DNA nanocrane-based catalysts for region-specific protein modification.

This paper describes the development and performance of catalytic DNA-based nanocranes for the controlled modification of wild-type proteins. We show that the position of the catalyst offers control over the region of modification, and that reversible interactions between the catalytic structure and thrombin enable trigger-responsive modification, even in cell lysate.

Precise and Controlled Modification of Proteins using Multifunctional Chemical Constructs.

Bioconjugation of chemical entities to biologically active proteins has increased our insight in the inner workings of a cell and resulted in novel therapeutic agents. A current challenge is the efficient generation of homogeneous conjugates of native proteins, not only when isolated, but also when still present in their native environment. To do this, various features of protein-modifying enzymes have been combined in artificial constructs. In this concept, the current status of this approach is evaluated, and the interplay between designs and protein modification will be discussed. Particular focus is directed on the protein-binding anchor, the chemistry that is used for the modification, and the linker that connects these two units. Suggestions how to include additional elements such as a trigger-responsive switch that regulated protein modification are also presented.

Rapid Access to Potent Bispecific T Cell Engagers Using Biogenic Tyrosine Click Chemistry.

Bispecific antibodies as T cell engagers designed to display binding capabilities to both tumor-associated antigens and antigens on T cells are considered promising agents in the fight against cancer. Even though chemical strategies to develop such constructs have emerged, a method that readily converts a therapeutically applied antibody into a bispecific construct by a fully non-genetic process is not yet available. Herein, we report the application of a biogenic, tyrosine-based click reaction utilizing chemoenzymatic modifications of native IgG1 antibodies to generate a synthetic bispecific antibody construct that exhibits tumor-killing capability at picomolar concentrations. Control experiments revealed that a covalent linkage of the different components is required for the observed biological activities. In view of the highly potent nature of the constructs and the modular approach that relies on convenient synthetic methods utilizing therapeutically approved biomolecules, our method expedites the production of potent bispecific antibody constructs with tunable cell killing efficacy with significant impact on therapeutic properties.

DNA G-quadruplex-stabilizing metal complexes as anticancer drugs.

Guanine quadruplexes (G4s) are important targets for cancer treatments as their stabilization has been associated with a reduction of telomere ends or a lower oncogene expression. Although less abundant than purely organic ligands, metal complexes have shown remarkable abilities to stabilize G4s, and a wide variety of techniques have been used to characterize the interaction between ligands and G4s. However, improper alignment between the large variety of experimental techniques and biological activities can lead to improper identification of top candidates, which hampers progress of this important class of G4 stabilizers. To address this, we first review the different techniques for their strengths and weaknesses to determine the interaction of the complexes with G4s, and provide a checklist to guide future developments towards comparable data. Then, we surveyed 74 metal-based ligands for G4s that have been characterized to the in vitro level. Of these complexes, we assessed which methods were used to characterize their G4-stabilizing capacity, their selectivity for G4s over double-stranded DNA (dsDNA), and how this correlated to bioactivity data. For the biological activity data, we compared activities of the G4-stabilizing metal complexes with that of cisplatin. Lastly, we formulated guidelines for future studies on G4-stabilizing metal complexes to further enable maturation of this field.

High Rates of Quinone-Alkyne Cycloaddition Reactions are Dictated by Entropic Factors.

Reaction rates of strained cycloalkynes and cycloalkenes with 1,2-quinone were quantified by stopped flow UV-Vis spectroscopy and computational analysis. We found that the strained alkyne BCN-OH 3 (k2 1824 M-1 s-1 ) reacts >150 times faster than the strained alkene TCO-OH 5 (k2 11.56 M-1 s-1 ), and that derivatization with a carbamate can lead to a reduction of the rate constant with almost half. Also, the 8-membered strained alkyne BCN-OH 3 reacts 16 times faster than the more strained 7-membered THS 2 (k2 110.6 M-1 s-1 ). Using the linearized Eyring equation we determined the thermodynamic activation parameters of these two strained alkynes, revealing that the SPOCQ reaction of quinone 1 with THS 2 is associated with ΔH≠ of 0.80 kcal/mol, ΔS≠ =-46.8 cal/K⋅mol, and ΔG≠ =14.8 kcal/mol (at 25 °C), whereas the same reaction with BCN-OH 3 is associated with, ΔH≠ =2.25 kcal/mol, ΔS≠ =-36.3 cal/K⋅mol, and ΔG≠ =13.1 kcal/mol (at 25 °C). Computational analysis supported the values obtained by the stopped-flow measurements, with calculated ΔG≠ of 15.6 kcal/mol (in H2 O) for the SPOCQ reaction with THS 2, and with ΔG≠ of 14.7 kcal/mol (in H2 O) for the SPOCQ reaction with BCN-OH 3. With these empirically determined thermodynamic parameters, we set an important step towards a more fundamental understanding of this set of rapid click reactions.

