A series of alpha-functional maleimide polymethacrylates (M(n) = 4.1-35.4 kDa, PDi = 1.06-1.27) have been prepared via copper-catalyzed living radical polymerization (LRP). Two independent synthetic protocols have been successfully developed and the polymers obtained in multigram scale, with an 80-100% content of maleimide reactive chain ends, depending on the method employed. A method for the synthesis of amino-terminated polymers, starting from Boc-protected amino initiators, has also been developed, as these derivatives are key intermediates in one of the two processes studied in the present work. The alternative synthetic pathway involves an initiator containing a maleimide unit "protected" as a Diels-Alder adduct. After the polymerization step, the maleimide functionality has been reintroduced by retro-Diels-Alder reaction, by simply refluxing those polymers in toluene for 7 h. These maleimido-terminated materials, poly(methoxyPEG((475))) methacrylates and poly(glycerol) methacrylates, differ for both the nature and size of the polymer side branches and showed an excellent solubility in water, a property that made them an ideal candidate for the synthesis of new polymer-(poly)peptide biomaterials. These functional polymers have been successfully employed in conjugation reactions in the presence of thiol-containing model substrates, namely, reduced glutathione (gamma-Glu-Cys-Gly) and the carrier protein, bovine serum albumin (BSA), in 100 mM phosphate buffer (pH 6.8-7.4) and ambient temperature.

Single-molecule techniques offer a unique tool for studying the dynamical behavior of individual molecules and provide the possibility to construct distributions from individual events rather than from a signal stemming from an ensemble of molecules. In biological systems, known for their complexity, these techniques make it possible to gain insights into the detailed spectrum of molecular conformational changes and activities. Here, we report on the direct observation of a single lipase-catalyzed reaction for extended periods of time (hours), by using confocal fluorescence microscopy. When adding a profluorescent substrate, the monitored enzymatic activity appears as a trajectory of ''on-state'' and ''off-state'' events. The waiting time probability density function of the off state and the state-correlation function fit stretched exponentials, independent of the substrate concentration in a certain range. The data analysis unravels oscillations in the logarithmic derivative of the off-state waiting time probability density function and correlations between off-state events. These findings imply that the fluctuating enzyme model, which involves a spectrum of enzymatic conformations that interconvert on the time scale of the catalytic activity, best describes the observed enzymatic activity. Based on this model, values for the coupling and reaction rates are extracted.single enzyme activity ͉ two-state trajectories D ynamics of chemical reactions are conventionally investigated by ensemble measurements. Recent advances in single-molecule spectroscopy have enabled the real-time study of biophysical processes (1-10) and conformational changes (11, 12) of single biomolecules. These studies have demonstrated that new information about such processes can be extracted from single-molecule measurements. In particular, deviations from the standard Michaelis-Menten behavior (13,14), which is expected for bulk enzymatic activity, have been observed (6 -8, 12).Motivated by these findings, we examined the enzymatic activity of individual molecules of the 33-kDa lipase B from Candida antarctica molecules (15, 16) by using confocal fluorescence microscopy. This lipase catalyzes the hydrolysis of esters in aqueous solution following the same reaction mechanism as that of a serine protease (17). To study the catalysis by single lipase, we used a fluorogenic substrate, namely the nonf luorescent ester 2Ј,7Ј-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, which upon hydrolysis forms a highly fluorescent carboxylic acid product (18,19). This method enabled us to probe the enzymatic activity by monitoring the fluorescence emission from single enzymes. The fluorescence emission displayed blinking of ''on'' and ''off'' events depending on the presence (or absence) of the fluorescent product in the confocal focus (20). By using this approach, we have been able to obtain long trajectories (for time periods of hours) suitable for reliable statistical analysis while varying the concentration of the substrate, thus ...

A new type of giant amphiphilic molecule has been synthesized by covalently connecting a lipase enzyme headgroup to a maleimide-functionalized polystyrene tail (40 repeat units). The resulting biohybrid forms catalytic micellar rods in water.

Spontane Vesikelbildung sowohl in organischen Lösungsmitteln als auch in Wasser wurde bei Stab‐Knäuel‐Diblockcopolymeren mit Thiophen‐Einheiten beobachtet. Die Thiophen‐Einheiten an der Oberfläche der Aggregate können unter Bildung „polymerisierter“ Vesikel verknüpft werden (siehe Bild und Titelbild). In die Vesikel können Enzyme eingeschlossen werden, wodurch katalytisch aktive Mikroreaktoren erhalten werden, deren Hülle für Substratmoleküle durchlässig ist.