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.

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.

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.

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.

Chemical Synthesis of Glycopeptides containing l-Arabinosylated Hydroxyproline and Sulfated Tyrosine.

Post-translationally modified peptides are important regulating molecules for living organisms. Here, we report the stereoselective total synthesis of ß-1,2-linked l-arabinosylated Fmoc-protected hydroxyproline building blocks and their incorporation, together with sulfated tyrosine and hydroxyproline, into the plant peptide hormone PSY1. Clean glycopeptides were obtained by performing acetyl removal from the l-arabinose groups prior to deprotection of the neopentyl-protected sulfated tyrosine.

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.

Analysis of Omega-3 Fatty Acid-Derived N-Acylethanolamines in Biological Matrices.

The adequate quantification of endocannabinoids and related N-acylethanolamines can be complex due to their low endogenous levels, structural diversity, and metabolism. Therefore, advanced analytical approaches, involving LC-MS, are required to quantify these molecules in plasma, tissues, and other matrices. It has been shown that endocannabinoid congeners synthesized from n-3 poly-unsaturated fatty acids (n-3 PUFAs), such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have interesting immunomodulatory and tumor-inhibiting properties. Recent work has shown that DHEA and EPEA can be further enzymatically metabolized by cyclo-oxygenase 2 (COX-2), forming oxygenated metabolites. Here, an LC-MS-based method for the quantification of the n-3 PUFA-derived endocannabinoid congeners DHEA and EPEA is described, which is also suited to measure a wider spectrum of endocannabinoids. The chapter contains a step-by-step protocol for the analysis of (n-3) endocannabinoids in plasma, including sample collection and solid phase extraction, LC-MS analysis, and data processing. In addition, protocol modifications are provided to allow quantification of n-3 PUFA-derived endocannabinoids and their COX-2 metabolites in tissues and cell culture media. Finally, conditions that alter endocannabinoid concentrations are briefly discussed.

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.

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.

PUFA-Derived N-Acylethanolamide Probes Identify Peroxiredoxins and Small GTPases as Molecular Targets in LPS-Stimulated RAW264.7 Macrophages.

We studied the mechanistic and biological origins of anti-inflammatory poly-unsaturated fatty acid-derived N-acylethanolamines using synthetic bifunctional chemical probes of docosahexaenoyl ethanolamide (DHEA) and arachidonoyl ethanolamide (AEA) in RAW264.7 macrophages stimulated with 1.0 µg mL-1 lipopolysaccharide. Using a photoreactive diazirine, probes were covalently attached to their target proteins, which were further studied by introducing a fluorescent probe or biotin-based affinity purification. Fluorescence confocal microscopy showed DHEA and AEA probes localized in cytosol, specifically in structures that point toward the endoplasmic reticulum and in membrane vesicles. Affinity purification followed by proteomic analysis revealed peroxiredoxin-1 (Prdx1) as the most significant binding interactor of both DHEA and AEA probes. In addition, Prdx4, endosomal related proteins, small GTPase signaling proteins, and prostaglandin synthase 2 (Ptgs2, also known as cyclooxygenase 2 or COX-2) were identified. Lastly, confocal fluorescence microscopy revealed the colocalization of Ptgs2 and Rac1 with DHEA and AEA probes. These data identified new molecular targets suggesting that DHEA and AEA may be involved in reactive oxidation species regulation, cell migration, cytoskeletal remodeling, and endosomal trafficking and support endocytosis as an uptake mechanism.

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.

Microsphere Peptide-Based Immunoassay for the Detection of Recombinant Bovine Somatotropin in Injection Preparations.

The use of peptides in immunoassays can be favored over the use of the full protein when more cost effective or less toxic approaches are needed, or when access to the full protein is lacking. Due to restricted access to recombinant bovine somatotropin (rbST), a protein enhancing growth and lactating performances of livestock, which use has been banned in the EU, Canada and Australia (amongst others), we developed a peptide-based biorecognition assay on an imaging planar array analyzer. For this, we identified the rbST epitope that is responsible for binding to the rbST-targeting monoclonal antibody 4H12 (MAb 4H12) to be 115DLEEGILALMR125. This linear peptide was synthesized and coupled to microspheres, after which it was tested in a biorecognition competitive inhibition assay format. We observed IC50 values of approximately 0.11 µg mL-1, which are lower than observed for the full rbST protein (IC50 = 0.20 µg mL-1). Importantly, there was no binding with the scrambled peptide. Preliminary results of directly coupled peptides in a microsphere biorecognition assay for detection of rbST are presented. Real-life applicability for detection of somatotropins (STs) in injection preparations of bovine-, porcine- and equine ST are shown. This newly developed immunoassay strongly supports future developments of peptide-based immunoassays to circumvent the limited access to the full protein.

Single-Molecule Force Spectroscopy of a Tetraaryl Succinonitrile Mechanophore.

Fluorescent damage reporters that use mechanochemical activation of a covalent bond to elicit an optical signal are emerging tools in material mechanics as a means to access the nanoscale distribution of forces inside materials under stress. A promising class of damage reporters are tetraaryl succinonitriles (TASN), whose mechanical activation results in stable fluorescent radical species. However, in-depth insights into the molecular mechanics of TASN activation are absent, precluding their use as quantitative mechanoprobes. Here we perform single-molecule force spectroscopy experiments to provide these insights. We use a bridged version of the TASN unit, embedded in multi-mechanophore polymer, to enable multiplexed mechanochemical measurements at the single-molecule level. Our experiments reveal that TASN activates at surprisingly low forces and short time scales compared to other covalent mechanophores. These results establish TASN as a promising candidate for reporting the lower end of relevant forces in material mechanics.

Molecular sensors reveal the mechano-chemical response of Phytophthora infestans walls and membranes to mechanical and chemical stress.

Phytophthora infestans, causal agent of late blight in potato and tomato, remains challenging to control. Unravelling its biomechanics of host invasion, and its response to mechanical and chemical stress, could provide new handles to combat this devastating pathogen. Here we introduce two fluorescent molecular sensors, CWP-BDP and NR12S, that reveal the micromechanical response of the cell wall-plasma membrane continuum in P. infestans during invasive growth and upon chemical treatment. When visualized by live-cell imaging, CWP-BDP reports changes in cell wall (CW) porosity while NR12S reports variations in chemical polarity and lipid order in the plasma membrane (PM). During invasive growth, mechanical interactions between the pathogen and a surface reveal clear and localized changes in the structure of the CW. Moreover, the molecular sensors can reveal the effect of chemical treatment to CW and/or PM, thereby revealing the site-of-action of crop protection agents. This mechano-chemical imaging strategy resolves, non-invasively and with high spatio-temporal resolution, how the CW-PM continuum adapts and responds to abiotic stress, and provides information on the dynamics and location of cellular stress responses for which, to date, no other methods are available.

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.

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 ).

Site-selective and inducible acylation of thrombin using aptamer-catalyst conjugates.

Two acyl-transfer catalysts were conjugated to thrombin-binding DNA aptamers to acylate thrombin. Modification occurred site-selectively on Lys (≫Ser) residues proximal to the respective aptamer-thrombin interface, was selective for thrombin in the presence of other proteins, and the activity of both DNA-catalysts could be controlled by an external trigger.

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.

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.

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.