Oxidation-Induced "One-Pot" Click Chemistry.

Click chemistry has been established rapidly as one of the most valuable methods for the chemical transformation of complex molecules. Due to the rapid rates, clean conversions to the products, and compatibility of the reagents and reaction conditions even in complex settings, it has found applications in many molecule-oriented disciplines. From the vast landscape of click reactions, approaches have emerged in the past decade centered around oxidative processes to generate in situ highly reactive synthons from dormant functionalities. These approaches have led to some of the fastest click reactions know to date. Here, we review the various methods that can be used for such oxidation-induced "one-pot" click chemistry for the transformation of small molecules, materials, and biomolecules. A comprehensive overview is provided of oxidation conditions that induce a click reaction, and oxidation conditions are orthogonal to other click reactions so that sequential "click-oxidation-click" derivatization of molecules can be performed in one pot. Our review of the relevant literature shows that this strategy is emerging as a powerful approach for the preparation of high-performance materials and the generation of complex biomolecules. As such, we expect that oxidation-induced "one-pot" click chemistry will widen in scope substantially in the forthcoming years.

Improved LC-MS identification of short homologous peptides using sequence-specific retention time predictors.

Peptides are an important group of compounds contributing to the desired, as well as the undesired taste of a food product. Their taste impressions can include aspects of sweetness, bitterness, savoury, umami and many other impressions depending on the amino acids present as well as their sequence. Identification of short peptides in foods is challenging. We developed a method to assign identities to short peptides including homologous structures, i.e. peptides containing the same amino acids with a different sequence order, by accurate prediction of the retention times during reversed phase separation. To train the method, a large set of well-defined short peptides with systematic variations in the amino acid sequence was prepared by a novel synthesis strategy called 'swapped-sequence synthesis'. Additionally, several proteins were enzymatically digested to yield short peptides. Experimental retention times were determined after reversed phase separation and peptide MS2 data was acquired using a high-resolution mass spectrometer operated in data-dependent acquisition mode (DDA). A support vector regression model was trained using a combination of existing sequence-independent peptide descriptors and a newly derived set of selected amino acid index derived sequence-specific peptide (ASP) descriptors. The model was trained and validated using the experimental retention times of the 713 small food-relevant peptides prepared. Whilst selecting the most useful ASP descriptors for our model, special attention was given to predict the retention time differences between homologous peptide structures. Inclusion of ASP descriptors greatly improved the ability to accurately predict retention times, including retention time differences between 157 homologous peptide pairs. The final prediction model had a goodness-of-fit (Q2) of 0.94; moreover for 93% of the short peptides, the elution order was correctly predicted.

Complete microviscosity maps of living plant cells and tissues with a toolbox of targeting mechanoprobes.

Mechanical patterns control a variety of biological processes in plants. The microviscosity of cellular structures effects the diffusion rate of molecules and organelles, thereby affecting processes such as metabolism and signaling. Spatial variations in local viscosity are also generated during fundamental events in the cell life cycle. While crucial to a complete understanding of plant mechanobiology, resolving subcellular microviscosity patterns in plants has remained an unsolved challenge. We present an imaging microviscosimetry toolbox of molecular rotors that yield complete microviscosity maps of cells and tissues, specifically targeting the cytosol, vacuole, plasma membrane, and wall of plant cells. These boron-dipyrromethene (BODIPY)-based molecular rotors are rigidochromic by means of coupling the rate of an intramolecular rotation, which depends on the mechanics of their direct surroundings, with their fluorescence lifetime. This enables the optical mapping of fluidity and porosity patterns in targeted cellular compartments. We show how apparent viscosity relates to cell function in the root, how the growth of cellular protrusions induces local tension, and how the cell wall is adapted to perform actuation surrounding leaf pores. These results pave the way to the noninvasive micromechanical mapping of complex tissues.

