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Carbohydrates will likely play a central role in the future production of organic chemicals, but the understanding of pathways that are accessible in carbohydrate upgrading has remained unsatisfactory. The polyfunctionality of carbohydrates and their complex appearance in linear and cyclic forms with different stereochemistries can lead to diverse and interconverting pools of isomers along the entire reaction pathway. Here, we identify avoidable pathways toward byproducts and humins using an experimental high-resolution investigation into the routes of carbohydrate conversion with an emphasis on the conversion of C5 carbohydrates by Lewis acidic SnCl4 in dimethyl sulfoxide (DMSO)/water. Reaction tracking with C5 carbohydrates and deoxy variants was used to evaluate the effects of loss of individual functional groups on the pathways that are accessible in carbohydrate conversion. Especially, the removal of the hydroxy group at C5 enhances (and accelerates) the selectivity of carbohydrate conversion through an acyclic pathway, not only relative to the formation of furanic compounds but also relative to the formation of humins. Blocking the primary alcohol in xylose may hence benefit future biomass conversion strategies. Even in the absence of intermolecular reactions toward humins, the conversion of pentose follows second-order kinetics.

The conversion of lignocellulosic biomass and its components (carbohydartes, lignin) to precursor chemcials is expected to both expand the range of industrial chemicals and to reduce the dependence on fossil resoucres. Challenges in the green transition arise from the multifunctionality of biosourced reactants and from the difficulty in identifying highly selective processes for their conversion to useful precursors. Among the promising precursor molecules that can be derived from carbohydrates through chemocatalysis are lactate and its ester variants. Chemocatalytic conversion of glucose byconcentrated aqueous solutions of barium hydroxide at ambient temperatures in the absence of oxygen has been described as highly selective, but high-resolution NMR or MS characterizations of the product mixture and mechanistic insights are sparse. Here, we employ sensitivity enhanced (“hyperpolarized”) NMR to track the reaction cascade of hexose conversion by barium hydroxide and directy visualize transient enol and dihydroxyacetone species as the intermeditaes in the chemocatalytic pathway, similar to biochemical glycolysis. Quantitative NMR indicates that the conversion of glucose bybarium hydroxide under anaerobic conditions indeed is highly selective for the formation 2-hydroxy-3-deoxy acids, which can include longer equivalents than lactate. C4-C6 analogues (metasaccharinic acids) can in total account for nearly the same carbon fraction as lactate in a reaction avoiding the formation of oxidation products.

Many enzymes have latent activities that can be used in the conversion of non-natural reactants for novel organic conversions. A classic example is the conversion of benzaldehyde to a phenylacetyl carbinol, a precursor for ephedrine manufacture. It is often tacitly assumed that purified enzymes are more promising catalysts than whole cells, despite the lower cost and easier maintenance of the latter. Competing substrates inside the cell have been known to elicit currently hard-to-predict selectivities that are not easily measured inside the living cell. We employ NMR spectroscopic assays to rationally combine isomers for selective reactions in commercial S. cerevisiae. This approach uses internal competition between alternative pathways of aldehyde clearance in yeast, leading to altered selectivities compared to catalysis with the purified enzyme. In this manner, 4-fluorobenzyl alcohol and 2-fluorophenylacetyl carbinol can be formed with selectivities on the order of 90%. Modification of the cellular redox state can be used to tune product composition further. Hyperpolarized NMR shows that cellular reaction and pathway usage are affected by the xenochemical. Overall, we find that the rational construction of ternary or more complex substrate mixtures can be used forIn-Cell NMR spectroscopy to optimize upgrading of similar xenochemicals to dissimilar products with cheap whole-cell catalysts.

The central carbon metabolism of microbes will likely be used in future sustainable bioproduction. A sufficiently deep understanding of central metabolism would advance the control of activity and selectivity in whole-cell catalysis. Opposite to the more obvious effects of adding catalysts through genetic engineering, the modulation of cellular chemistry through effectors and substrate mixtures remains less clear. NMR spectroscopy is uniquely suited for in-cell tracking to advance mechanistic insight and to optimize pathway usage. Using a comprehensive and self-consistent library of chemical shifts, hyperpolarized NMR, and conventional NMR, we probe the versatility of cellular pathways to changes in substrate composition. Conditions for glucose influx into a minor pathway to an industrial precursor (2,3-butanediol) can thus be designed. Changes to intracellular pH can be followed concurrently, while mechanistic details for the minor pathway can be derived using an intermediate-trapping strategy. Overflow at the pyruvate level can be induced in nonengineered yeast with suitably mixed carbon sources (here glucose with auxiliary pyruvate), thus increasing glucose conversion to 2,3-butanediol by more than 600-fold. Such versatility suggests that a reassessment of canonical metabolism may be warranted using in-cell spectroscopy.

Electrochemical reduction can capture and utilize CO2 through its conversion to small chemicals and fuels. Analytical standard operating procedures for the accurate quantification of short chain acids, aldehydes, and alcohols by HPLC, GC-MS or NMR are not well established. Electrochemical reduction of CO2 produces the smallest conceivable organic compounds, which necessitates slow NMR quantifications due to slow T1 relaxation towards equilibrium magnetization. It is shown that the use of paramagnetic contrast agents or cooling of water/DMSO provides T1-optimized measurements with attractive sensitivity and speed. 1H NMR experiments that are widely used in the study of biological mixtures are found unsuitable for quantitative analyses of T1-optimized samples.