Highly selective chemical modification of poly-His tagged peptides and proteins.

Chemical modifications to proteins have wide applications. They may be used in, for example, the production of biopharmaceuticals and fluorescent probes. Despite their importance, highly regioselective chemical protein modifications are often challenging to achieve. We have developed two highly selective methods for protein acylation using poly-His tags inserted either at the N-terminus or in combination with a specific Lys residue. For this, we used an N-terminal Gly-His6 (Gly-His tag) or the sequence Hism-Lys-Hisn (Lys-His tag), respectively. The Gly-His tag directed the acylation to the N-terminal Nα-amine when reacted with 4-methoxyphenyl esters to yield stable conjugates. Next, the Lys-His tag was developed to allow modifications at the C-terminus or in loop regions of proteins. This gave a high selectivity of acylation of the designated Lys Nε-amine in the tag over native Lys residues in the protein under mild conditions. Here, we describe the synthesis of aromatic esters carrying different functionalities and reactivity tuning substituents on the phenol. The expression of poly-His tagged proteins, and the procedure for the highly selective peptide and protein acylations are detailed in this contribution. The versatility of these methods has been demonstrated by the attachment of different functionalities such as fluorophores, biotin, and azides to different proteins and an antibody.

Venom-inspired somatostatin receptor 4 (SSTR4) agonists as new drug leads for peripheral pain conditions.

Persistent pain affects one in five people worldwide, often with severely debilitating consequences. Current treatment options, which can be effective for mild or acute pain, are ill-suited for moderate-to-severe persistent pain, resulting in an urgent need for new therapeutics. In recent years, the somatostatin receptor 4 (SSTR 4 ), which is expressed in sensory neurons of the peripheral nervous system, has emerged as a promising target for pain relief. However, the presence of several closely related receptors with similar ligand-binding surfaces complicates the design of receptor-specific agonists. In this study, we report the discovery of a potent and selective SSTR 4 peptide, consomatin Fj1, derived from extensive venom gene datasets from marine cone snails. Consomatin Fj1 is a mimetic of the endogenous hormone somatostatin and contains a minimized binding motif that provides stability and drives peptide selectivity. Peripheral administration of synthetic consomatin Fj1 provided analgesia in mouse models of postoperative and neuropathic pain. Using structure-activity studies, we designed and functionally evaluated several Fj1 analogs, resulting in compounds with improved potency and selectivity. Our findings present a novel avenue for addressing persistent pain through the design of venom-inspired SSTR 4 -selective pain therapeutics. One Sentence Summary: Venom peptides from predatory marine mollusks provide new leads for treating peripheral pain conditions through a non-opioid target.

Bioconjugation of a Near-Infrared DNA-Stabilized Silver Nanocluster to Peptides and Human Insulin by Copper-Free Click Chemistry.

DNA-stabilized silver nanoclusters (DNA-AgNCs) are biocompatible emitters with intriguing properties. However, they have not been extensively used for bioimaging applications due to the lack of structural information and hence predictable conjugation strategies. Here, a copper-free click chemistry method for linking a well-characterized DNA-AgNC to molecules of interest is presented. Three different peptides and a small protein, human insulin, were tested as labeling targets. The conjugation to the target compounds was verified by MS, HPLC, and time-resolved anisotropy measurements. Moreover, the spectroscopic properties of DNA-AgNCs were found to be unaffected by the linking reactions. For DNA-AgNC-conjugated human insulin, fluorescence imaging studies were performed on Chinese hamster ovary (CHO) cells overexpressing human insulin receptor B (hIR-B). The specific staining of the CHO cell membranes demonstrates that DNA-AgNCs are great candidates for bioimaging applications, and the proposed linking strategy is easy to implement when the DNA-AgNC structure is known.

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies.

Insulin formulations with diverse oligomerization states are the hallmark of interventions for the treatment of diabetes. Here using single-molecule recordings we firstly reveal that insulin oligomerization can operate via monomeric additions and secondly quantify the existence, abundance and kinetic characterization of diverse insulin assembly and disassembly pathways involving addition of monomeric, dimeric or tetrameric insulin species. We propose and experimentally validate a model where the insulin self-assembly pathway is rerouted, favoring monomeric or oligomeric assembly, by solution concentration, additives and formulations. Combining our practically complete kinetic characterization with rate simulations, we calculate the abundance of each oligomeric species from nM to mM offering mechanistic insights and the relative abundance of all oligomeric forms at concentrations relevant both for secreted and administrated insulin. These reveal a high abundance of all oligomers and a significant fraction of hexamer resulting in practically halved bioavailable monomer concentration. In addition to providing fundamental new insights, the results and toolbox presented here can be universally applied, contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for additional proteins.

