A Versatile Mechanistic Probe for Three EnzymesIn an earlier publication (la) we described an experiment involving the enzymatically catalyzed hydrolysis of p-nitrophenyl acetate (PNPA) by bovine carbonic anhydrase II (BCA).Since that time, many student projects have been derived from these experiments at Seattle University and the University of Washington. They have allowed this part of our biochemistry laboratory activities to be expanded to include enzyme assays for two additional enzymes that also exhibit esterase activity. The reaction of eq 1 is versatile: It is catalyzed not only by carbonic anhydrase but also by a-chymotrypsin (a-CT) and acetylcholinesterase (AcCE). Thus, a single reaction, conveniently monitored, serves as a pedagogic tool and a mechanistic probe for
The ionic environment within solutions of lithium perchlorate-diethyl ether (LPDE) was probed by utilizing the extraordinary spectral shifts these media impart on various nitroanilines at 25 degrees C. These compounds all have UV-visible spectra that are sensitive to the polarity of the medium and the nitroanilines investigated all exhibited varying degrees of solvatochromatic behavior in LPDE solutions. In all cases, the low-energy absorbance band exhibited a dependence upon LiClO(4) concentration throughout the entire solubility range investigated. For 4-nitroaniline and N,N-dimethyl-4-nitroaniline bathochromic shifts of 51.3 and 62.0 nm, respectively, were observed on going from pure ether to a 5.7 M LPDE solution, corresponding to a stabilization of 10.55 and 11.13 kcal mol(-1), respectively, for this transition. Thus, as the medium changes from diethyl ether to one containing ionic clusters of lithium perchlorate-diethyl ether, less energy is required to transfer the molecules from their ground states to their first excited states. For 2,6-dibromo- and 2,6-diiodo-4-nitroaniline smaller red shifts of 19.0 and 9.0 nm, respectively, were noted over the same concentration range of LPDE, resulting in stabilizations of 4.45 and 2.11 kcal mol(-1), respectively. Analysis of the observed molar transition energies indicates that for 4-nitroaniline and N,N-dimethyl-4-nitroaniline the stabilization of the zwitterionic excited states of such push-pull molecules is on the order of 2.0 kcal mol(-1) per mol of added salt. Furthermore, such stabilization is independent of the composition of the media. Thus these compounds can act as solvent polarity indicators for LPDE solutions throughout the entire solubility range of LiClO(4) in diethyl ether. As such, linear relationships are seen between the E(T) values of 4-nitroaniline and N,N-dimethyl-4-nitroaniline and the log of the second-order rate constants for the [4+2] cycloaddition reaction of 9,10-dimethylanthracene and acrylonitrile in LPDE. We also observe linear relationships between the E(T) values of 4-nitroaniline and N,N-dimethyl-4-nitroaniline and the keto-enol ratio of acetylacetone in LPDE.
Acetylacetone (2,4-pentanedione, 1) is a molecule whose tautomeric forms are in dynamic equilibrium. Concentrated salt solutions in nonaqueous solvents exert a remarkable influence on the keto-enol ratio of this beta-diketone. The keto content of 1 increases from 5% in pure diethyl ether to 84.5% in a 4.14 M lithium perchlorate-diethyl ether (LPDE) solution, a nearly 17-fold increase. The equilibrium expression, K = [keto]/[enol] = k(f)/k(r), exhibits a linear dependence on [LiClO(4)], with the formal order of participation of lithium ion in the equilibrium being 1.0. A kinetic analysis reveals that k(f) is independent of LPDE concentration, whereas k(r) displays an inverse dependence on salt concentration, indicating preferential coordination of the keto tautomer with Li(+). Although 1 exits as the enol in water only to the extent of 16%, the addition of lithium perchlorate further reduces this figure. In an aqueous 4.02 M LiClO(4) solution, acetylacetone enol accounts for only 4.6% of the total amount of 2,4-pentanedione present. It has also been found that acetylacetone itself is an excellent solvent for LiClO(4) as well as for NaClO(4) with solutions containing up to 7.5 M LiClO(4) attainable. The enol content of 1 decreases dramatically from 81% to 7.4% on going from the neat liquid to a solution of 6.39 M LiClO(4) in acetylacetone.
ABSTRACT:Estimates of excited-state dipole moments for various 1,3-disubstituted azulenes possessing electron-withdrawing substituents, using the CIS/6-31ϩG(d,p) model, reveal that unlike azulene, the excited-state dipole moments are generally in the same direction as, although smaller in magnitude than, their ground-state counterparts. This gives rise to hypsochromic shifts in the UV-visible spectrum of these dipolar indicator molecules as solvent polarity is increased. Furthermore, B3LYP density functional calculations on several 1,3-diacylazulenes indicate that the s-cis conformation is preferred over s-trans.
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