Intraligand Charge Transfer Enables Visible‐Light‐Mediated Nickel‐Catalyzed Cross‐Coupling Reactions

We demonstrate that several visible‐light‐mediated carbon−heteroatom cross‐coupling reactions can be carried out using a photoactive NiII precatalyst that forms in situ from a nickel salt and a bipyridine ligand decorated with two carbazole groups (Ni(Czbpy)Cl2). The activation of this precatalyst towards cross‐coupling reactions follows a hitherto undisclosed mechanism that is different from previously reported light‐responsive nickel complexes that undergo metal‐to‐ligand charge transfer. Theoretical and spectroscopic investigations revealed that irradiation of Ni(Czbpy)Cl2 with visible light causes an initial intraligand charge transfer event that triggers productive catalysis. Ligand polymerization affords a porous, recyclable organic polymer for heterogeneous nickel catalysis of cross‐coupling reactions. The heterogeneous catalyst shows stable performance in a packed‐bed flow reactor during a week of continuous operation

Unnatural amino acids as novel probes for ultrafast 2D-IR spectroscopy of proteins : towards real-time investigation of biomolecular dynamics and vibrational energy flow

Ultrafast protein dynamics are of great interest for understanding the molecular basis of biochemical function. One method to study structural changes with highest time-resolution starting in the femtosecond regime is 2D-IR spectroscopy. However its application to investigate protein dynamics both with high temporal and spatial resolution is currently limited to few biological systems with intrinsic chromophores. Spectral congestion, the contribution of many similar oscillators to the same signals, makes it difficult to draw conclusions about local structural dynamics in most other proteins. The aim of this thesis is to extend the application of 2D-IR spectroscopy to a wider range of proteins by introducing unnatural amino acids (UAAs) with azide or nitrile groups as site-specific vibrational probes, which absorb in the free spectral window between 1800 to 3000 cm-1 by using methods from chemical biology. In a comparative experimental study using FTIR and 2D-IR spectroscopy of single amino acids azidohomoalanine (Aha), a methionine analogue, was identified as preferred label. To demonstrate the application potential of UAAs as site-specific probes, Aha was then incorporated into different positions in a small globular protein. By using both FTIR and ultrafast 2D-IR it was shown, that indeed the local microenvironment as well as conformational fluctuations on picosecond timescale could be monitored with high spatial information. The azide moiety shows a shift of its absorption frequency depending on the polarity of its surrounding. Using this approach, different subensembles for the protein conformations with more polar and less polar environment around the vibrational probe can be distinguished. A second major application of site-specific labels is the study of vibrational energy transfer processes (VET), predicted to be relevant for allosteric communication in protein domains such as the PDZ domain. VET can be tracked with high spatial resolution using time-resolved IR spectroscopy by exciting a localized vibrational mode and probing separate modes in a two-colour 2D-IR experiment. To extend this kind of experiment to proteins, a specific donor-acceptor pair of two UAAs was introduced. It uses an azulene moiety as donor that can be excited in the visible range but deposits the excess energy by internal conversion into the vibrational modes of the ground state. In small peptides this VET pair was applied successfully, showing a distance-dependent energy transfer induced signal for VET through covalent bonds. These findings bare great promise for the direct observation of vibrational energy flow in proteins in real-time. Overall this thesis is the basis for extending the usability of 2D-IR spectroscopy to study structural dynamics in a wide range of proteins systems both with high temporal and spatial resolution

pH dependency of photolabile protection groups used for applications in dynamic structural biology

Photolabile protection groups (PPGs) are important tools in biophysical experiments and allow light-induced spatial and temporal control of compound release. Photocaged substrates are recently driving new experiments in dynamic structural biology, in particular time-resolved serial crystallography (TR-SX) at XFELs and synchrotrons. PPGs are seldom characterized in aqueous conditions, despite their application in biological experiments. Several common PPGs exhibit a strong pH dependency of their absorption spectrum and extinction coefficient, which needs to be considered in experimental planning. We report here the static pH dependent photophysical properties (key absorption bands and corresponding extinction coefficients) of four common PPGs as initial guide for planning time-resolved experiments in dynamic structural biology

