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Water is essential to the hydrolysis and conversion of lignocellulosic materials as it is both the medium through which enzymes diffuse to and products diffuse away from the reaction sites and a reactant in the hydrolysis reaction of the glycosidic bonds within the polysaccharides. However, little is known about how water interactions with the biomass change with solids content and how this affects mass transfer resistances during high solids saccharification. Nuclear magnetic resonance spectroscopy measurements of the T 2 relaxation times of water in cellulose suspensions were used to demonstrate that increases in solids content led to increases in the physical constraint of water in the suspensions. Moreover, the addition of either glucose (a monosaccharide which end-product inhibits b-glucosidase) or mannose (a stereoisomer of glucose that does not end-product inhibit b-glucosidase) further increased water constraint at all solids contents. The presence of either monosaccharide constrained water and inhibited saccharification rates to similar extents. This observation, coupled with the absence of cellobiose produced in the reactions, demonstrated that the presence of soluble sugars can negatively impact saccharification efficiency simply by increasing water constraint in cellulose suspensions before impacting enzyme activity. Furthermore, results are presented that demonstrate strong correlations between water constraint in cellulose suspensions with diffusivities of enzyme and monosaccharides within the system. These results are discussed in the context of increased viscosity of the aqueous fraction in the suspension resulting from increased watercellulose and water-solute interactions that ultimately increases diffusion resistances and decreases saccharification rates.

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Approximately 15% of all human tumors harbor mutant KRAS, a membrane-associated small GTPase and notorious oncogene. Mutations that render KRAS constitutively active will lead to uncontrolled cell growth and cancer. However, despite aggressive efforts in recent years, there are no drugs on the market that directly target KRAS and inhibit its aberrant functions. In the current work, we combined structure-based design with a battery of cell and biophysical assays to discover a novel pyrazolopyrimidine-based allosteric KRAS inhibitor that binds to activated KRAS with sub-micromolar affinity and disrupts effector binding, thereby inhibiting KRAS signaling and cancer cell growth. These results show that pyrazolopyrimidine-based compounds may represent a first-in-class allosteric noncovalent inhibitors of KRAS. Moreover, by studying two of its analogues, we identified key chemical features of the compound that interact with a set of specific residues at the switch regions of KRAS and play critical roles for its high-affinity binding and unique mode of action, thus providing a blueprint for future optimization efforts.