Sanne Schoffelen

Recent advances in covalent, site-specific protein immobilization

The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. Covalent, site-specific immobilization strategies are robust and can provide the level of control that is desired in this kind of application. Recent advances include the use of enzymes, such as sortase A, to couple proteins in a site-specific manner to materials such as microbeads, glass, and hydrogels. Also, self-labeling tags such as the SNAP-tag can be employed. Last but not least, chemical approaches based on bioorthogonal reactions, like the azide–alkyne cycloaddition, have proven to be powerful tools. The lack of comparative studies and quantitative analysis of these immobilization methods hampers the selection process of the optimal strategy for a given application. However, besides immobilization efficiency, the freedom in selecting the site of conjugation and the size of the conjugation tag and the researcher’s expertise regarding molecular biology and/or chemical techniques will be determining factors in this regard.

Covalent and Stable CuAAC Modification of Silicon Surfaces for Control of Cell Adhesion

Stable primary functionalization of metal surfaces plays a significant role in reliable secondary attachment of complex functional molecules used for the interfacing of metal objects and nanomaterials with biological systems. In principle, this can be achieved through chemical reactions either in the vapor or liquid phase. In this work, we compared these two methods for oxidized silicon surfaces and thoroughly characterized the functionalization steps by tagging and fluorescence imaging. We demonstrate that the vapor‐phase functionalization only provided transient surface modification that was lost on extensive washing. For stable surface modification, a liquid‐phase method was developed. In this method, silicon wafers were decorated with azides, either by silanization with (3‐azidopropyl)triethoxysilane or by conversion of the amine groups of an aminopropylated surface by means of the azido‐transfer reaction. Subsequently, D‐amino acid adhesion peptides could be immobilized on the surface by use of CuI‐catalyzed click chemistry. This enabled the study of cell adhesion to the metal surface. In contrast to unmodified surfaces, the peptide‐modified surfaces were able to maintain cell adhesion during significant flow velocities in a microflow reactor.

Click chemistry mediated functionalization of vertical nanowires for biological applications

Semiconductor nanowires (NWs) are gaining significant importance in various biological applications, such as biosensing and drug delivery. Efficient and controlled immobilization of biomolecules on the NW surface is crucial for many of these applications. Here, we present for the first time the use of the Cu(I) -catalyzed alkyne-azide cycloaddition and its strain-promoted variant for the covalent functionalization of vertical NWs with peptides and proteins. The potential of the approach was demonstrated in two complementary applications of measuring enzyme activity and protein binding, which is of general interest for biological studies. The attachment of a peptide substrate provided NW arrays for the detection of protease activity. In addition, green fluorescent protein was immobilized in a site-specific manner and recognized by antibody binding to demonstrate the proof-of-concept for the use of covalently modified NWs for diagnostic purposes using minute amounts of material.

Click-Chemistry-Mediated Synthesis of Selective Melanocortin Receptor 4 Agonists

The melanocortin receptor 4 (MC4R) subtype of the melanocortin receptor family is a target for therapeutics to ameliorate metabolic dysfunction. Endogenous MC4R agonists possess a critical pharmacophore (HFRW), and cyclization of peptide agonists often enhances potency. Thus, 17 cyclized peptides were synthesized by solid phase click chemistry to develop novel, potent, selective MC4R agonists. Using cAMP measurements and a transcriptional reporter assay, we observed that several constrained agonists generated by a cycloaddition reaction displayed high selectivity (223- to 467-fold) toward MC4R over MC3R and MC5R receptor subtypes without compromising agonist potency. Significant variation was also observed between the EC50 values for the two assays, with robust levels of reporter expression measured at lower concentrations than those effecting appreciable increases in cAMP levels for the majority of the compounds tested. Collectively, we characterized significant elements that modulate the activity of the core pharmacophore for MC4R and provide a rationale for careful assay selection for agonist screening.

