Frozen aqueous systems, reactions

Paramagnetic species formed by reactions of the radiation-produced transient species in ice and frozen aqueous systems have been studied by ESR technique. The radiation-produced electrons have been found to react e.g. with acidic solutes to form H-atoms and with group 11(b) metal ions to give the corresponding univalent radical ions, while the holes can react with anions such as S04 2 and H2P04 giving the radical ions S04 and HP04. Evidence that the electron and hole are coupled to each other, and may in fact exist in irradiated pure ice primarily in an (exciton-like) bound state has been discussed. The present work provides evidence for the reactions of the radiation-produced positive holes apart from the reactions of the electrons. 

Table III. Comparison of the Relative Rates of Reactions of the Electron with Different Scavengers in Liquid and Frozen Aqueous Systems...

The reverse action of a trypsin-free elastase isolated from porcine pancreas was studied in frozen aqueous systems and was found to catalyze peptide bond formation more effectively than in solution at room temperature (Haensler, 1998). The acceptance of free amino acids as nucleophilic amino components indicates a changed specificity of the endoprotease in frozen reaction mixtures. In elastase-catalyzed formation of Ser-, lie-, and Val-X-bonds in frozen aqueous reaction mix-... 

S. Gerisch, G. Ullmann, K. Stubenrauch, and H.-D. Jakubke, Enzymatic peptide synthesis in frozen aqueous systems influence of modified reaction conditions on the peptide yield, Biol. Chem. Hoppe Seyler, 1994, 375, 825-828. 

A step-by-step peptide synthesis from the N- to the C-terminus is not possible with chemical methods as it risks partial epimerization due to the repeated carboxy activation procedures, In constrast, the stereo- and regiospecificity of serine and cysteine proteases ensures integrity of the stereogenic center and allows ecological reaction conditions without side-chain protection. Scheme 4 shows the synthesis scheme using clostripain and chymo-trypsin as catalysts.The second coupling reaction was carried out by enzyme catalysis in a frozen aqueous system (see Section 4.2.3.1). 

Some new approaches to suppress competitive reactions in protease-catalyzed peptide synthesis have been developed in our group [14], namely leaving group manipulations at the acyl donor in kinetically controlled reactions, enzymatic synthesis in organic solvent-free microaqueous systems, cryoenzymatic peptide synthesis, and biotransformations in frozen aqueous systems using the reverse hydrolysis potential of proteases and other hydrolases... 

Wojnarovits L, Takacs E (2008) Radiat Phys Chem 77 225 Wypych M (1999) Pulse radiolysis induced transients in frozen aqueous systems at low temperatures. In Mayer J (ed) Properties and reactions of radiation induced transients. Selected topics. Polish Scientific Publishers, Warszava, pp 5—37 Yang K, Manno PJ (1959) J Am Chem Soc 81 3507 Zador E, Warman JM, Hummel A (1973) Chem Phys Lett 23 363... 

In attempts to isolate the aforementioned irradiated products of thymine derivatives at lower temperature, the photochemical reactions were carried out in frozen aqueous solutions containing either thymine or 1,3-dimethylthymine. The resulting products were not hydrates, but had elementary analyses corresponding to the starting material. Molecular weight determination indicated that the products were dimers, and infrared and ultraviolet spectral data suggested cyclo addition across the 5,6-double bond to form a cyclobutane system... 

A major point was made after a critical review of numerous reports on reaction rates in frozen systems (50). Kinetic-mechanistic surprises in frozen systems may not require exceptional hypotheses. Concentration effects may account for them. Even if a system appears to be completely solidified and, therefore, not amenable to analysis in terms of unfrozen liquid puddles (51), a liquid phase should still be considered a possibility. A case in point is provided by the efficient electron transfer observed between ferrous and ferric ions in an aqueous system frozen below its putative eutectic point (52). This seemed to require an ice structure in order to bridge the distance between reactants that were calculated to be too far apart for significant reactivity. However, it was pointed out that the assumed eutectic point was based only on the major... 

Oxidation of the amino acid moieties in irradiated aqueous systems by reaction with OH is well established for fluid systems, but it is not likely to be encountered in frozen systems. Being a strong oxidant, the OH reacts by electron transfer. It also adds readily to double bonds and abstracts H from C—H, N—H, and S—H bonds, but with lower reaction rate constants. A compendium of rate constants for aqueous solution has been published (52) and a few representative values for amino acids are shown in Table I. As discussed by Simic (53), the predominant sites for reaction in amino acids and peptides can be inferred from these values, which indicate that the ring groups are favored, while abstraction from the peptide backbone is less likely. Hydroxylation of the phenylalanine ring also occurs as was found for the prototype reaction with benzene (54). Formation of phenoxyl radical following OH addition to tyrosine should be similar to the mechanism established for phenol (55) in which elimination of water occurs as is shown in reaction 12 ... 

The best explanation of the good results for peptide syntheses in ice-water mixtures are based on the freeze-concentration-model, which just provides for a volume-reducing function for the ice while the liquid aqueous part is still the only relevant phase for the reaction. All observed enhancements of reaction rate would then have to be attributed to an increase in effective concentration. H-NMR relaxation time measurements have been used to determine the amount of unfrozen water in partially frozen systems, thus quantifying the extent of the freeze-concentration effect (Ullmann, 1997). Comparative studies in ice and at room temperature verify the importance of freeze-concentration which, however, is not sufficient for a complete understanding of the observed effects. 

The major advantages unique to cryoenzymology stem from the potential to accumulate essentially all of the enzyme in the form of a particular intermediate. The large rate reductions allow the most specific substrates to be used and hence provide the most accurate model for the in vivo catalyzed reactions. Virtually all the standard chemical and biophysical techniques used in studying proteins and enzymes under normal conditions may be used at subzero temperatures. The main limitations of the technique are the necessity to use aqueous organic cryosolvent systems to prevent the inherent rate-limiting enzyme-substrate diffusion of frozen solutions, and the possibility that the potential-energy surface for the reaction may be such that conditions in which an intermediate accumulates cannot be attained. 

Cyclodextrin-appended porphyrins are one of the suitable types of compound for ET models, since cyclodextrin (CD) has a hydrophobic cavity able to capture a small nonpolar molecule such as a quinone in aqueous solution. The fir.st example of a porphyrin- -CD photochemical system 137 was reported by Bolton, Weedon and coworkers in 1984. They monitored the photoinduced ET reaction in a frozen mixture of porphyrin 137 and p-benzoquinone by ESR spectroscopy. Irradiation of the solution showed the characteristic single ESR signal due to the generation of porphyrin cation radical and quinone anion radical species. The signal intensities in the ESR spectra, using a variety of quinones, indicated that the efficiency of the ET depended upon the reduction potential of the quinone acceptors. 

Figure 128 shows the apparatus used by Braman and Tomkins which consists of a sample reaction chamber, U-trap, the flame emission type detector, and conventional type photometric readout and recording system. Inorganic and methyltin compounds in aqueous solution in the reaction chamber are reduced to stannane or the corresponding methylstannanes by treatment with sodium borohydride solution buffered at pH 6.5. Helium carrier gas scrubs the volatile stannanes out of solution and into the liquid nitrogen-cooled U-trap where they are frozen out. Upon removal of the liquid nitrogen and warming, the stannanes are separated and carried into the detector. 

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