A study of the hydrolysis of formamide is reported with the aims of isolating the water reaction for hydrolysis from the acid and base hydrolysis terms and determining the solvent deuterium kinetic isotope effect (dkie) on base-catalyzed hydrolysis. Respective activation parameters (ΔH and ΔS) of (17.0 ± 0.4) kcal mol1 and (18.8 ± 1.3) cal mol1 K1 for the acid reaction and (17.9 ± 0.2) kcal mol1 and (11.1 ± 0.5) cal mol1 K1 for the base reaction were determined from Eyring plots of the second-order rate constants over the range of 27120°C. Kinetic studies at the minima of the pH/rate profiles in the pH range from 5.6 to 6.2 in MES buffers at 56°C, and in the pH range of 4.256.87 in acetate and phosphate buffers at 120°C are reported. At 56°C the available data fit the expression k56obs = 0.00303[H3O+] + 0.032[HO] + (3.6 ± 0.1) × 109, while at 120°C the data fit k120obs = (0.15 ± 0.02)[H3O+] + (3.20 ± 0.24)[HO] + (1.09 ± 0.29) × 106. Preliminary experimental estimates of Ea (ln A) of 22.5 kcal mol1 (15.03) for the water rate constant (kw) are calculated from an Arrhenius plot of the 56 and 120°C data giving an estimated kw of 1.1 × 1010 s1 (t1/2 = 199 years) at 25°C. Solvent dkie values of kOH/kOD = 1.15 and 0.77 ± 0.06 were determined at [OL] = 0.075 and 1.47 M, respectively. The inverse value is determined under conditions where the the first step of the reaction dominates and is analyzed in terms of a rate-limiting attack of OL.Key words: formamide, activation parameters, water reaction, acid and base hydrolysis, solvent kinetic isotope effect.
Alzheimer’s disease is characterized by the misfolding and self-assembly of the amyloidogenic protein amyloid-β (Aβ). The aggregation of Aβ leads to diverse oligomeric states, each of which may be potential targets for intervention. Obtaining insight into Aβ oligomers at the atomic level has been a major challenge to most techniques. Here, we use magic angle spinning recoupling 1H-1H NMR experiments to overcome many of these limitations. Using 1H-1H dipolar couplings as a NMR spectral filter to remove both high and low molecular weight species, we provide atomic-level characterization of a non-fibrillar aggregation product of the Aβ1-40 peptide using non-frozen samples without isotopic labeling. Importantly, this spectral filter allows the detection of the specific oligomer signal without a separate purification procedure. In comparison to other solid-state NMR techniques, the experiment is extraordinarily selective and sensitive. A resolved 2D spectra could be acquired of a small population of oligomers (6 micrograms, 7% of the total) amongst a much larger population of monomers and fibers (93% of the total). By coupling real-time 1H-1H NMR experiments with other biophysical measurements, we show that a stable, primarily disordered Aβ1-40 oligomer 5–15 nm in diameter can form and coexist in parallel with the well-known cross-β-sheet fibrils.
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