Impact of azidohomoalanine incorporation on protein structure and ligand binding

The impact of the incorporation of a non-natural amino acid (NNAA) on protein structure, dynamics, and ligand binding has not been studied rigorously so far. NNAAs are regularly used to modify proteins post-translationally in vivo and in vitro through click chemistry. Herein, structural characterisation of the impact of the incorporation of azidohomoalanine (AZH) into the model protein domain PDZ3 is examined by means of NMR spectroscopy and X-ray crystallography. The structure and dynamics of the apo state of AZH-modified PDZ3 remain mostly unperturbed. Furthermore, the binding of two PDZ3 binding peptides are unchanged upon incorporation of AZH. The interface of the AZH-modified PDZ3 and an azulene-linked peptide for vibrational energy transfer studies has been mapped by means of chemical shift perturbations and NOEs between the unlabelled azulene-linked peptide and the isotopically labelled protein. Co-crystallisation and soaking failed for the peptide-bound holo complex. NMR spectroscopy, however, allowed determination of the protein-ligand interface. Although the incorporation of AZH was minimally invasive for PDZ3, structural analysis of NNAA-modified proteins through the methodology presented herein should be performed to ensure structural integrity of the studied target

Time-resolved crystallography reveals allosteric communication aligned with molecular breathing

A comprehensive understanding of protein function demands correlating structure and dynamic changes. Using time-resolved serial synchrotron crystallography, we visualized half-of-the-sites reactivity and correlated molecular-breathing motions in the enzyme fluoroacetate dehalogenase. Eighteen time points from 30 milliseconds to 30 seconds cover four turnover cycles of the irreversible reaction. They reveal sequential substrate binding, covalent-intermediate formation, setup of a hydrolytic water molecule, and product release. Small structural changes of the protein mold and variations in the number and placement of water molecules accompany the various chemical steps of catalysis. Triggered by enzyme-ligand interactions, these repetitive changes in the protein framework’s dynamics and entropy constitute crucial components of the catalytic machinery

Femtosecond Electron Diffraction Study of the Spin Crossover Dynamics of Single Crystal [Fe(PM-AzA)2](NCS)2

International audienceThe atomic motions involved in spin crossover photo-switching dynamics of single crystal [Fe(PM-AzA)2](NCS)2 are investigated by femtosecond electron diffraction (FED). The experiment was performed with an ultrabright femtosecond electron source using 8.5 × 104 electrons per pulse with 400 fs temporal instrument response function

Antimicrobial Polymers of Linear and Bottlebrush Architecture: Probing the Membrane Interaction and Physicochemical Properties

Polymeric antimicrobial peptide mimics are a promising alternative for the future management of the daunting problems associated with antimicrobial resistance. However, the development of successful antimicrobial polymers (APs) requires careful control of factors such as amphiphilic balance, molecular weight, dispersity, sequence, and architecture. While most of the earlier developed APs focused on random linear copolymers, the development of APs with advanced architectures proved to be more potent in the mimicry of antimicrobial peptides. We recently developed multivalent bottlebrush APs with improved antibacterial and hemocompatibility profiles, outperforming their linear counterparts. Understanding the rationale behind the outstanding biological activity of these newly developed antimicrobials is vital to further improving their performance. This work investigates the physicochemical properties governing the differences in activity between linear and bottlebrush architectures using diverse spectroscopic and microscopic techniques. Linear copolymers are more solvated, thermo-responsive and possess facial amphiphilicity resulting in random aggregations when interacting with liposomes mimicking E. coli-membranes. The bottlebrush copolymers adopt a more stable secondary conformation in aqueous solution in comparison to linear copolymers, conferring rapid and more specific binding mechanism to membranes. The advantageous physicochemical properties of the bottlebrush topology seem to be a determinant factor in the activity of these promising APs

Direct observation of structural dynamics upon photo-excitation in a spin crossover crystal with femtosecond electron diffraction

Photoinduced spin transitions are studied by femtosecond electron diffraction to understand ultrafast structural dynamics associated with intersystem crossing. The results indicate the structural reorganization occurs within 2.3 ps, as the metal-ligand bond distribution narrows during intramolecular vibrational energy redistribution

Direct observation of structural dynamics upon photo-excitation in a spin crossover crystal with femtosecond electron diffraction

Photoinduced spin transitions are studied by femtosecond electron diffraction to understand ultrafast structural dynamics associated with intersystem crossing. The results indicate the structural reorganization occurs within 2.3 ps, as the metal-ligand bond distribution narrows during intramolecular vibrational energy redistribution