N(2)-isomer ratio from ∼2 : 1 to ∼1 : 2. 2-Methyl-4-nitro- and 2,4-dinitroimidazoles are unreactive in this reaction. The structure of N(1)/N(2) isomers was elucidated by NOE measurements and by the comparison of experimental and DFT calculated 13C NMR chemical shifts.

The anti–cancer nano drug delivery 13C–edited/13C–filtered transferred dynamic 15N{1H} NOE measurements for studying DNA/RNA interactions with short non–linear motifs: A modern tool for studying DNA/RNA dynamics in gum cancer cells

The anti–cancer nano drug delivery 13C–edited/13C–filtered transferred dynamic 15N{1H} NOE measurements for studying DNA/RNA interactions with short non–linear motifs: A modern tool for studying DNA/RNA dynamics in gum cancer cells

Lithium–Olefin π-Complexes and the Mechanism of Carbolithiation: Synthesis, Solution Behavior, and Crystal Structure of (2,2-Dimethylpent-4-en-1-yl)lithium

We describe the first crystallographically characterized example of a nonconjugated olefin bound in a simple dihapto fashion to a lithium center, as part of a study of two alkyllithium compounds that contain C═C double bonds at the alkyl chain terminus: (2,2-dimethylbut-3-en-1-yl)lithium (1) and the related pentenyl compound (2,2-dimethylpent-4-en-1-yl)lithium (2). The Li–olefin interactions in the crystal structure of 2 serve as a model for those proposed to be present in the [RLi···olefin] intermediate in olefin carbolithiation reactions. As seen in other systems, the Li–olefin interaction is correlated with deshielding of the 1H NMR resonances of the olefinic hydrogen atoms. DOSY and NOE measurements show that 1 and 2 remain tetrameric in cyclohexane and that the lithium–olefin interactions persist in solution. Addition of a Lewis base such as THF to these ω-alkenyllithium species has two effects: the THF displaces the lithium–olefin interactions while accelerating the rate of carbolithiation. A deuteration experiment shows that compound 2 undergoes reversible carbolithiation to the corresponding cyclobutylmethyllithium species in the presence of Lewis bases, but this transformation is thermodynamically uphill owing to ring strain. In comparison, the longer chain hexenyl species (2,2-dimethylhex-5-en-1-yl)lithium is thermodynamically unstable with respect to the intramolecular carbolithiation product [(3,3-dimethylcyclopentyl)methyl]lithium (3). We suggest that rate-determining step in carbolithiation reactions may not always be formation of the C–C bond, as is often assumed, but in some cases may be formation of the lithium–olefin complex; the coordination of the olefin to lithium may occur in a concerted fashion with disaggregation of lithium clusters. Finally, we point out that activation enthalpies can be obtained solely from NMR line shapes above the coalescence point.

A Physical Modeling Approach to Determine Protein Structures from Paramagnetic NMR Measurements

Paramagnetic NMR approaches to protein structure determination can provide critical long-range information that may be difficult to obtain using standard NOE measurements, particularly for larger proteins. The paramagnetic relaxation enhancement (PRE) effect can be used to infer distances between residues and site-specifically engineered paramagnetic spin labels. These measurements are both sparse, due to the cost of making spin-labeled mutants, and ambiguous, due to the inherent flexibility of the spin-label. Here, we demonstrate the application of MELD (Modeling Employing Limited Data), a Bayesian approach combing sparse and ambiguous data with physical modeling, to the structure determination of a Calmodulin-peptide complex. Ten single-cysteine mutants were labeled with the common MTSL spin label, and PRE measurements were taken. Our approach can identify dominant conformations within 4 Å RMSD of the reference crystal structure using an implicit solvent model, while simulations without PRE data yield no near-native conformations. Inclusion of an explicit solvent significantly improves the model in comparison to the reference.

Lepadins I-K, 3- O-(3'-Methylthio)acryloyloxy-decahydroquinoline Esters from a Bahamian Ascidian Didemnum sp. Assignment of Absolute Stereostructures.

