Self-association In Solution Research Articles

Improved Diffusion Interaction Parameter Measurement to Predict the Viscosity of Concentrated mAb Solutions.

During the developability assessment of therapeutic monoclonal antibody (mAb) candidates, utilization of robust high-throughput predictive assays enables rapid selection of top candidates with low risks for late-stage development. Predicting the viscosities of highly concentrated mAbs using limited materials is an important aspect of developability assessment because high viscosity can complicate manufacturability, stability, and administration. Here, we report a high-throughput assay measuring protein-protein interactions to predict mAb viscosity. The diffusion interaction parameter (kD) measures colloidal self-association in dilute solutions and has been reported to be predictive of the mAb viscosity at high concentrations. However, kD of Amgen early stage IgG1 mAb candidates measured in 10 mM acetate at pH 5.2 containing sucrose and polysorbate (denoted A52SuT) shows only weak correlation to their viscosities at 140 mg/mL in A52SuT. We hypothesize that kD measured in A52SuT reflects primarily long-range electrostatic repulsions because most of these mAb candidates carry strong net positive charges in this low ionic strength formulation with pH (5.2) well below pI values of mAb candidates. However, the viscosities of high concentration mAbs depend heavily on short-range molecular interactions. We propose an improved kD method in which salt is added to suppress charge repulsions and to allow for detection of key short-range interactions in dilute solutions. Salt types and salt concentrations were screened, and an optimal salt condition was identified. This optimized method was further validated using two test mAb sets. Overall, the method improves the Pearson R2 between kD and viscosity (6-230 cP) from 0.24 to 0.80 for a data set consisting of 37 mAbs.

Self-diffusion of aromatic compounds in the carbon tetrachloride – acetone binary system. NMR experiment and MD simulation

In this work, we studied the influence of the structure of molecules of aromatic compounds (methylparaben (MP), propylparaben (PP), syringic acid (SA), gallic acid (GA), protocatechualdehide (PAL), p-coumaric acid (p-CA), and caffeic acid (CA)) on their molecular association and self-diffusion in the binary carbon tetrachloride – acetone solvent. The self-diffusion coefficients of PAL, SA, and GA in the binary carbon tetrachloride – acetone‑d6 solvent were measured by the pulsed gradient spin-echo nuclear magnetic resonance (PGSE NMR) method at temperatures of 288, 298, and 308 K. The data obtained at 298 K were also compared with the data on the self-diffusion coefficients of MP, PP, CFA, and p-CA in the binary solvent available in the literature. According to the presented data, the self-diffusion coefficients of all the substances under consideration become greater with an increase in the acetone‑d6 concentration and decrease in the MP > PAL > PP > p-CA > SA > CA > GA series of compounds. Quantitative assessment of the degree of influence of molecular association on solute self-diffusion was carried out within the framework of a method based on the ratio of the self-diffusion coefficient of a solute to the self-diffusion coefficient of its monomers. The study shows that the degree of influence of molecular association of the solute on its self-diffusion is determined by the number and type of active functional groups in the structure of the solute molecules (MP < PP < PAL < SA < p-CA < CA < GA), but does not depend on the composition of the binary solvent. According to the results of molecular dynamics (MD) simulation, this effect correlates with the increase in the degree of self-association of solutes and decrease in the degree of solute – acetone heteroassociation observed when the carbon tetrachloride concentration becomes higher. This effect is also responsible for the similar concentration dependencies of the self-diffusion coefficients of active and inert compounds.