Calibrating Catalytic DNA Nanostructures for Site-Selective Protein Modification.

Many biomedical fields rely on proteins that are selectively modified. These can be attached using reactive or catalytic moieties, but the position where these moieties are attached is often poorly controlled. We assessed how catalyst position affects the efficiency and selectivity of protein modification. For this, we anchored a template DNA strand to three different proteins, which were subsequently hybridized to DNA strands that contained catalysts at different positions. We found a strong correlation between the catalyst-to-protein distance and the efficiency of protein modification for acyl transfer catalysts, which operate via a covalently bound reactant intermediate. Additionally, we found that the catalyst's distance and orientation with respect to the protein surface, also influences its site-selectivity. A catalyst operating with unbound reactant intermediates showed only enhanced efficiency. Our results are rationalized using computational simulations, showing that one-point anchoring of the DNA construct leads to notable differences in the site of modification.

DNA-assisted site-selective protein modification.

Protein modification is important for various types of biomedical research, including proteomics and therapeutics. Many methodologies for protein modification exist, but not all possess the required level of efficiency and site selectivity. This review focuses on the use of DNA to achieve the desired conversions and levels of accuracy in protein modification by using DNA (i) as a template to help concentrate dilute reactants, (ii) as a guidance system to achieve selectivity by binding specific proteins, and (iii) even as catalytic entity or construct to enhance protein modification reactions.

Design of Polypeptides Self-Assembling into Antifouling Coatings: Exploiting Multivalency.

We propose to exploit multivalent binding of solid-binding peptides (SBPs) for the physical attachment of antifouling polypeptide brushes on solid surfaces. Using a silica-binding peptide as a model SBP, we find that both tandem-repeated SBPs and SBPs repeated in branched architectures implemented via a multimerization domain work very well to improve the binding strength of polypeptide brushes, as compared to earlier designs with a single SBP. At the same time, for many of the designed sequences, either the solubility or the yield of recombinant production is low. For a single design, with the domain structure B-M-E, both solubility and yield of recombinant production were high. In this design, B is a silica-binding peptide, M is a highly thermostable, de novo-designed trimerization domain, and E is a hydrophilic elastin-like polypeptide. We show that the B-M-E triblock polypeptide rapidly assembles into highly stable polypeptide brushes on silica surfaces, with excellent antifouling properties against high concentrations of serum albumin. Given that SBPs attaching to a wide range of materials have been identified, the B-M-E triblock design provides a template for the development of polypeptides for coating many other materials such as metals or plastics.

Vectorial Catalysis in Surface-Anchored Nanometer-Sized Metal-Organic Frameworks-Based Microfluidic Devices.

Vectorial catalysis-controlling multi-step reactions in a programmed sequence and by defined spatial localization in a microscale device-is an enticing goal in bio-inspired catalysis research. However, translating concepts from natural cascade biocatalysis into artificial hierarchical chemical systems remains a challenge. Herein, we demonstrate integration of two different surface-anchored nanometer-sized metal-organic frameworks (MOFs) in a microfluidic device for modelling vectorial catalysis. Catalyst immobilization at defined sections along the microchannel and a two-step cascade reaction was conducted with full conversion after 30 seconds and high turnover frequencies (TOF≈105  h-1 ).

Non-Genetic Generation of Antibody Conjugates Based on Chemoenzymatic Tyrosine Click Chemistry.

The availability of tools to generate homogeneous and stable antibody conjugates without recombinant DNA technology is a valuable asset in fields spanning from in vitro diagnostics to in vivo imaging and therapeutics. We present here a general approach for the conjugation to human IgG1 antibodies, by employing a straightforward two-stage protocol based on antibody deglycosylation followed by tyrosinase-mediated ortho-quinone strain-promoted click chemistry. The technology is validated by the efficient and clean generation of highly potent DAR2 and DAR4 antibody-drug conjugates (ADCs) with cytotoxic payloads MMAE or PBD dimer, and their in vitro evaluation.