Ca2+-Responsive Glyco-insulin.

Chemical modification of peptides and proteins, such as PEGylation and lipidation, creates conjugates with new properties. However, they are typically not dynamic or stimuli-responsive. Self-assembly controlled by a stimulus will allow adjusting properties directly. Here, we report that conjugates of oligogalacturonic acids (OGAs), isolated from plant-derived pectin, are Ca2+-responsive. We report the conjugation of OGA to human insulin (HI) to create new glyco-insulins. In addition, we coupled OGA to model peptides. We studied their self-assembly by dynamic light scattering, small-angle X-ray scattering, and circular dichroism, which showed that the self-assembly to form nanostructures depended on the length of the OGA sequence and Zn2+ and Ca2+ concentrations. Subcutaneous administration of OGA12-HI with Zn2+ showed a stable decrease in blood glucose over a longer period of time compared to HI, despite the lower receptor binding affinity.

Selective Acylation of Proteins at Gly and Lys in His Tags.

The chemical modification of proteins is of great importance in chemical biology, biotechnology, and for the production of modified biopharmaceuticals, as it enables introduction of fluorophores, biotin, half-life extending moieties, and more. We have developed two methods that use poly-His sequences to direct the highly selective acylation of proteins, either at the N-terminus or at a specific Lys residue. For the former, we used an N-terminal Gly-His6 segment (Gly-His tag) that directed acylation of the N-terminal Nα -amine with 4-methoxyphenyl esters, resulting in stable conjugates. Next, we developed the peptide sequences Hisn -Lys-Hism (Lys-His tags) that direct the acylation of the designated Lys Nϵ -amine under mild conditions and with high selectivity over native Lys residues. Both the Gly-His and Lys-His tags maintain the capacity for immobilized metal ion affinity chromatography. We have demonstrated the robustness of these methods by attaching different moieties such as azides, fluorophores, and biotin to different proteins, including antibodies.

Highly Selective Lysine Acylation in Proteins Using a Lys-His Tag Sequence.

Chemical modification of proteins has numerous applications, but it has been challenging to achieve the required high degree of selectivity on lysine amino groups. Recently, we described the highly selective acylation of proteins with an N-terminal Gly-His6 segment. This tag promoted acylation of the N-terminal Nα -amine resulting in stable conjugates. Herein, we report the peptide sequences Hisn -Lys-Hism , which we term Lys-His tags. In combination with simple acylating agents, they facilitate the acylation of the designated Lys Nϵ -amine under mild conditions and with high selectivity over native Lys residues. We show that the Lys-His tags, which are 7 to 10 amino acids in length and still act as conventional His tags, can be inserted in proteins at the C-terminus or in loops, thus providing high flexibility regarding the site of modification. Finally, the selective and efficient acylation of the therapeutic antibody Rituximab, pure or mixed with other proteins, demonstrates the scope of the Lys-His tag acylation method.

Chirality transmission in macromolecular domains.

Chiral communications exist in secondary structures of foldamers and copolymers via a network of noncovalent interactions within effective intermolecular force (IMF) range. It is not known whether long-range chiral communication exists between macromolecular tertiary structures such as peptide coiled-coils beyond the IMF distance. Harnessing the high sensitivity of single-molecule force spectroscopy, we investigate the chiral interaction between covalently linked DNA duplexes and peptide coiled-coils by evaluating the binding of a diastereomeric pair of three DNA-peptide conjugates. We find that right-handed DNA triple helices well accommodate peptide triple coiled-coils of the same handedness, but not with the left-handed coiled-coil stereoisomers. This chiral communication is effective in a range (<4.5 nm) far beyond canonical IMF distance. Small-angle X-ray scattering and molecular dynamics simulation indicate that the interdomain linkers are tightly packed via hydrophobic interactions, which likely sustains the chirality transmission between DNA and peptide domains. Our findings establish that long-range chiral transmission occurs in tertiary macromolecular domains, explaining the presence of homochiral pairing of superhelices in proteins.

Metal-ion coordinated self-assembly of human insulin directs kinetics of insulin release as determined by preclinical SPECT/CT imaging.