Corrosion inhibitor distribution on abrasive-blasted steels

HypothesisAbrasive-blasted steel surfaces exhibit a complex, multi-substrate environment. Adsorption to contaminant substrates can reduce the amount of available corrosion inhibitor and decrease its efficiency. Knowledge of where inhibitors preferentially adsorb is required. ExperimentsThe quantitative extent and strength of adsorption of the representative corrosion inhibitor benzotriazole (BTAH) from toluene to particular substrates is given, including corrections for solution self-association, and complemented by X-ray photoelectron spectroscopy (XPS), sum-frequency generation spectroscopy (SFG), and quartz crystal microbalance (QCM) measurements. FindingsAll substrates show adsorbed BTAH layers. Based on the adsorption strength, preferential adsorption is found to be in the order steel > iron oxide > calcium carbonate and garnet > silica – this is relevant when there is limited BTAH. However, with ample BTAH, the amounts adsorbed in the plateau regions of the isotherm are more relevant and the order is calcium carbonate and silica > iron oxide > garnet > steel. Although the contaminant substrates deplete the BTAH concentration, the steel should still have a complete monolayer of BTAH inhibitor. This work is part of a larger initiative developing novel methods of corrosion inhibitor delivery via the blasting process, to prevent corrosion between blasting and repainting.

Synthesis, electronic properties, and self-assembly of an alkylated dibenzo(biscorannulene)

The synthesis and properties of a novel fully-conjugated biscorannulene derivative were reported. This biscorannulene derivative shows self-association in solution and adopts a stacked geometry in crystal.

Use of Peptide Microarrays for Fast and Informative Profiling of Therapeutic Antibody Formulation Conditions.

Methods to optimize the solution behavior of therapeutic proteins are frequently time-consuming, provide limited information, and often use milligram quantities of material. Here, we present a simple, versatile method that provides valuable information to guide the identification and comparison of formulation conditions for, in principle, any biopharmaceutical drug. The subject protein is incubated with a designed synthetic peptide microarray; the extent of binding to each peptide is dependent on the solution conditions. The array is washed, and the adhesion of the subject protein is detected using a secondary antibody. We exemplify the method using a well-characterized human single-chain Fv and a selection of human monoclonal antibodies. Correlations of peptide adhesion profiles can be used to establish quantitative relationships between different solution conditions, allowing subgrouping into dendrograms. Multidimensional reduction methods, such as t-distributed stochastic neighbor embedding, can be applied to compare how different monoclonals vary in their adhesion properties under different solution conditions. Finally, we screened peptide binding profiles using a selection of monoclonal antibodies for which a range of biophysical measurements were available under specified buffer conditions. We used a neural network method to train the data against aggregation temperature, kD, percentage recovery after incubation at 25 °C, and melting temperature. The results demonstrate that peptide binding profiles can indeed be effectively trained on these indicators of protein stability and self-association in solution. The method opens up multiple possibilities for the application of machine learning methods in therapeutic protein formulation.

Molecular relaxation dynamics and self-association of dexamethasone sodium phosphate solutions.

Detailed concentration-dependent measurements of sound absorption and velocity have been performed in dexamethasone sodium phosphate (DSP) aqueous solutions in the MHz frequency range. A single well-resolved relaxation process dominates the experimental acoustic spectra following a Debye-type distribution function. The analysis of the temperature-dependent ultrasonic relaxation data also revealed analogous effect with concentration on the relaxation spectra. All acoustic parameters were estimated by means of a fitting procedure. The behavior of the relaxation frequency and amplitude with concentration allowed us to assign the observed process to self-association mechanism. Combining the ultrasonic and electric conductivity data, the self-association scheme has been established. The thermodynamic constants and the rate of the aggregation due to hydrophobic interactions have been estimated in view of the Eyring’s theory. The concentration dependence of relaxation amplitude and characteristic frequency revealed that the presence of additional relaxation processes in the spectra related to additional mechanisms, such as conformational changes and proton-transfer reaction is excluded and the self-association process considered here was found to dominate in this frequency range. The results have been discussed in view of the fair ability of DSP for hydrophobic interactions and aggregate formation in aqueous environment.Graphic abstract

A key point mutation (V156E) affects the structure and functions of human apolipoprotein A-I.