Alizarin Grafting onto Ultrasmall ZnO Nanoparticles: Mode of Binding, Stability, and Colorant Studies.

The demand is rising for colorants that are obtained from natural resources, tolerant to industrial processing methods, and meet color quality demands. Herein, we report how relevant properties such as thermal stability and photostability of the natural colorant alizarin can be improved by grafting it onto ZnO nanoparticles (NPs), allowing application in a warm extrusion process for the fabrication of polyamide fibers. For this study, ZnO NPs (diameter 2.0 ± 0.6 nm) were synthesized and subsequently functionalized with alizarin. The alizarin-coated ZnO NPs (i.e., dyed nanoparticles, DNPs) were characterized. Thermogravimetric analysis and ultraviolet-visible (UV-vis) studies revealed that alizarin coating accounts for ∼65% (w/w) of the total mass of the DNPs. A subsequent detailed characterization with Fourier transform infrared (FT-IR), 1H nuclear magnetic resonance (NMR), 13C cross-polarization magic angle spinning (CP-MAS) NMR, X-ray photoelectron spectroscopy (XPS), and quantum chemistry studies using various density functional theory (DFT) functionals and basis sets indicated that binding onto the ZnO NPs occurred predominantly via the catechol moiety of alizarin. Importantly, this grafting increased the thermal stability of alizarin with >100 °C, which allowed the processing of the DNPs into polyamide fibers by warm extrusion at 260 °C. Evaluation of the lightfastness of the DNP-dyed nylon fibers revealed that the changes in color quantified via the distance metric ΔE* of alizarin when embedded in a hybrid material were 2.6-fold better compared to nylon fibers that were directly dyed with alizarin. This reveals that the process of immobilization of a natural dye onto ZnO nanoparticles indeed improves the dye properties significantly and opens the way for a wide range of further studies into surface-immobilized dyes.

Detection of methionine- and alanine-recombinant bovine somatotropins and their induced antibodies in serum and milk of cows suggests blood-milk barrier specificity for these compounds.

The elimination of recombinant bovine somatotropin (rbST) and its induced antibodies through milk of 2 formulations is studied to propose a control strategy for its use or abuse. Two dairy cows were treated with alanine-rbST (Ala-rbST), which is identical to endogenous bovine somatotropin, and ten dairy cows were treated with methionine-rbST (Met-rbST), which differs by 1 amino acid from endogenous bovine somatotropin. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method able to measure rbST at a decision limit (CCα) of 0.8 ng/mL and 2.3 ng/mL for serum and milk, respectively. The results show that the administered Ala-rbST is transferred from blood to milk but that this is not the case for Met-rbST. This suggests a blood-milk barrier-related specificity for these compounds. In addition, rbST-induced antibodies were formed in animals treated with Ala-rbST and those treated with Met-rbST. In both treatments, the rbST-induced antibodies were transferred from blood to milk, showing no blood-milk barrier specificity for these antibodies. These elimination patterns show that, for enforcement purposes, the detection of rbST-induced antibodies in tank milk can serve to screen for rbST administration, and subsequent confirmatory serum analysis by LC-MS/MS is needed to identify whether Ala-rbST or Met-rbST has been used.

Comparison of Proteomic Responses as Global Approach to Antibiotic Mechanism of Action Elucidation.

New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology.

A Bis-Zn2+ -Pyridyl-Salen-Type Complex Conjugated to the ATP Aptamer: An ATPase-Mimicking Nucleoapzyme.

