Structural cosolvent effects

While it is tempting to explain regulatory and cosolvent effects on the basis of conformational changes favorable or unfavorable to enzyme activity, it is much more difficult to demonstrate the actual involvement, amount, and structural details of such changes. Experimental evidence consists in most cases of bits and pieces provided by techniques such as absorption and fluorescence spectroscopy, circular dichroism, and magnetic circular dichroism. These tools work in solution (and, when desired, at subzero temperatures) to investigate not simply empty enzymes but enzyme—substrate intermediates. However, even with this information, the conformational basis of enzyme activity remains more postulated than demonstrated at the ball and stick level, and in spite of data about the number and sequence of intermediates, definition of their approximate nature, rate constants, and identification of the types of catalysis involved, full explanation of any particular reaction cannot be given and rests on speculative hypothesis. 

Proton transfer is sensitive to the local solute environment in liquid solutions as evidenced by the water quenching curves for 2-naphthol and its cyano- derivatives. We have used proton transfer as a mechanism to probe the cosolvent composition around a solute in supercritical fluids to discern any difference between local and bulk concentrations. No proton transfer was observed from either 2-naphthol or 5-cyano-2-naphthol, presumably indicating insufficient structure in the SCF to solvate the proton. Although significant cosolvent effects on the fluorescence emission were observed, these appear to be independent of the thermodynamic variables. 

Furthermore, the system of water, sec-butanol, and acetone was also used to investigate the cosolvent effect on the formation of hierarchically structured aluminas [123]. The resulting materials for sec-butanol and acetone had macropores and textural properties same as those when ethanol was used as the cosolvent... 

We have reported the first example of a ring-opening metathesis polymerization in C02 [144,145]. In this work, bicyclo[2.2.1]hept-2-ene (norbornene) was polymerized in C02 and C02/methanol mixtures using a Ru(H20)6(tos)2 initiator (see Scheme 6). These reactions were carried out at 65 °C and pressure was varied from 60 to 345 bar they resulted in poly(norbornene) with similar conversions and molecular weights as those obtained in other solvent systems. JH NMR spectroscopy of the poly(norbornene) showed that the product from a polymerization in pure methanol had the same structure as the product from the polymerization in pure C02. More interestingly, it was shown that the cis/trans ratio of the polymer microstructure can be controlled by the addition of a methanol cosolvent to the polymerization medium (see Fig. 12). The poly(norbornene) prepared in pure methanol or in methanol/C02 mixtures had a very high trans-vinylene content, while the polymer prepared in pure C02 had very high ds-vinylene content. These results can be explained by the solvent effects on relative populations of the two different possible metal... 

Miscible organic solutes modify the solvent properties of the solution to decrease the interfacial tension and give rise to an enhanced solubility of organic chemicals in a phenomenon often called cosolvency . According to theory, a miscible organic chemical such as a short chain alcohol will have the effect of modifying the structure of the water in which it is dissolved. On the macroscopic scale, this will manifest itself as a decrease in the surface tension of the solution [238,246]. 

Because cryosolvents must be used in studies of biochemical reactions in water, it is important to recall that the dielectric constant of a solution increases with decreasing temperature. Fink and Geeves describe the following steps (1) preliminary tests to identify possible cryosolvent(s) (2) determination of the effect of cosolvent on the catalytic properties (3) determination of the effect of cosolvent on the structural properties (4) determination of the effect of subzero temperature on the catalytic properties (5) determination of the effect of subzero temperature on the structural properties (6) detection of intermediates by initiating catalytic reaction at subzero temperature (7) kinetic, thermodynamic, and spectral characterization of detected intermediates (8) correlation of low-temperature findings with those under normal conditions and (9) structural studies on trapped intermediates. 

The second approach that has been rather popular with mixed aqueous solvents is to assume that the mixture is more or less structured than that of pure water. There is much evidence to show that the particular hydrogen-bonded structure of water influences many of the properties of electrolytes in water (15). If nonelectrolytes can modify the structure of water (15), they can have an indirect effect on the properties of electrolytes. This explanation has been particularly successful in the case of U + W mixtures (1,2). Such a simple approach is not as successful with hydrophobic cosolvents. For example, AHe°(W — W + TBA) are positive for both alkali halides (16) and tetraalkylammonium bro-... 

As a consequence of the model employed, values of Hb(H20) and N ought to be independent of the choice of the cosolvent as long as specific structural effects are absent. Therefore, we applied Equation 3 to the enthalpies of solution of n-Bu4NBr in DMSO-water mixtures (iO), since DMSO is a dipolar aprotic solvent like DMF. The best fit of the AHE values in this mixture yields Hb(H20) = —49.2 kj mol-1 and N/4 = 6.4, in excellent agreement with our values at 25°C given in Table III. 

Poly(vinylidene chloride) also dissolves readily in certain solvent mixtures. One component must be a sulfoxide or AlfV-dialkylamide. Effective cosolvents are less polar and have cyclic structures. 

In supercritical fluids, the possibility of local composition enhancements of cosolvent about a solute suggests that we should see enhancement of anion fluorescence if the water cosolvent clusters effectively about the 2-naphthol solute. Although in liquids the water concentration must be >30% to see anion emission, the higher diffusivity and density fluctuations in SCFs could allow stabilization of the anion at much lower water concentrations provided that the water molecules provide sufficient structure. Therefore the purpose of these experiments was to investigate 2-naphthol fluorescence in supercritical CO 2 with water cosolvent in the highly compressible region of the mixture to probe the local environment about the solute. 

The effect of water cosolvent on the emission of 2-naphthol is shown in Figure 2. There is some loss of fine structure but no detectable peak in the 400-500 nm range indicative of the anion species. This lack of anion emission is likely due to the low concentration of water in the solution, and since the concentration is limited by the water/CC>2 phase equilibria (18), there is a need for a more sensitive probe. 

The effect of water (0.003 mole fraction) and methanol (0.02 mole fraction) cosolvents on the emission of 5CN2N is shown in Figure 4. For this probe there is complete loss of fine structure, a significant red shift in the neutral emission with methanol cosolvent, but again, no detectable anion emission. 

ID NMR experiments are often used for quality-control purposes and can readily be used to confirm the purity of peptides. Simple ID spectra are also often used to determine whether a peptide is structured or unstructured or whether aggregation is present. Dispersion of the amide chemical shifts is an indicator of the former, whereas a narrow distribution, in the range of 7.5-8.5 ppm, is characteristic of unstructured peptides. Aggregation leads to broad peaks and spectra of poor quality. Adjustment of conditions by varying pH, buffer, cosolvent (e.g., acetonitrile for hydrophobic peptides), or peptide concentration and monitoring the effects on ID spectra is often used to find optimum conditions. 

The most commonly used generic term for a dissolved substance is solute, and this is the term that we will employ in most contexts, for both large and small compounds, that is, for macromolecules and micromolecules. A closely related term, cosolvent, is often used by physical chemists when the issue in question involves a dissolved substance that either stabilizes or destabilizes the structures of macromolecules. For instance, cosolvent is often used in literature on the effects of solutes on protein stability. A more restrictive and specific term that will be employed when we discuss the osmotic relationships of organisms is osmolyte. 

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