Human insulin (HI) has fascinating metal-facilitated self-assembly properties that are essential for its biological function. HI has a natural Zn2+ binding site and we have previously shown that covalently attached abiotic ligands (e.g., bipyridine, terpyridine) can lead to the formation of nanosized oligomeric structures through the coordination of metal ions. Here we studied the hypothesis that metal ions can be used to directly control the pharmacokinetics of insulin after covalent attachment of an abiotic ligand that binds metal ions. We evaluated the pharmacokinetics (PK) and biodistribution of HI self-assemblies directed by metal ion coordination (i.e., Fe2+/Zn2+, Eu3+/Zn2+, Fe2+/Co3+) using preclinical SPECT/CT imaging and ex vivo gamma counting. HI was site-specifically modified with terpyridine (Tpy) at the PheB1 or LysB29 position to create conjugates that bind either Fe2+ or Eu3+, while its natural binding site (HisB10) preferentially coordinates with either Zn2+ or Co3+. HI was also functionalized with trans-cyclooctene (TCO) opposite to Tpy at PheB1 or LysB29, respectively, to allow for tetrazine-TCO coupling via a tetrazine-modified DTPA followed by 111In-radiolabeling for SPECT/CT imaging. When the 111In-B29Tpy-HI conjugate was coordinated with Fe2+/Zn2+, its retention at the injection site 6 h after injection was ~8-fold higher than the control without the metal ions, while its kidney accumulation was lower. 111In-B1Tpy-HI showed comparable retention at the injection site 6 h after injection and slightly increased retention at 24 h. However, higher kidney accumulation and residence time of degraded 111In-B1Tpy-HI was observed compared to that of 111In-B29Tpy-HI. Quantitative PK analysis based on SPECT/CT images confirmed slower distribution from the injection site of the HI-metal ion assemblies compared to control HI conjugates. Our results show that the Tpy-binding site (i.e., PheB1 or LysB29) on HI and its coordination with the added metal ions (i.e., Fe2+/Zn2+ or Fe2+/Co3+) directed the distribution half-life of HI significantly. This clearly indicates that the PK of insulin can be controlled by complexation with different metal ions.

Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors.

Plants and animals use cell surface receptors to sense and interpret environmental signals. In legume symbiosis with nitrogen-fixing bacteria, the specific recognition of bacterial lipochitooligosaccharide (LCO) signals by single-pass transmembrane receptor kinases determines compatibility. Here, we determine the structural basis for LCO perception from the crystal structures of two lysin motif receptor ectodomains and identify a hydrophobic patch in the binding site essential for LCO recognition and symbiotic function. We show that the receptor monitors the composition of the amphiphilic LCO molecules and uses kinetic proofreading to control receptor activation and signaling specificity. We demonstrate engineering of the LCO binding site to fine-tune ligand selectivity and correct binding kinetics required for activation of symbiotic signaling in plants. Finally, the hydrophobic patch is found to be a conserved structural signature in this class of LCO receptors across legumes that can be used for in silico predictions. Our results provide insights into the mechanism of cell-surface receptor activation by kinetic proofreading of ligands and highlight the potential in receptor engineering to capture benefits in plant-microbe interactions.

Safety-catch linkers for Fmoc solid-phase synthesis of peptide thioesters and hydrazides by amide-to-imide activation.

The use of C-terminal peptide thioesters and hydrazides in synthetic protein chemistry has inspired the search for optimal solid-phase peptide synthesis (SPPS) strategies for their assembly. However, peptide thioesters are not directly accessible by conventional Fmoc-SPPS owing to the nucleophilicity of the secondary amine required for Fmoc removal. Here, we report the mild and effective activation of the pGlu linker and a new safety-catch linker that was used for the convenient synthesis of peptide thioesters and hydrazides via efficient amide-to-imide activation followed by nucleophilic displacement.

IRDye800CW labeled uPAR-targeting peptide for fluorescence-guided glioblastoma surgery: Preclinical studies in orthotopic xenografts.

