Carbon Kinetic Isotope Effects and the Mechanisms of Acid-Catalyzed Decarboxylation of 2,4-Dimethoxybenzoic Acid and CO2 Incorporation into 1,3-Dimethoxybenzene.

The rate of decarboxylation of 2,4-dimethoxybenzoic acid (1) is accelerated in parallel to the extent that the carboxyl group acquires a second proton (1H+). However, the conjugate acid would resist C-C bond breaking as that would lead to formation of doubly protonated CO2. An alternative via formation of a higher-energy protonated phenyl tautomer (2H+) prior to C-C bond breaking would produce protonated CO2, an energetically inaccessible species that can be avoided by transfer of the carboxyl proton to water in the same step. Headspace sampling of CO2 that evolves in the acid-catalyzed process and analysis by GC-IRMS gives a smaller than expected value of 1.022 for the carbon kinetic isotope (CKIE), k12/k13. While this value establishes that C-C cleavage is part of the rate-determining process, intrinsic CKIEs for decarboxylation reactions are typically greater than 1.03. Computational analysis of the C-C bond cleavage from 2H+ gives an intrinsic CKIE of 1.051 and suggests two partially rate-determining steps in the decarboxylation of 1: transfer of the second carboxyl proton to the adjacent phenyl carbon and C-C cleavage in which the carboxyl proton is also transferred to water. Applying the principle of microscopic reversibility to fixation of CO2 in acidic solutions reveals the importance of proton transfers to both carbon and oxygen in the overall fixation process.

How Acid-Catalyzed Decarboxylation of 2,4-Dimethoxybenzoic Acid Avoids Formation of Protonated CO2.

The decarboxylation of 2,4-dimethoxybenzoic acid (1) is accelerated in acidic solutions. The rate of reaction depends upon solution acidity in a manner that is consistent with the formation of the conjugate acid of 1 (RCO2H2(+)), with its higher energy ring-protonated tautomer allowing the requisite C-C bond cleavage. However, this would produce the conjugate acid of CO2, a species that would be too energetic to form. Considerations of mechanisms that fit the observed rate law were supplemented with DFT calculations. Those results indicate that the lowest energy pathway from the ring-protonated reactive intermediate involves early proton transfer from the carboxyl group to water along with C-C bond cleavage, producing 1,3-dimethoxybenzene and CO2 directly.

Carbon kinetic isotope effects reveal variations in reactivity of intermediates in the formation of protonated carbonic acid.

Kinetic evidence suggests that acid-catalyzed decarboxylation reactions of aromatic carboxylic acids can occur by a hydrolytic process that generates protonated carbonic acid (PCA) as the precursor of CO2. Measurements of reaction rates and carbon kinetic isotope effects (CKIE) for decarboxylation of isomeric sets of heterocyclic carboxylic acids in acidic solutions reveal that C-C cleavage to form PCA is rate-determining with significant variation in the magnitude of the observed CKIE (1.018-1.043). Larger values are associated with the more reactive member in each isomeric pair. This variation is consistent with stepwise mechanisms in which C-C cleavage is competitive with C-O cleavage, leading to reversion to the protonated reactant to varying degrees with an invariant intrinsic CKIE for C-C cleavage. Thus, the relative barriers to reversion and formation of PCA control the magnitude of the observed CKIE in a predictable manner that correlates with reactivity. Application of the proposed overall mechanism reveals that carboxylation reactions in acidic solutions will proceed by way of initial formation of PCA.

Pressure-monitored headspace analysis combined with compound-specific isotope analysis to measure isotope fractionation in gas-producing reactions.

RATIONALE: Processes that lead to pressure changes in closed experimental systems can dramatically increase the total uncertainty in enrichment factors (ε) based on headspace analysis and compound-specific isotope analysis (CSIA). We report: (1) A new technique to determine ε values for non-isobaric processes, and (2) a general approach to evaluate the experimental error in calculated ε values. METHODS: ε values were determined by monitoring the change in headspace pressure from the production of CO2 in a decarboxylation reaction using a pressure gauge and measuring the δ(13) C values using CSIA. The statistical error was assessed over shorter reaction progress intervals to evaluate the impact of experimental error on the total uncertainty associated with calculated ε values. RESULTS: As an alternative to conventional compositional analysis, calculation of CO2 produced during the reaction monitored with a pressure gauge resulted in rate constants and ε values with improved correlation coefficients and confidence intervals for a non-isobaric process in a closed system. Further, statistical evaluation of the ε values as a function of reaction progress showed that uncertainty in data points for reaction progress (f) at late stages of the reaction can have a significant impact on the reported ε value. CONCLUSIONS: Pressure-monitored headspace analysis reduces the uncertainty associated with monitoring the reaction progress (f) based on estimating substrate removal and headspace dilution during sampling. Statistical calculations over shorter intervals should be used to evaluate the total error for reported ε values.

Protonated carbonic acid and reactive intermediates in the acidic decarboxylation of indolecarboxylic acids.

Elucidation of the mechanism for decarboxylation of indolecarboxylic acids over a wide range of solution acidity reveals the importance of protonated carbonic acid (PCA) as a reaction intermediate. In concentrated acid, the initial addition of water to the carboxyl group of the indolecarboxylic acid leads to a hydrated species that is capable of releasing PCA upon rate-determining carbon-carbon bond cleavage. The overall process is catalytic in water and acid, implicating PCA as a potential carboxylating reagent in the microscopic reverse reaction.

Prodrugs of a 1'-CN-4-Aza-7,9-dideazaadenosine C-Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV.

A discovery program targeting respiratory syncytial virus (RSV) identified C-nucleoside 4 (RSV A2 EC50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (1, GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1-NTP, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4. A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.

Protein-protein coupling and its application to functional red cell substitutes.

The need for an alternative to red cells for oxygen transport in transfusions has led to the creation of hemoglobin-based oxygen carriers, materials produced by chemical modification or genetic engineering of human or bovine hemoglobin. Modifications of the native proteins are necessitated by the spontaneous dissociation of the functional hemoglobin tetramers (alpha(2)beta(2)) into non-functional alphabeta dimers. Based on clinical observations of hypertension resulting from some of these materials, it was proposed that the stabilized tetramers are sufficiently small to extravasate through blood vessels and scavenge nitric oxide, depleting the endothelium of the signal for smooth muscle relaxation. In order to increase size and minimize extravasation while maintaining structure and function, methods for producing larger entities through protein-protein conjugation were developed. Approaches have included the use of nonspecific reagents that polymerize proteins (e.g., polyglutaraldehyde), conjugation to polyethylene glycol, expression of naturally occurring multimers and the use of selective reagents, which is the focus of this article.

Unprotected vinyl aziridines: facile synthesis and cascade transformations.

Functionalized vinylaziridines, readily available from water-stable aziridine aldehydes have led to the construction of a variety of stereochemically rich heterobicycles. A cascade ring-opening/ring-contraction mechanism operates in the course of the process. These results underscore the notion that interesting and useful nitrogen-mediated relay processes can arise when elements of strain are merged with the manifolds of enamine/iminium ion reactivity.