A naturally occurring point mutant of human apolipoprotein A-I (apoA-I), V156E, which is associated with extremely low plasma apoA-I and high density lipoprotein (HDL) levels, and coronary artery disease (Huang, W., Sasaki, J., Matsunaga, A., Nanimatsu, H., Moriyama, K., Han, H. Kugi, M., Koga, T., Yamaguchi, K., and Arakawa, K. (1998) Arterioscler. Throm. Vasc. Biol. 18, 389-396), was produced in an Escherichia coli expression system. The purified recombinant proapoA-I V156E mutant was examined in its structural and functional properties, both, in the lipid-free and lipid-bound states. In the lipid-free form the mutant protein exhibited small changes in conformation, but was more stable, and quite resistant to self-association, compared with control apoA-I. The V156E mutant was able to interact with phospholipid (PL) at high PL:protein ratios (95:1, mol/mol), but was inefficient in forming reconstituted HDL (rHDL) complexes at lower PL:protein ratios (40:1). In the lipid-bound, rHDL state, the mutant protein was somewhat more alpha-helical and formed a larger complex (110 A) than control apoA-I (97 A). Furthermore, the rHDL particles containing the V156E mutant did not rearrange to smaller particles in the presence of low density lipoproteins, and had minimal reactivity with lecithin-cholesterol acyltransferase (LCAT), compared with rHDL particles made with control apoA-I. These results suggest a key role for Val-156, or the adjacent central region of apoA-I in the modulation of apoA-I conformation, stability, and self-association in solution, and in the formation of small HDL, the conformational adaptability of apoA-I leading to structural rearrangements of HDL, and the activation of LCAT.

Polymorph-Selective Role of Hydrogen Bonding and π–π Stacking in p-Aminobenzoic Acid Solutions

Understanding molecular self-association in solution is vital for uncovering polymorph-selective crystal nucleation pathways. In this paper, we combine solution NMR spectroscopy and molecular dynamics simulations to shed light on the structural and dynamical features of p-aminobenzoic acid (pABA) in solution, and on their role in pABA crystals nucleation. pABA is known to yield different crystal forms (α, and β) depending on solvent choice and supersaturation conditions. NMR reveals that dominant interactions stabilizing pABA oligomers are markedly solvent-dependent: in organic solvents, hydrogen bonds dominate, while water promotes π-πstacking. Despite this clear preference, both types of interactions contribute to the variety of self-associated species in all solvents considered. MD simulations support this observation and show that pABA oligomers are short-lived and display a fluxional character, therefore indicating that the growth unit involved in pABA crystallization is likely to be a single molecule. Nevertheless, we note that the interactions dominating in pABA oligomers are indicative of the polymorph obtained from precipitation. In water, at low pABA concentrations - conditions that are known to yield crystals of the β form - carboxylic-carboxylic hydrogen bonds are exclusively asymmetric. At higher pABA concentration conditions in which the crystallization is known to yield the α form - a small but statistically significant fraction of symmetric carboxylic-carboxylic hydrogen-bonded dimers is present. We interpret the presence of these interactions in solvated pABA oligomers as indicative of the fact that a simultaneous and complete desolvation of two carboxylic groups, necessary to form the symmetric hydrogen-bonded dimer typical of the α crystal form, is accessible, therefore directing the nucleation pathway toward the nucleation of α-pABA.

Bifunctional Peptide-Polymer Conjugate-Based Fibers via a One-Pot Tandem Disulfide Reduction Coupled to a Thio-Bromo "Click" Reaction.

In view of the potential applications of fibers in material sciences and biomedicine, an effective synthetic strategy is described to construct peptide-based bifunctional polymeric conjugates for supramolecular self-association in solution. A direct coupling method of an α-acyl-brominated peptide Phe-Phe-Phe-Phe (FFFF) with a disulfide-bridged polymeric scaffold of poly(ethylene glycol) (PEG) (Mn,GPC = 8700 g mol–1, Đ = 2.02) is reported to readily prepare the bi-headed conjugate FFFF-PEG-FFFF (Mn,GPC = 3800 g mol–1, Đ = 1.10) via a one-pot, tandem disulfide reduction (based on tris(2-carboxyethyl)phosphine hydrochloride (TCEP)) coupled to a thio-bromo “click” reaction. The conjugate was investigated via transmission electron microscopy to exploit supramolecular fibril formation and solvent-dependent structuring into macroscale fibers via fibril–fibril interactions and interfibril cross-linking-induced bundling. Circular dichroism spectroscopic analysis is further performed to investigate β-sheet motifs in such fibrous scaffolds. Overall, this synthetic approach opens an attractive approach for a simplified synthesis of PEG-containing peptide conjugates.