Catalytic nucleic acids consisting of a bis-Zn2+ -pyridyl-salen-type ([di-ZnII 3,5 bis(pyridinylimino) benzoic acid]) complex conjugated to the ATP aptamer act as ATPase-mimicking catalysts (nucleoapzymes). Direct linking of the Zn2+ complex to the 3'- or 5'-end of the aptamer (nucleoapzymes I and II) or its conjugation to the 3'- or 5'-end of the aptamer through bis-thymidine spacers (nucleoapzymes III and IV) provided a set of nucleoapzymes exhibiting variable catalytic activities. Whereas the separated bis-Zn2+ -pyridyl-salen-type catalyst and the ATP aptamer do not show any noticeable catalytic activity, the 3'-catalyst-modified nucleoapzyme (nucleoapzyme IV) and, specifically, the nucleoapzyme consisting of the catalyst linked to the 3'-position through the spacer (nucleoapzyme III) reveal enhanced catalytic features in relation to the analogous nucleoapzyme substituted at the 5'-position (kcat =4.37 and 6.88 min-1 , respectively). Evaluation of the binding properties of ATP to the different nucleoapzyme and complementary molecular dynamics simulations suggest that the distance separating the active site from the substrate linked to the aptamer binding site controls the catalytic activities of the different nucleoapzymes.

Site-Specific and Trigger-Activated Modification of Proteins by Means of Catalytic Hemin/G-quadruplex DNAzyme Nanostructures.

Catalytic nanostructures have the potency to mimic enzymatic features. In this paper, we show that the complex between hemin and G-quadruplex DNA efficiently catalyzes the modification of proteins with N-methyl luminol derivatives. Final conversions are reached within 15-30 min, and LC-MS analysis of tryptic digests of the proteins shows that the reaction proceeds with chemoselectivity for electron-rich aromatic residues (Tyr ≫ Trp), and the site-specificity of the modification depends on the sequence and secondary structure folding of the G-quadruplex nanostructure. Furthermore, the modification can be applied on proteins with different biomedical functions, and the nanostructure can be designed to contain a regulatory element in order to regulate protein modification by an external stimulus.

Identification of Chaoborus kairomone chemicals that induce defences in Daphnia.

Infochemicals play important roles in aquatic ecosystems. They even modify food web interactions, such as by inducing defenses in prey. In one classic but still not fully understood example, the planktonic freshwater crustacean Daphnia pulex forms specific morphological defenses (neckteeth) induced by chemical cues (kairomones) released from its predator, the phantom midge larva Chaoborus. On the basis of liquid chromatography, mass spectrometry, and chemical synthesis, we report here the chemical identity of the Chaoborus kairomone. The biologically active cues consist of fatty acids conjugated to the amino group of glutamine via the N terminus. These cues are involved in Chaoborus digestive processes, which explains why they are consistently released despite the disadvantage for its emitter. The identification of the kairomone may allow in-depth studies on multiple aspects of this inducible defense system.

Novel COX-2 products of n-3 polyunsaturated fatty acid-ethanolamine-conjugates identified in RAW264.7 macrophages.

Cyclooxygenase 2 (COX-2) plays a key role in the regulation of inflammation by catalyzing the oxygenation of PUFAs to prostaglandins (PGs) and hydroperoxides. Next to this, COX-2 can metabolize neutral lipids, including endocannabinoid-like esters and amides. We developed an LC-HRMS-based human recombinant (h)COX-2 screening assay to examine its ability to also convert n-3 PUFA-derived N-acylethanolamines. Our assay yields known hCOX-2-derived products from established PUFAs and anandamide. Subsequently, we proved that eicosapentaenoylethanolamide (EPEA), the N-acylethanolamine derivative of EPA, is converted into PGE3-ethanolamide (PGE3-EA), and into 11-, 14-, and 18-hydroxyeicosapentaenoyl-EA (11-, 14-, and 18-HEPE-EA, respectively). Interestingly, we demonstrated that docosahexaenoylethanolamide (DHEA) is converted by hCOX-2 into the previously unknown metabolites, 13- and 16-hydroxy-DHEA (13- and 16-HDHEA, respectively). These products were also produced by lipopolysaccharide-stimulated RAW267.4 macrophages incubated with DHEA. No oxygenated DHEA metabolites were detected when the selective COX-2 inhibitor, celecoxib, was added to the cells, further underlining the role of COX-2 in the formation of the novel hydroxylated products. This work demonstrates for the first time that DHEA and EPEA are converted by COX-2 into previously unknown hydroxylated metabolites and invites future studies toward the biological effects of these metabolites.