Measurement and modelling solubility of amino acids and peptides in aqueous 2-propanol solutions.

Amino acids and peptides are essential components in the biochemical industry. The final products are employed in a wide range of applications and are often synthesized by fermentation and purified in a complex downstream process. One possible separation step is using an additional solvent to lower the solubility of the desired product and, thus, promote the crystallization of the particular component. Therefore, it is crucial to have accurate knowledge of the solubility of these components. In this work, the solubilities of 20 proteinogenic amino acids and 21 peptides in aqueous 2-propanol solutions were gravimetrically determined. Additionally, the pH values of the saturated liquid phases were measured and the crystal structures of solid crystals were analysed using X-ray diffraction. The anti-solvent 2-propanol caused a decrease in the solubilities of the amino acids and peptides upon increasing its mass fraction. Exceptions were found for amino acids with aromatic substituents, l-phenylalanine and l-tyrosine. The solubility of 15 amino acids and 18 peptides was successfully modelled using the equation of state PC-SAFT that used recently determined melting properties of the amino acids and peptides as input data.

Unravelling the nature of citric acid:L-arginine:water mixtures: the bifunctional role of water.

The use of water as a component of deep eutectic systems (DES) has raised some questions regarding its influence on the nature of the mixture. Does it form a DES or an aqueous solution and what is the role of water? In this work, the nature of citric acid:l-arginine:water mixtures was explored through phase equilibria studies and spectroscopic analysis. In a first step, PC-SAFT was validated as a predictive tool to model the water influence on the solid liquid equilibria (SLE) of the DES reline using the individual-component approach. Hence, activity coefficients in the ternary systems citric acid:l-arginine:water and respective binary combinations were studied and compared using ePC-SAFT. It was observed that the water-free mixtures citric acid:l-arginine showed positive deviation from Raoult's law, while upon addition of water strong negative deviation from Raoult's law was found, yielding melting depressions around 100 K. Besides these strong interactions, pH was found to become acidic (pH = 3.5) upon water addition, which yields the formation of charged species ([H2Cit]- and [l-arg]+). Thus, the increased interactions between the molecules upon water addition might be caused by several mechanisms such as hydrogen bonding or ionic forces, both being induced by water. For further investigation, the liquid mixtures citric acid:l-arginine:water were studied by FTIR and NMR spectroscopy. FTIR spectra disproved a possible solubility enhancement caused by salt formation between citric acid and l-arginine, while NMR spectra supported the formation of a hydrogen bonding network different from the binary systems citric acid:water and l-arginine:water. Either being a DES or other type of non-ideal solution, the liquefaction of the studied systems is certainly caused by a water-mediator effect based on the formation of charged species and cross interactions between the mixture constituents.

Melting properties of amino acids and their solubility in water.

The state-of-the-art unit operation for separation and purification of amino acids is still crystallization, which requires solubility data and melting properties of pure compounds. Since measuring solubility is time-consuming, prediction tools are desired. Further, melting properties are not yet available due to decomposition of amino acids upon slow heating. In this work, melting properties of twenty amino acids (except Met) were measured by Fast Scanning Calorimetry (FSC) with heating rates up to 20 000 K s-1. PC-SAFT was used to predict interactions in amino acid + water systems. Additionally, solubility, pH, and PXRD was measured. By combining FSC and PC-SAFT, the solubility of 15 amino acids was successfully predicted in a wide temperature range in good agreement with the experimental data. Thus, this work provides melting properties of amino acids for the first time and highlights the usefulness of such data to predict material properties such as aqueous solubility of amino acids.

Melting properties of peptides and their solubility in water. Part 1: dipeptides based on glycine or alanine.