Computational Study of Association of Dihydroxybenzoic Acids in Solution: Testing the Molecular Self-Association Computational Methodology for Formation of Binary Systems

Already validated methodology for self-association in solutions were used to evaluate if this method could also be used in other studies regarding association in solution. Three isomeric dihydroxybenzoic acids were used to calculate Gibbs free energies of association for homodimers, heterotetramers and associates with solvent molecules by using DFT calculations in multiple environments and having different conformations.

Synthesis and structural features of N-[(2-(trimethylsilyl)oxy)phenyl]-arylsulfonamides

N-[(2-(Trimethylsilyl)oxy)phenyl]-4-methylbenzenesulfonamide and N-[(2-trimethylsilyloxy)phenyl]-4-chlorobenzenesulfonamide were prepared by two different methods. Their structures were studied by X-ray single-crystal analysis and DFT calculations including MO and NBO analyses. Self-association in solutions was shown by FTIR-spectroscopy.

Pre-nucleation aggregation based on solvent microheterogeneity.

Investigation into pre-nucleation aggregates indicating hydrate formation of caffeine and theophylline in aqueous acetonitrile showed hydrate crystallisation at much lower water fraction than significant solute self-association. Molecular dynamics simulations indicate that the solvent separates on the molecular scale and that solute molecules preferentially localise on the phase interface.

Solution effects on the self-association of a water-soluble peptoid.

A desire to replicate the structural and functional complexity of proteins with structured, sequence-specific oligomers motivates study of the structural features of water-soluble peptoids (N-substituted glycine oligomers). Understanding the molecular-level details of peptoid self-assembly in water is essential to advance peptoids' application as novel materials. Peptoid 1, an amphiphilic, putatively helical peptoid previously studied in our laboratory, shows evidence of self-association in aqueous solution. In this work, we evaluate how changes to aqueous solution conditions influence the self-association of 1. We report that changes to pH influence the fluorescence and CD spectroscopic features as well as the peptoid's interaction with a solvatochromic fluorophore and its apparent size as estimated by size exclusion chromatography. Addition of guanidine hydrochloride and ammonium sulfate also modulate spectroscopic features of the peptoid, its interaction with a solvatochromic fluorophore, and its elution in size exclusion chromatography. These data suggest that the ordering of the self-assembly changes in response to pH and with solvent additives and is more ordered at higher pH and in the presence of guanidine hydrochloride. The deeper understanding of the self-association of 1 afforded by these studies informs the design of new stimuli-responsive peptoids with stable tertiary or quaternary structures.

Effect of solute aggregation on solubilization

Strong self-association of hydrophobic solutes takes place in water. However, solute self-association has often been neglected in understanding the aqueous solubility of drugs as well as their solubilization by excipients, cosolvents and hydrotropes. Based on a rigorous statistical thermodynamic foundation, here we show how to estimate the contribution from solute self-association to solubility and solubilization, based on experimental data such as solubility and the osmotic second virial coefficients. Such data show that solute self-association can indeed be negligible in most common cases of hydrotropic solubilization, Setschenow coefficients and the hydrophobic hydration.

