Controlled manipulation of enzyme specificity through immobilization-induced flexibility constraints

It is thought that during immobilization enzymes, as dynamic biomolecules, may become distorted and this may alter their catalytic properties. However, the effects of different immobilization strategies on enzyme rigidity or flexibility and their consequences in specificity and stereochemistry at large scale has not been yet clearly evaluated and understood. This was here investigated by using as model an ester hydrolase, isolated from a bacterium inhabiting a karstic lake, with broad substrate spectrum (72 esters being converted; 61.5 U mg$^{-1}$ for glyceryl tripropionate) but initially non-enantiospecific. We found that the enzyme (7 nm × 4.4 nm × 4.2 nm) could be efficiently ionic exchanged inside the pores (9.3 nm under dry conditions) of amino-functionalized ordered mesoporous material (NH$_{2}$-SBA-15), achieving a protein load of 48 mg g−1, and a specific activity of 4.5 ± 0.1 U mg$^{-1}$. When the enzyme was site-directed immobilized through His interaction with an immobilized cationon the surface of two types of magnetic micro-particles through hexahistidine-tags, protein loads up to 10.2 μg g$^{-1}$ and specific activities of up to 29.9 ± 0.3 U mg$^{-1}$, were obtained. We found that ionically exchanged enzyme inside pores of NH2-SBA-15 drastically narrowed the substrate range (17 esters), to an extent much higher than ionically exchanged enzyme on the surface of magnetic micro-particles (up to 61 esters). This is attributed to differences in surface chemistry, particle size, and substrate accessibility to the active site tunnel. Our results also suggested, for the first time, that immobilization of enzymes in pores of similar size may alter the enzyme structures and produce enzyme active centers with different configuration which promote stereochemical conversions in a manner different to those arising from surface immobilization, where the strength of the ionic exchange also has an influence. This was shown by demonstrating that when the enzyme was introduced inside pores with a diameter (under dry conditions) slightly higher than that of the enzyme crystal structure a biocatalyst enantiospecific for ethyl (R)-4-chloro-3-hydroxybutyrate was produced, a feature not found when using wider pores. By contrast, immobilization on the surface of ferromagnetic microparticles produced selective biocatalysts for methyl (S)-(+)-mandelate or methyl (S)-lactate depending on the functionalization. This study illustrates the benefits of extensive analysis of the substrate spectra to better understand the effects of different immobilization strategies on enzyme flexibility/rigidity, as well as substrate specificity and stereochemistry. Our results will help to design tunable materials and interfaces for a controlled manipulation of specificity and to transform non-enantiospecific enzymes into stereo-chemically substrate promiscuous biocatalysts capable of converting multiple chiral molecules

Hetero-Diels–Alder Cycloaddition with RAFT Polymers as Bioconjugation Platform

We introduce the bioconjugation of polymers synthesized by RAFT polymerization, bearing no specific functional end group, by means of hetero‐Diels–Alder cycloaddition through their inherent terminal thiocarbonylthio moiety with a diene‐modified model protein. Quantitative conjugation occurs over the course of a few hours, at ambient temperature and neutral pH, and in the absence of any catalyst. Our technology platform affords thermoresponsive bioconjugates, whose aggregation is solely controlled by the polymer chains

Hetero-Diels-Alder-Cycloaddition mit RAFT-Polymeren als Biokonjugationsplattform

Wir stellen die Biokonjugation von Polymeren vor, die durch RAFT‐Polymerisation mittels Hetero‐Diels‐Alder‐Cycloaddition durch ihren inhärenten terminalen Thiocarbonylthiorest mit einem dienmodifizierten Modellprotein synthetisiert wurden und keine spezifische funktionelle Endgruppe tragen. Die quantitative Konjugation erfolgt im Verlauf einiger Stunden bei Raumtemperatur und nahezu neutralem pH‐Wert und in Abwesenheit jeglichen Katalysators. Unsere Technologieplattform liefert thermoresponsive Biokonjugate, deren Aggregation allein durch die Polymerketten gesteuert wird

Polymerization-Induced Self-Assembly of Block Copolymer Nano-objects via RAFT Aqueous Dispersion Polymerization

In this Perspective, we discuss the recent development of polymerization-induced self-assembly mediated by reversible addition–fragmentation chain transfer (RAFT) aqueous dispersion polymerization. This approach has quickly become a powerful and versatile technique for the synthesis of a wide range of bespoke organic diblock copolymer nano-objects of controllable size, morphology, and surface functionality. Given its potential scalability, such environmentally-friendly formulations are expected to offer many potential applications, such as novel Pickering emulsifiers, efficient microencapsulation vehicles, and sterilizable thermo-responsive hydrogels for the cost-effective long-term storage of mammalian cells

Ambient temperature polymer modification by in situ phototriggered deprotection and thiol-ene chemistry

A novel and efficient methodology for the light-triggered release of thiols at ambient temperature is presented, which can be utilized for the in situ modification of polymeric backbones prepared via radical polymerization. Initially, a model reaction on poly(ethylene glycol) methyl ether was examined via size-exclusion chromatography coupled with electrospray ionization-mass spectrometry (SEC/ESI-MS) to establish the photodeprotection feasibility of 2-nitrobenzyl thioether moieties in the presence of variable activators or catalysts employed are Michael-type or radical thiol-ene chemistries, respectively. When 0.01 eq. of dimethylphenylphosphine is employed, disulfide coupling is reduced to its minimum and quantitative phototriggered formation of thiol-capped poly(ethylene glycol) methyl ether species is observed after a 16 hour irradiation period at 320 nm by a low-cost light source. The concept is extended to polymer backbone modification by atom transfer radical polymerization of the novel photosensitive monomer: 2-((3-((2-nitrobenzyl)thio) propanoyl)oxy)ethyl methacrylate containing the 2-nitrobenzyl thioether moiety. Well-defined homopolymers (4700 g·mol -1 ≤ M n ≤ 20000 g·mol -1, 1.29 ≤ PDI ≤ 1.40) containing one protected thiol per repeating unit were obtained and, upon a light stimulus (λ max = 320 nm), thiol entities are released along the lateral polymer chain. The photodeprotection process is mapped by exploiting the increased absorbance of photocleaved o-nitrosobenzaldehyde molecules at 345 nm and UV-Vis data suggests a quantitative backbone deprotection after a 16 hour irradiation time period. Further in situ functionalization of polymeric backbone is achieved via base-catalyzed maleimide-thiol addition at ambient temperature and its outcome is evidenced by a re-increased molecular weight in SEC, by virtue of decreased signal intensity of the 2-nitrobenzyl thioether moiety and the appearance of characteristic product protons in NMR spectroscopy (the polymer backbone functionalization is estimated as >90% by NMR analysis). © 2012 The Royal Society of Chemistry

Formation of polymer vesicles by simultaneous chain growth and self-assembly of amphiphilic block copolymers

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Chemical approaches to synthetic polymer surface biofunctionalization for targeted cell adhesion using small binding motifs

In the present review the principal strategies to chemically modify the surface of synthetic polymeric materials with small molecules for targeted cell adhesion are collated and critically discussed. The focus is purposely oriented on the chemistry involved in these modifications and neither the physical characterizations nor the activity evaluations resulting from these modifications are addressed in depth, although most reviewed examples demonstrate cell adhesion. Particularly, the introduction of a chemical anchor onto the polymeric substrate, the spacing via a linker between the polymer surface and the cell-binding motif, as well as the linkage generated on this cell-binding motif are discussed. Particular cases where variable substrate geometries or spatial patterning are achieved are additionally highlighted. © 2012 The Royal Society of Chemistry