Bioresponsive hydrogels

We highlight recent developments in hydrogel materials with biological responsiveness built in. These ‘smart’ biomaterials change properties in response to selective biological recognition events. When exposed to a biological target (nutrient, growth factor, receptor, antibody, enzyme, or whole cell), molecular recognition events trigger changes in molecular interactions that translate into macroscopic responses, such as swelling/collapse or solution-to-gel transitions. The hydrogel transitions may be used directly as optical readouts for biosensing, linked to the release of actives for drug delivery, or instigate biochemical signaling events that control or direct cellular behavior. Accordingly, bioresponsive hydrogels have gained significant interest for application in diagnostics, drug delivery, and tissue regeneration/wound healing

Pathway-dependent gold nanoparticle formation by biocatalytic self-assembly

YesWe report on the use of non-equillibrium biocatalytic self-assembly and gelation to guide the reductive synthesis of gold nanoparticles. We show that biocatalytic rates simultaneously dictate supramolecular order and presentation of reductive phenols which in turn results in size control of nanoparticles that are formed.BBSRC funding (BB/K007513/1); European Research Council under the European Union’s Seventh Framework Programme, ERC (Starting Grant EMERgE) grant agreement no. 258775

Tunable supramolecular gel properties by varying thermal history

YesThe possibility of using differential pre‐heating prior to supramolecular gelation to control the balance between hydrogen‐bonding and aromatic stacking interactions in supramolecular gels and obtain consequent systematic regulation of structure and properties is demonstrated. Using a model aromatic peptide amphiphile, Fmoc‐tyrosyl‐leucine (Fmoc‐YL) and a combination of fluorescence, infrared, circular dichroism and NMR spectroscopy, it is shown that the balance of these interactions can be adjusted by temporary exposure to elevated temperatures in the range 313–365 K, followed by supramolecular locking in the gel state by cooling to room temperature. Distinct regimes can be identified regarding the balance between H‐bonding and aromatic stacking interactions, with a transition point at 333 K. Consequently, gels can be obtained with customizable properties, including supramolecular chirality and gel stiffness. The differential supramolecular structures also result in changes in proteolytic stability, highlighting the possibility of obtaining a range of supramolecular architectures from a single molecular structure by simply controlling the pre‐assembly temperature.FP7 Ideas: European Research Council. Grant Number: 25877

Solid-to-solid biocatalysis: thermodynamic feasibility and energy efficiency

Enzymes can catalyse solid-to-solid condensation reactions in highly concentrated aqueous substrate suspensions. Reaction products precipitate from the reaction mixture and very high conversion yields can be obtained in low volume reactors. Solid-to-solid biocatalysis combines the advantages of using enzymes in aqueous media with the high conversion yields that are typically associated with non-aqueous biocatalysis. In this article, methods are presented for the calculation of the Gibbs free energy changes and heats of reaction of condensation reactions to form amides. The overall enthalpy change of the enzymatic reaction was compared to that of the conventional chemical methods and it was found that the enzymatic reaction produces a third of the heat with better atom efficiency

Understanding enzyme action on immobilised substrates

With increasing interest in automated synthesis and screening protocols, solid supported chemistry and biochemistry are attractive technologies. Studies with surface-immobilised substrates have been carried out to analyse enzyme accessibility, kinetics and thermodynamics. Several interesting new methods have been developed to monitor enzyme action on substrates attached to a solid phase such as polymer beads glass or gold surfaces. These include fluorescence measurements, MALDI-TOF mass spectrometry, and the use of quartz crystal microbalances to measure weight changes of immobilised molecules directly on the surface. Approaches that allow spatial resolution in single beads have also been reported. The ability of enzymes to reach the inside of beads is becoming better characterised and new supports have been developed that allow improved accessibility. The equilibrium position of reactions on the solid surface can be substantially shifted compared with reactions in solution, and this can be usefully exploited using hydrolases in reverse. Research is also starting to tackle the way in which kinetics are modified when the substrates are surface immobilised

Enzyme responsive polymer hydrogel beads

We report on a new class of enzyme responsive polymer hydrogels, the molecular accessibility of which can be changed selectively by enzymes present in a sample fluid

Understanding protease catalysed solid phase peptide synthesis

A protease (thermolysin) was used to directly synthesise a number of dipeptides from soluble Fmoc-amino acids onto a solid support (PEGA1900) in bulk aqueous media, often in very good yields. This shift in equilibrium toward synthesis is remarkable because for soluble dipeptides in aqueous solution hydrolysis rather than synthesis is observed. Three possible reasons for the equilibrium shift were considered: (i) using a solid support makes it easy to use an excess of reagents, so mass action contributes towards synthesis; (ii) reduction in the unfavourable hydrophobic hydration of the Fmoc group within the solid support compared with the free amino acid in solution and (iii) suppression of the ionization of amino groups linked to the solid phase due to mutual electrostatic repulsion. It was found that under the conditions studied the second effect was most important

Protease-catalyzed peptide synthesis on solid support

The direct enzymatic synthesis of peptides from amino acids is widely used as a useful alternative to chemical synthesis. However, good yields of such enzyme-catalyzed reactions require altered reaction conditions to overcome the bias for hydrolysis in aqueous medium. We argue that the synthesis/hydrolysis equilibrium can be shifted toward synthesis in aqueous medium by immobilizing the amine on solid support. In this report, we show the first examples of solid-phase peptide synthesis catalyzed by a protease in bulk aqueous buffer

A single aqueous reference equilibrium constant for amide synthesis-hydrolysis

Experimentally measured equilibrium constants at a given pH in part reflect the contributions of ionisation of acidic and basic groups present. These contributions can be isolated from the equilibrium constant by expressing all reactant concentrations in terms of the uncharged forms only. This article presents methods to calculate uncharged reference equilibrium constants for amide synthesis/hydrolysis reactions. For zwitterions in particular these methods are not always straightforward. It is explained how (microscopic) pK(a) values can be estimated where experimental values are not available. A large number of equilibrium data are analysed for hydrolysis or synthesis of protected and unprotected di- and tri-peptides, beta-lactam antibiotics, and acyl acids and amides. This reveals just how similar the reference equilibrium constants are when ionisation is properly accounted for (K-ref(0) = 10(3.6) M-1) regardless of the molecular form of the reactants involved

Enzyme-responsive hydrogels for biomedical applications

This chapter highlights recent developments in enzyme-responsive gels. The focus is on peptide-based small-molecule hydrogels, for biomedical applications. The use of enzymes in this context provides a powerful methodology for controlled assembly, taking advantage of both biological selectivity and catalytic amplification. The building blocks for self-assembly and basic design rules for small molecule peptide gelators are discussed first. This is followed by a discussion of key features of biocatalytic self-assembly of hydrogels, focusing on control of nanoscale organization and consequent function. Finally, the potential applications of the enzyme-responsive hydrogels as biomaterials are discussed in the areas of cell culture, drug delivery, biosensing, and control of cell fate