Single-Molecule Observations Provide Mechanistic Insights into Bimolecular Knoevenagel Amino Catalysis

Abstract

While single-molecule (SM) methods have provided new insights to various catalytic processes, bimolecular reactions have been particularly challenging to study. Here, the fluorogenic Knoevenagel condensation of an aromatic aldehyde with methyl cyanoacetate promoted by surface-immobilized piperazine is quantitatively characterized using super-resolution fluorescence imaging and stochastic analysis using hidden Markov modeling (HMM). Notably, the SM results suggest that the reaction follows the iminium intermediate pathway before the formation of a fluorescent product with intramolecular chargetransfer character. Moreover, the overall process is limited by the turnover rate of the catalyst, which is involved in multiple steps along the reaction coordinate.

Surface Preparation for Single-Molecule Fluorescence Imaging in Organic Solvents

“…Because the intensity of each frame in the trajectory was calculated by averaging the intensities of the 3 × 3 pixels (330 × 330 nm 2 ) area around the center position of the bright spot, the result indicated that repetitive nonspecific adsorptions could occur within the 3 × 3 pixels area. Thus, to minimize the influence of nonspecific adsorption on the analysis of single-molecule events, e.g., the fluorogenic reactions or the turnovers involving fluorescently labeled reactants, , it is better to make sure that the time intervals between target events are much shorter than the average interval τ i of nonspecific adsorptions.…”

“…The applications of single-molecule fluorescence imaging have been growing fast in the past decades, particularly in biochemical research, including biochemical reactions, − biomolecule interactions, − and super-resolution imaging. − Single-molecule imaging provided insights into the heterogeneity and dynamic properties of individual molecules that were usually hidden in bulk measurements due to the averaging effect. Because of the advantages, single-molecule imaging has also been used to study heterogeneous catalysis of nanomaterials. ,− Recently, there has been a growing interest in studying organic reactions by single-molecule fluorescence imaging. − To achieve that, it is critical to keep the fluorescent molecules well separated in space with low fluorescence background. The surface preparation procedure has been well developed for single-molecule fluorescence imaging of biomolecules in aqueous solutions. − However, little has been done to optimize the surface preparation procedure for single-molecule fluorescence imaging in organic solvents. , Compared to water, organic solvents typically contain higher percentage of impurities that are almost impossible to remove completely.…”

Exploring chemistry with single-molecule and -particle fluorescence microscopy

“…Another case study wherein single-molecule fluorescence microscopy techniques were used to distinguish between two plausible reaction mechanisms is the Knoevenagel condensation reaction, which is the condensation of an active methylene with an aldehyde in the presence of amine catalyst [34]. There are two plausible options for the mechanistic role of the amine: base catalyst Preparative synthetic chemistry: preparative chemistry, synthetic chemistry, or bench-scale chemistry is chemistry on the macroscale; chemistry on a sufficiently large scale for product isolation and use.…”

“…In organic solvents [3,44] Under air-free conditions (glovebox setup and adding solid reagents during imaging) [4,5] Imaging in flow cells [9,32,52] Higher temperature imaging (diffusion at 135°C, chemical reaction at 45°C) [24,52] Types of reactions and processes imaged Hydrolysis [6] Proton-transfer [53] Epoxidation [26,33] Solubilization [4,36] Deallylation [5] Redox (nanoparticle and photocatalysis) [10,11,17,[19][20][21]54] Cross-coupling [46] Oligomerization [14] Polymerization [40][41][42]55] Hydrogenation [56] Cycloaddition [47][48][49][50] Condensation [34,57] Corrosion [15,16] Molecular metal-ligand exchange/dissociation [37][38][39]44] Oxidative addition [4,35,46] Mechanistic questions answered Identification of previously unknown organic and organometallic intermediates …”

“…The first approach is 'participant', wherein the core of the fluorescent probe participates in the reaction; the chemical reaction occurs directly on the probe core molecule, leading to change in the fluorescence properties (switch on/off, or color change). The type of reactions associated with this approach are typically redox reactions [9,31], hydrolysis reactions [6], and other reactions that can produce fluorescent dyes in situ [33,34]. A popular probe in this category is the nonfluorescent resazurin that can be reduced to the highly fluorescent resorufin in the presence of a reducing agent (e.g., Figure IA,B).…”