Three decahydroisoquinoline alkaloids, lepadins I-K, were isolated from a specimen of Didemnum sp. collected in the Bahamas. The structures of the new compounds were assigned by an integrated analysis of MS, IR, and 1H, 13C, and 2D NMR spectra. Like previously reported lepadins, the structures of the new compounds contain a decahydroquinoline heterocyclic core in lepadin I, and a new variation, an octahydroquinoline in lepadin J, but differ from earlier reported compounds by acylation of the 3-hydroxyl group by a rare 3'-methylthioacrylate. The absolute configuration of lepadin I was solved by interpretation of NOE measurements, and exciton coupled circular dichroism (ECCD) of the corresponding N- p-bromobenzoyl derivative. The latter constitutes a general method for determination of absolute configuration of the entire lepadin family. The configuration of the remote side-chain secondary carbinol was solved by the modified Mosher's esters method. Lepadin I inhibited butyrylcholineesterase (BuChE, IC50 3.1 μM), but only weakly inhibited acetylcholineesterase (AChE) (10% at 100 μM).

Highly regio- and Stereoselective Diels–Alder Cycloadditions of α-Alkoxyvinyl(ethoxy)carbene complexes with exo-heterocyclic dienes

A comparative study of reactivity and selectivity was carried out between the α-alkoxyvinyl(ethoxy)carbene complexes of group 6 metals (CO)5M = C[C(=CH2)(OR)]OEt [1a–c: M = Cr; 6a–c: M = Mo; 7a–c: M = W; a, R = Et; b, R = n–Pr; c, R = n–Bu] and exo-heterocyclic dienes 5a–e in Diels–Alder reactions. The cycloadditions of complexes 1, 6 and 7 with the unsubstituted diene 5a (R1 = H) were performed at 25 °C, proving to be highly regioselective in favor of the para pentacarbonyl cycloadducts 8, 10 and 12. On the other hand, the cycloadditions of complexes 1a–c and 6a–c with the substituted dienes 5b–e (R1 = Me or Et) took place at a higher temperature (50 °C) to afford the novel para-endo C,O-chelated tetracarbonyl cycloadducts 15a–f and 17a–d, respectively, in high regio- and stereoselectivity. DFT calculations of the transition states rationalized their selectivity. Under the same reaction conditions, the W(0) carbene complexes 7a–c produced the para-endo non-chelated pentacarbonyl complex adducts 19a–e. The stereochemical assignment of the cycloadducts was supported by NOE measurements. The C,O-chelated tetracarbonyl cycloadduct 15b and non-chelated cycloadduct 19d were further characterized by single-crystal X-ray diffraction.

Structural and conformational studies of the heparan sulfate mimetic PI-88.

The heparan sulfate mimetic PI-88 is a complex mixture of sulfated oligosaccharides with anti-metastatic and anti-angiogenic activity due to its potent inhibition of heparanase and heparan sulfate-dependent angiogenic growth factors. It was recently in Phase III clinical trials for postresection hepatocellular carcinoma. The major oligosaccharide constituents of PI-88 were prepared for the first time by sulfonation of individually purified phosphorylated oligosaccharides isolated from the PI-88 precursor. PI-88 and its components were subjected to detailed 1D and 2D NMR spectroscopic analysis. The spectra of the individual components greatly assisted the assignment of minor resonances in the 1H NMR spectrum of PI-88. The data also showed that the majority of the oligosaccharides in PI-88 are fully sulfated and that undersulfated species present are largely due to anomeric desulfation. The solution conformation of the phosphomannopentaose sulfate (major component) of PI-88 was then determined by a combination of molecular dynamics simulations and NOE measurements which may provide insights into its binding interactions with target proteins.

Structure and dynamics of the extended-helix state of alpha-synuclein: Intrinsic lability of the linker region.