Glioblastoma (GBM) is a devastating cancer with basically no curative treatment. Even with aggressive treatment, the median survival is disappointing 14 months. Surgery remains the key treatment and the postoperative survival is determined by the extent of resection. Unfortunately, the invasive growth with irregular infiltrating margins complicates an optimal surgical resection. Precise intraoperative tumor visualization is therefore highly needed and molecular targeted near-infrared (NIR) fluorescence imaging potentially constitutes such a tool. The urokinase-type Plasminogen Activator Receptor (uPAR) is expressed in most solid cancers primarily at the invading front and the adjacent activated peritumoral stroma making it an attractive target for targeted fluorescence imaging. The purpose of this study was to develop and evaluate a new uPAR-targeted optical probe, IRDye800CW-AE344, for fluorescence guided surgery (FGS). Methods: In the present study we characterized the fluorescent probe with regard to binding affinity, optical properties, and plasma stability. Further, in vivo imaging characterization was performed in nude mice with orthotopic human patient derived glioblastoma xenografts, and we performed head-to-head comparison within FGS between our probe and the traditional procedure using 5-ALA. Finally, the blood-brain barrier (BBB) penetration was characterized in a 3D BBB spheroid model. Results: The probe effectively visualized GBM in vivo with a tumor-to-background ratio (TBR) above 4.5 between 1 to 12 h post injection and could be used for FGS of orthotopic human glioblastoma xenografts in mice where it was superior to 5-ALA. The probe showed a favorable safety profile with no evidence of any acute toxicity. Finally, the 3D BBB model showed uptake of the probe into the spheroids indicating that the probe crosses the BBB. Conclusion: IRDye800CW-AE344 is a promising uPAR-targeted optical probe for FGS and a candidate for translation into human use.

Controlling the fractal dimension in self-assembly of terpyridine modified insulin by Fe2+ and Eu3+ to direct in vivo effects.

Metal ion-induced self-assembly (SA) of proteins into higher-order structures can provide new, dynamic nano-assemblies. Here, the synthesis and characterization of a human insulin (HI) analog modified at LysB29 with the tridentate chelator 2,2':6',2''-terpyridine (Tpy) is described. SA of this new insulin analog (LysB29Tpy-HI) in the presence of the metal ions Fe2+ and Eu3+ at different concentrations was studied in solution by fluorescence luminescence and CD spectroscopy, dynamic light scattering, and small-angle X-ray scattering, while surface assembly was probed by AFM. Unique oligomerization was observed in solution, as Fe2+ yielded small magenta-colored discrete non-native assemblies, while Eu3+ caused the formation of large fractal assemblies. Binding of both metal ions to Tpy was demonstrated spectroscopically, and emission lifetime experiments revealed a distinct Eu3+ coordination geometry that included two water molecules. SAXS suggested that LysB29Tpy-HI with Fe2+ oligomerized to a discrete, roughly octameric species, while LysB29Tpy-HI with Eu3+ gave very large assemblies that could be modelled as fractals. The fractal dimensionality increased with the Eu3+ concentration. We propose that this is a consequence of Eu3+ binding to both Tpy and to free carboxylic acid groups on the insulin surface. LysB29Tpy-HI maintained insulin receptor affinity, and showed extended blood glucose lowering and plasma concentration after subcutaneous injection in rats. The combination of metal ion directed SA and native SA provides control of nano-scale fractal dimensionality and points towards use in therapeutics.

High-Performance Reversed-Phase Flash Chromatography Purification of Peptides and Chemically Modified Insulins.

Preparative reversed-phase HPLC is the established method for the purification of peptides, but has significant limitations. We systematically investigated the use of high-performance reversed-phase flash chromatography (HPFC) to rapidly purify laboratory-scale quantities of crude, synthetic peptides and chemically modified insulins. We demonstrated these methods for a diverse set of peptides, including short, medium, and long peptides. Depending on the purity profile of the peptide, HPFC can be used either as the sole purification method, or as a pre-purification method prior to final HPLC purification. Furthermore, HPFC is suitable for the purification of peptides that are not fully in solution. We provide guidelines for the HPFC of synthetic peptides and small proteins, including the choice of columns, eluents, and gradients. We believe that HPFC is a valuable alternative to HPLC purification of peptides and small proteins.

An Aldehyde Responsive, Cleavable Linker for Glucose Responsive Insulins.

A glucose responsive insulin (GRI) that responds to changes in blood glucose concentrations has remained an elusive goal. Here we describe the development of glucose cleavable linkers based on hydrazone and thiazolidine structures. We developed linkers with low levels of spontaneous hydrolysis but increased level of hydrolysis with rising concentrations of glucose, which demonstrated their glucose responsiveness in vitro. Lipidated hydrazones and thiazolidines were conjugated to the LysB29 side-chain of HI by pH-controlled acylations providing GRIs with glucose responsiveness confirmed in vitro for thiazolidines. Clamp studies showed increased glucose infusion at hyperglycemic conditions for one GRI indicative of a true glucose response. The glucose responsive cleavable linker in these GRIs allow changes in glucose levels to drive the release of active insulin from a circulating depot. We have demonstrated an unprecedented, chemically responsive linker concept for biopharmaceuticals.

Polyamine-Functionalized 2'-Amino-LNA in Oligonucleotides: Facile Synthesis of New Monomers and High-Affinity Binding towards ssDNA and dsDNA.

