Is the single transition-state model appropriate for the fundamental reactions of organic chemistry?

In recent years, we have reported that a number of organic reactions generally believed to follow simple second-order kinetics actually follow a more complex mechanism. This mechanism, the reversible consecutive second-order mechanism, involves the reversible formation of a kinetically significant reactant complex intermediate followed by irreversible product formation. The mechanism is illustrated for the general reaction between reactant and excess reagent under pseudo-first-order conditions in eq. i where kf' is the pseudo-first-order rate constant equal to kf[Excess Reagent].Reactant + Excess reagent = Reactant complex = Products (i)The mechanisms are determined for the various systems, and the kinetics of the complex mechanisms are resolved by our "non-steady-state kinetic data analysis". The basis for the non-steady-state kinetic method will be presented along with examples. The problems encountered in attempting to identify intermediates formed in low concentration will be discussed.

Analytical Derivatization Reactions Based on Changes in Fluorescence Polarization: A Worthwhile Idea?

When a fluorophor is coupled to an analyte it will necessarily increase in size and, therefore, will depolarize fluorescence less rapidly than fluorophor by itself. Thus, it is possible to distinguish analyte fluorescence from excess reagent fluorescence on the basis of polarization even if the analytical reaction does not otherwise cause a change in fluorescence behavior. This would permit the use of sensitive fluorogenic reagents. However, sensitivity is lost when analyte is distinguished from excess reagent on the basis of polarization because the measured analytical signal is a relatively small difference between large signals. We have calculated detection limits for analyte resolved from excess reagent on the basis of fluorescence polarization relative to the detection limit for fluorophor by itself. Detection limits are calculated as a function of rotational relaxation times for analyte and excess reagent, intrinsic polarization, excess reagent concentrations, and nature of the noise. It is assumed that coupling the fluorophor to analyte does not affect fluorescence efficiency or wavelength distribution. The calculations serve to define the conditions under which it may be practical to resolve analyte from excess reagent on the basis of polarization.

A radiochemical titrant for the determination of the operational molarity of solutions of acid proteinases

N-Diazoacetyl-L-phenylalanine 3-phenyl[2,3-3H]propylamide was synthesized and shown to inhibit pepsin A (EC3,4,23.1) and cathepsin D (EC 3.4.23.5) irreversibly and stoicheiometrically in the presence of Cu2+. Quantitative separation of the inhibited enzyme from excess reagent by gel filtration followed by measurement of the radioactivity of the protein peak provided a method for determining the operational molarity of these enzymes. Several other putative active-site-directed irreversible inhibitors were synthesized, but were inactive. Data on the synthesis of these compounds have been deposited as Supplementary Publication SUP50096 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

QUANTITATIVE PRECIPITATION OF VARIOUS 3β-HYDROXYSTEROLS WITH DIGITONIN

Conditions are described for the quantitative precipitation of nine 3β-hydroxysterols with digitonin. After removal of excess reagent, the sterol and digitonin moieties are dissolved in a known volume of dry pyridine. An aliquot of the pyridine solution is processed to give a measure of total 3β-hydroxysterol by estimating the digitonin moiety with the use of anthrone. From another aliquot, the free sterols may be regenerated for differential-photometric and gas–liquid or thin-layer chromatographic analysis.Recoveries for each sterol at three levels of concentration were as follows: β-sitosterol, 98.6 ± 1.8% (mean and S.D.); campesterol, 99.1 ± 1.9%; stigmasterol, 98.9 ± 1.7%; stigmastanol, 99.1 ± 1.9%; cholesterol, 100.1 ± 1.5%; cholestanol, 100.8 ± 1.3%; coprostanol, 93.6 ± 1.6%; Δ7-cholestenol, 98.5 ± 1.8%; and 7-dehydrocholesterol, 91.9 ± 1.8%.

Determination of Choline in Feeds

The "free" and "bound" forms of choline were determined in feeds by a rapid method in which extraction and hydrolysis were accomplished simultaneously with a barium hydroxidemethanol-chloroform mixture. The choline was isolated from the hydrolysate by adsorption on a Florisil column. Passing ammonium reineckate through the column formed the choline reineckate which appeared as a pink band after excess reagent was washed out. The concentration of the choline was determined by eluting the reineckate band and measuring the absorbance at 526 mμ. Recoveries of added choline ranged from 98.9 to 100.8%.