The Parkinson's protein alpha-synuclein binds to synaptic vesicles in vivo and adopts a highly extended helical conformation when binding to lipid vesicles in vitro. High-resolution structural analysis of alpha-synuclein bound to small lipid or detergent micelles revealed two helices connected by a non-helical linker, but corresponding studies of the vesicle-bound extended-helix state are hampered by the size and heterogeneity of the protein-vesicle complex. Here we employ fluorinated alcohols (FAs) to induce a highly helical aggregation-resistant state of alpha-synuclein in solution that resembles the vesicle-bound extended-helix state but is amenable to characterization using high-resolution solution-state NMR. Analysis of chemical shift, NOE, coupling constant, PRE and relaxation measurements shows that the lipid-binding domain of alpha-synuclein in FA solutions indeed adopts a single continuous helix and that the ends of this helix do not come into detectable proximity to each other. The helix is well ordered in the center, but features an increase in fast internal motions suggestive of helix fraying approaching the termini. The central region of the helix exhibits slower time scale motions that likely result from flexing of the highly anisotropic structure. Importantly, weak or missing short- and intermediate-range NOEs in the region corresponding to the non-helical linker of micelle-bound alpha-synuclein indicate that the helical structure in this region of the protein is intrinsically unstable. This suggests that conversion of alpha-synuclein from the extended-helix to the broken-helix state represents a functionally relevant structural transition.

Existence of a Preferred Orientation for the Methoxy Group on an Extended Aromatic System.

AbstractThe conformations of a methoxy group introduced on naphthalene in position 2 has been investigated both experimentally (searches in crystallographic data, NOE measurements by NMR) and theoretically with ab initio calculations. While expected at first glance as being equivalent and predicted as such by force field methods, the two conformations where the methoxy is in the plane of the aromatic ring and turned either towards one or the other proton of naphthalene are actually very different in energy : ΔE=1.2 to 1.8 kcal mol−1, a surprisingly high value! The delocalization of the oxygen lone pair on the π‐system of the aromatic ring differs between the two conformers and is proposed to account predominantly for such unexpectedly large ΔE. It is our opinion that given the magnitude of the energies involved, force field methods should be corrected to take into account this conformational effect.

Integrating NOE and RDC using sum-of-squares relaxation for protein structure determination.

We revisit the problem of protein structure determination from geometrical restraints from NMR, using convex optimization. It is well-known that the NP-hard distance geometry problem of determining atomic positions from pairwise distance restraints can be relaxed into a convex semidefinite program (SDP). However, often the NOE distance restraints are too imprecise and sparse for accurate structure determination. Residual dipolar coupling (RDC) measurements provide additional geometric information on the angles between atom-pair directions and axes of the principal-axis-frame. The optimization problem involving RDC is highly non-convex and requires a good initialization even within the simulated annealing framework. In this paper, we model the protein backbone as an articulated structure composed of rigid units. Determining the rotation of each rigid unit gives the full protein structure. We propose solving the non-convex optimization problems using the sum-of-squares (SOS) hierarchy, a hierarchy of convex relaxations with increasing complexity and approximation power. Unlike classical global optimization approaches, SOS optimization returns a certificate of optimality if the global optimum is found. Based on the SOS method, we proposed two algorithms-RDC-SOS and RDC-NOE-SOS, that have polynomial time complexity in the number of amino-acid residues and run efficiently on a standard desktop. In many instances, the proposed methods exactly recover the solution to the original non-convex optimization problem. To the best of our knowledge this is the first time SOS relaxation is introduced to solve non-convex optimization problems in structural biology. We further introduce a statistical tool, the Cramér-Rao bound (CRB), to provide an information theoretic bound on the highest resolution one can hope to achieve when determining protein structure from noisy measurements using any unbiased estimator. Our simulation results show that when the RDC measurements are corrupted by Gaussian noise of realistic variance, both SOS based algorithms attain the CRB. We successfully apply our method in a divide-and-conquer fashion to determine the structure of ubiquitin from experimental NOE and RDC measurements obtained in two alignment media, achieving more accurate and faster reconstructions compared to the current state of the art.