Glycine’s pH-Dependent Polymorphism: A Perspective from Self-Association in Solution

As a simple amino acid, glycine (Gly)’s polymorphism is pH-dependent. The α form is typically obtained from aqueous solution between pH of 4 and 9, while the γ is produced at either lower or higher pH. Formation of cyclic, hydrogen-bonded dimer in water is debated as a possible cause for the formation of the α form. To further understand the pH-dependent polymorphism, our current study examined the self-association of Gly in aqueous solutions under a wide range of pH, utilizing NMR, FTIR, and electronic calculation. The results indicate that glycine molecules form open, not cyclic, hydrogen-bonded dimers in water. It is revealed that the dimerization becomes significant between pH of 4 and 8 but remains trivial at the two pH extremes. The apparent connection between the pH-dependent polymorphism and self-association in solution implies that formation of the α form is driven by the dimerization, and moreover, charged molecular species at the extreme pH facilitate stabilization of γ nuclei.

A peptoid supramolecular host for benzo[a]pyrene in water

Studies that identify a new supramolecular host for the highly toxic and prevalent carcinogen benzo[a]pyrene (BP) are reported here. By monitoring changes to absorption and fluorescence spectra, the interaction between BP and two peptoids (N-substituted glycine oligomers) of different length and self-association propensities were compared. Peptoid length and solvent choice influence spectral features. Observed trends support that peptoid self-association in aqueous solution generates a hydrophobic core to which BP can associate.

‘Frustrated’ hydrogen-bonded self-associated systems as templates towards DNA incorporated nanostructure formation

Herein, we present the synthesis of a thymine nucleobase-appended ‘frustrated’ monomer, which exhibits self-association in DMSO solutions through the formation of hydrogen bonds. This self-association process has been explored in both competitive DMSO solutions and the solid state, using a combination of NMR and single crystal X-ray diffraction techniques. The self-associative equilibria within the solution state are balanced in such a way that the hydrogen bond donating and accepting thymine residue present within the monomeric structure is free to coordinate further guest species such as the complimentary DNA base adenine. The adenine simulants, 2-aminopyridine and 2,6-diaminopyridine have been used to explore the potential of these self-associated structures towards the coordination of complimentary DNA base pairs.

Phenanthrylene-butadiynylene and Phenanthrylene-thienylene Macrocycles: Synthesis, Structure, and Properties.

A series of macrocycles consisting of 9,10-substituted phenanthrenes connected by butadiynylene linkers in positions 3 and 6 has been described as well as their transformation into the corresponding phenanthrylene-thienylene macrocycles. Structure and properties of the macrocycles, such as self-association in solution and optical and electrochemical properties, were studied and reported in a comparative manner with respect to the effects of the different sizes and shapes of the macrocycles and the character and length of their side chains.

Thermodynamics and kinetics of amphotericin B self-association in aqueous solution characterized in molecular detail.

Amphotericin B (AmB) is a potent but toxic drug commonly used to treat systemic mycoses. Its efficiency as a therapeutic agent depends on its ability to discriminate between mammalian and fungal cell membranes. The association of AmB monomers in an aqueous environment plays an important role in drug selectivity, as oligomers formed prior to membrane insertion – presumably dimers – are believed to act differently on fungal (ergosterol-rich) and mammalian (cholesterol-rich) membranes. In this work, we investigate the initial steps of AmB self-association by studying the structural, thermodynamic and spectral properties of AmB dimers in aqueous medium using molecular dynamics simulations. Our results show that in water, the hydrophobic aggregation of AmB monomers yields almost equiprobable populations of parallel and antiparallel dimers that rapidly interconvert into each other, and the dipole-dipole interaction between zwitterionic head groups plays a minor role in determining the drug’s tendency for self-aggregation. A simulation of circular dichroism (CD) spectra indicates that in experimental measurements, the signature CD spectrum of AmB aggregates should be attributed to higher-order oligomers rather than dimers. Finally, we suggest that oligomerization can impair the selectivity of AmB molecules for fungal membranes by increasing their hydrophobic drive for non-specific membrane insertion.

Synthesis and characterization of novel dibenz[a,c]anthracenedicarboxythioimides: the effect of thionation on self-assembly

The effect of thionation on the formation of columnar liquid crystalline phases of dibenzanthracenedicarboximides as well as their self-association in solution is described.