Quantitative Online Monitoring of an Immobilized Enzymatic Network by Ion Mobility-Mass Spectrometry.

The forward design of in vitro enzymatic reaction networks (ERNs) requires a detailed analysis of network kinetics and potentially hidden interactions between the substrates and enzymes. Although flow chemistry allows for a systematic exploration of how the networks adapt to continuously changing conditions, the analysis of the reaction products is often a bottleneck. Here, we report on the interface between a continuous stirred-tank reactor, in which an immobilized enzymatic network made of 12 enzymes is compartmentalized, and an ion mobility-mass spectrometer. Feeding uniformly 13C-labeled inputs to the enzymatic network generates all isotopically labeled reaction intermediates and products, which are individually detected by ion mobility-mass spectrometry (IMS-MS) based on their mass-to-charge ratios and inverse ion mobilities. The metabolic flux can be continuously and quantitatively monitored by diluting the ERN output with nonlabeled standards of known concentrations. The real-time quantitative data obtained by IMS-MS are then harnessed to train a model of network kinetics, which proves sufficiently predictive to control the ERN output after a single optimally designed experiment. The high resolution of the time-course data provided by this approach is an important stepping stone to design and control sizable and intricate ERNs.

Generation, Spectroscopic Characterization, and Computational Analysis of a Six-Coordinate Cobalt(III)-Imidyl Complex with an Unusual S = 3/2 Ground State that Promotes N-Group and Hydrogen Atom-Transfer Reactions with Exogenous Substrates.

We report the synthesis and characterization of a mononuclear nonheme cobalt(III)-imidyl complex, [Co(NTs)(TQA)(OTf)]+ (1), with an S = 3/2 spin state that is capable of facilitating exogenous substrate modifications. Complex 1 was generated from the reaction of CoII(TQA)(OTf)2 with PhINTs at -20 °C. A flow setup with ESI-MS detection was used to explore the kinetics of the formation, stability, and degradation pathway of 1 in solution by treating the Co(II) precursor with PhINTs. Co K-edge XAS data revealed a distinct shift in the Co K-edge compared to the Co(II) precursor, in agreement with the formation of a Co(III) intermediate. The unusual S = 3/2 spin state was proposed based on EPR, DFT, and CASSCF calculations and Co Kß XES results. Co K-edge XAS and IR photodissociation (IRPD) spectroscopies demonstrate that 1 is a six-coordinate species, and IRPD and resonance Raman spectroscopies are consistent with 1 being exclusively the isomer with the NT ligand occupying the vacant site trans to the TQA aliphatic amine nitrogen atom. Electronic structure calculations (broken symmetry DFT and CASSCF/NEVPT2) demonstrate an S = 3/2 oxidation state resulting from the strong antiferromagnetic coupling of an •NTs spin to the high-spin S = 2 Co(III) center. Reactivity studies of 1 with PPh3 derivatives revealed its electrophilic characteristic in the nitrene-transfer reaction. While the activation of C-H bonds by 1 was proved to be kinetically challenging, 1 could oxidize weak O-H and N-H bonds. Complex 1 is, therefore, a rare example of a Co(III)-imidyl complex capable of exogenous substrate transformations.

Computing Arithmetic Functions Using Immobilised Enzymatic Reaction Networks.

Living systems use enzymatic reaction networks to process biochemical information and make decisions in response to external or internal stimuli. Herein, we present a modular and reusable platform for molecular information processing using enzymes immobilised in hydrogel beads and compartmentalised in a continuous stirred tank reactor. We demonstrate how this setup allows us to perform simple arithmetic operations, such as addition, subtraction and multiplication, using various concentrations of substrates or inhibitors as inputs and the production of a fluorescent molecule as the readout.