Melting properties (melting temperature, melting enthalpy and heat capacity difference between liquid and solid phase) of biomolecules are indispensable for natural and engineering sciences. The direct determination of these melting properties by using conventional calorimeters for biological compounds is often not possible due to decomposition during slow heating. In the current study this drawback is overcome by using fast scanning calorimetry (FSC) to directly measure the melting properties of five dipeptides (glycyl-glycine, glycyl-l-alanine, l-alanyl-glycine, l-alanyl-l-alanine and cyclo(l-alanyl-glycine)). The experimental melting properties were used as inputs into a thermodynamic solid-liquid equilibrium relation to predict solubility of the dipeptides in water. The required activity coefficients were predicted with PC-SAFT using solubility-independent model parameters. PC-SAFT predicted different solubility profiles (solubility vs. temperature) of isomers. The predictions were validated by new experimental solubility data, and the crystal structure of the dipeptides in saturated solution was verified by X-ray diffraction. The different water solubility profiles of isomers (glycyl-l-alanine and l-alanyl-glycine) were found to be caused by the big difference in the melting enthalpy of the two dipeptides. To conclude, combining the PC-SAFT and FSC methods allows for accurate prediction of dipeptide solubility in water in a wide temperature range without the need to fit any model parameters to experimental solubility data.

New experimental melting properties as access for predicting amino-acid solubility.

The properties of melting are required for the prediction of solubility of solid compounds. Unfortunately, direct determination of the enthalpy of fusion and melting temperature by using conventional DSC or adiabatic calorimetry is often not possible for biological compounds due to decomposition during the measurement. To overcome this, fast scanning calorimetry (FSC) with scanning rates up to 2 × 104 K s-1 was used in this work to measure the melting parameters for l-alanine and glycine. The enthalpy of fusion and melting temperature (extrapolated to zero heating rate) were Δfus H = (22 ± 5) kJ mol-1 and T fus = (608 ± 9) K for l-alanine, and Δfus H = (21 ± 4) kJ mol-1 and T fus = (569 ± 7) K for glycine. These melting properties were used in the modeling framework PC-SAFT to predict amino-acid solubility in water. The pure-component PC-SAFT parameters and one binary parameter were taken from literature, in which these parameters were fitted to solubility-independent thermodynamic properties such as osmotic coefficients or mixture densities. It was shown that this allowed accurately predicting amino-acid solubility in water over a broad temperature range. The combined methodology of PC-SAFT and FSC proposed in this work opens the door for predicting solubility of molecules that decompose before melting.

Standard Gibbs energy of metabolic reactions: II. Glucose-6-phosphatase reaction and ATP hydrolysis.

ATP (adenosine triphosphate) is a key reaction for metabolism. Tools from systems biology require standard reaction data in order to predict metabolic pathways accurately. However, literature values for standard Gibbs energy of ATP hydrolysis are highly uncertain and differ strongly from each other. Further, such data usually neglect the activity coefficients of reacting agents, and published data like this is apparent (condition-dependent) data instead of activity-based standard data. In this work a consistent value for the standard Gibbs energy of ATP hydrolysis was determined. The activity coefficients of reacting agents were modeled with electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT). The Gibbs energy of ATP hydrolysis was calculated by combining the standard Gibbs energies of hexokinase reaction and of glucose-6-phosphate hydrolysis. While the standard Gibbs energy of hexokinase reaction was taken from previous work, standard Gibbs energy of glucose-6-phosphate hydrolysis reaction was determined in this work. For this purpose, reaction equilibrium molalities of reacting agents were measured at pH7 and pH8 at 298.15K at varying initial reacting agent molalities. The corresponding activity coefficients at experimental equilibrium molalities were predicted with ePC-SAFT yielding the Gibbs energy of glucose-6-phosphate hydrolysis of -13.72±0.75kJ·mol-1. Combined with the value for hexokinase, the standard Gibbs energy of ATP hydrolysis was finally found to be -31.55±1.27kJ·mol-1. For both, ATP hydrolysis and glucose-6-phosphate hydrolysis, a good agreement with own and literature values were obtained when influences of pH, temperature, and activity coefficients were explicitly taken into account in order to calculate standard Gibbs energy at pH7, 298.15K and standard state.