Advances in Merging Triazoles with Peptides and Proteins

Five-membered heterocycles have found extensive use as peptide- and disulfide-bond mimics in peptidomimetics. The application of 1,4- and 1,5-substituted 1,2,3-triazoles has been particularly favored due to their ease of preparation by a variety of “click” methods including the copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) and the ruthenium-catalyzed azide–alkyne cycloaddition (RuAAC) reactions. These heterocycles are electronically similar to amide bonds and have provided both functional and structural analogues of biologically active peptides. One advantage of triazole ring amide surrogates is their stability toward natural enzyme activity. The quantitative and orthogonal nature of the CuAAC reaction has facilitated its use in peptide macrocyclization reactions. The CuAAC reaction is particularly useful to replace disulfide bonds in order to stabilize bioactive conformations of biologically active peptides. Metal-free cycloadditions promoted by ring strain (SPAAC) have been favored for labeling in living systems in which transition metals are poorly tolerated. A range of in vivo biomolecular “click” reactions have demonstrated the versatility of SPAAC reactions in living cells and even multicellular organisms. Although azides and alkynes can conveniently be introduced in peptides during synthesis, site-specific incorporation of these functional groups into proteins is more challenging. A variety of methods has been developed to make these reactive precursors, including residue-specific replacement and genetic code expansion. Recent developments of new ligands and catalysts for the CuAAC reaction have further contributed to the promising possibilities that triazoles provide for future applications in the peptide and protein field.

Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence

Methods for site-selective chemistry on proteins are in high demand for the synthesis of chemically modified biopharmaceuticals, as well as for applications in chemical biology, biosensors and more. Inadvertent N-terminal gluconoylation has been reported during expression of proteins with an N-terminal His tag. Here we report the development of this side-reaction into a general method for highly selective N-terminal acylation of proteins to introduce functional groups. We identify an optimized N-terminal sequence, GHHHn− for the reaction with gluconolactone and 4-methoxyphenyl esters as acylating agents, facilitating the introduction of functionalities in a highly selective and efficient manner. Azides, biotin or a fluorophore are introduced at the N-termini of four unrelated proteins by effective and selective acylation with the 4-methoxyphenyl esters. This Gly-Hisn tag adds the unique capability for highly selective N-terminal chemical acylation of expressed proteins. We anticipate that it can find wide application in chemical biology and for biopharmaceuticals.His-tagged proteins can undergo N-terminal acylation as an undesired side-reaction. Here, the authors utilize this to develop a method for highly selective acylation and further modification of peptides and proteins using an optimized His sequence and 4-methoxyphenyl esters as acyl donors.

Rational Tuning of Fluorobenzene Probes for Cysteine‐Selective Protein Modification

Fluorobenzene probes for protein profiling through selective cysteine labeling have been developed by rational reactivity tuning. Tuning was achieved by selecting an electron-withdrawing para substituent in combination with variation of the number of fluorine substituents. Optimized probes chemoselectively arylated cysteine residues in proteins under aqueous conditions. Probes linked to azide, biotin, or a fluorophore were applicable to labeling of eGFP and albumin. Selective inhibition of cysteine proteases was also demonstrated with the probes. Additionally, probes were tuned for site-selective labeling of cysteine residues and for activity-based protein profiling in cell lysates.

Sustainable Flow Synthesis of Encoded Beads for Combinatorial Chemistry and Chemical Biology

Monosized beads of polar resins were synthesized for combinatorial chemistry and chemical biology by sustainable microchannel flow synthesis. Regular, biocompatible, and optically encoded beads could be efficiently prepared on large scale and in high yield. In a preparative flow polymerization instrument, taking advantage of a designed T-connector for droplet formation, quality beads were synthesized with accurate size control using a minimal amount of recirculating silicon oil as suspension medium. Bead-size was controlled through shear imposed by the silicon oil flow rate. This process provided 86% yield of ∼500 μm macrobeads beads within a 20 μm size range with no deformities or vacuoles, ideally suited for combinatorial chemistry and protein binding studies. The simple flow equipment consisted of a syringe pump for monomer and initiator delivery, a T-connector, a gear pump for oil recirculation, a long, heated coil of Teflon tubing and a collector syringe. The method was used for preparation of PEGA1900 beads, optically encoded with fluorescent microparticles. The microparticle matrix (MPM) encoded beads were tested in a MPM-decoder showing excellent recognition in bead decoding.

Specific Electrostatic Molecular Recognition in Water.

The identification of pairs of small peptides that recognize each other in water exclusively through electrostatic interactions is reported. The target peptide and a structure-biased combinatorial ligand library consisting of ≈78 125 compounds were synthesized on different sized beads. Peptide-peptide interactions could conveniently be observed by clustering of the small, fluorescently labeled target beads on the surface of larger ligand-containing beads. Sequences of isolated hits were determined by MS/MS. The interactions of the complex showing the highest affinity were investigated by a novel single-bead binding assay and by 2D NMR spectroscopy. Molecular dynamics (MD) studies revealed a putative mode of interaction for this unusual electrostatic binding event. High binding specificity occurred through a combination of topological matching and electrostatic and hydrogen-bond complementarities. From MD simulations binding also seemed to involve three tightly bound water molecules in the interface between the binding partners. Binding constants in the submicromolar range, useful for biomolecular adhesion and in nanostructure design, were measured.