Attachment of cationic moieties to oligonucleotides (ONs) promises not only to increase the binding affinity of antisense ONs by reducing charge repulsion between the two negatively charged strands of a duplex, but also to augment their in vivo stability against nucleases. In this study, polyamine functionality was introduced into ONs by means of 2'-amino-LNA scaffolds. The resulting ONs exhibited efficient binding towards ssDNA, ssRNA and dsDNA targets, and the 2'-amino-LNA analogue carrying a triaminated linker showed the most pronounced duplex- and triplex-stabilizing effect. Molecular modelling revealed that favourable conformational and electrostatic effects led to salt-bridge formation between positively charged polyamine moieties and the Watson-Hoogsteen groove of the dsDNA targets, resulting in the observed triplex stabilization. All the investigated monomers showed increased resistance against 3'-nucleolytic digestion relative to the non-functionalized controls.

Ligand-recognizing motifs in plant LysM receptors are major determinants of specificity.

Plants evolved lysine motif (LysM) receptors to recognize and parse microbial elicitors and drive intracellular signaling to limit or facilitate microbial colonization. We investigated how chitin and nodulation (Nod) factor receptors of Lotus japonicus initiate differential signaling of immunity or root nodule symbiosis. Two motifs in the LysM1 domains of these receptors determine specific recognition of ligands and discriminate between their in planta functions. These motifs define the ligand-binding site and make up the most structurally divergent regions in cognate Nod factor receptors. An adjacent motif modulates the specificity for Nod factor recognition and determines the selection of compatible rhizobial symbionts in legumes. We also identified how binding specificities in LysM receptors can be altered to facilitate Nod factor recognition and signaling from a chitin receptor, advancing the prospects of engineering rhizobial symbiosis into nonlegumes.

How Membrane Geometry Regulates Protein Sorting Independently of Mean Curvature.

Biological membranes have distinct geometries that confer specific functions. However, the molecular mechanisms underlying the phenomenological geometry/function correlations remain elusive. We studied the effect of membrane geometry on the localization of membrane-bound proteins. Quantitative comparative experiments between the two most abundant cellular membrane geometries, spherical and cylindrical, revealed that geometry regulates the spatial segregation of proteins. The measured geometry-driven segregation reached 50-fold for membranes of the same mean curvature, demonstrating a crucial and hitherto unaccounted contribution by Gaussian curvature. Molecular-field theory calculations elucidated the underlying physical and molecular mechanisms. Our results reveal that distinct membrane geometries have specific physicochemical properties and thus establish a ubiquitous mechanistic foundation for unravelling the conserved correlations between biological function and membrane polymorphism.

Bioimaging and Biodistribution of the Metal-Ion-Controlled Self-Assembly of PYY3-36 Studied by SPECT/CT.

The controlled self-assembly of peptide- and protein-based pharmaceuticals is of central importance for their mode of action and tuning of their properties. Peptide YY3-36 (PYY3-36 ) is a 36-residue peptide hormone that reduces food intake when peripherally administered. Herein, we describe the synthesis of a PYY3-36 analogue functionalized with a metal-ion-binding 2,2'-bipyridine ligand that enables self-assembly through metal complexation. Upon addition of CuII , the bipyridine-modified PYY3-36 peptide binds stoichiometric quantities of metal ions in solution and contributes to the organization of higher-order assemblies. In this study, we aimed to explore the size effect of the self-assembly in vivo by using non-invasive quantitative single-photon emission computed tomography/computed tomography (SPECT/CT) imaging. For this purpose, bipyridine-modified PYY3-36 was radiolabeled with a chelator holding 111 InIII , followed by the addition of CuII to the bipyridine ligand. SPECT/CT imaging and biodistribution studies showed fast renal clearance and accumulation in the kidney cortex. The radiolabeled bipyridyl-PYY3-36 conjugates with and without CuII presented a slightly slower excretion 1 h post injection compared to the unmodified-PYY3-36 , thus demonstrating that higher self-assemblies of the peptide might have an effect on the pharmacokinetics.

The ABC of Insulin: The Organic Chemistry of a Small Protein.

Insulin is a small protein crucial for regulating the blood glucose level in all animals. Since 1922 it has been used for the treatment of patients with diabetes. Despite consisting of just 51 amino acids, insulin contains 17 of the proteinogenic amino acids, A- and B-chains, three disulfide bridges, and it folds with 3 α-helices and a short ß-sheet segment. Insulin associates into dimers and further into hexamers with stabilization by Zn2+ and phenolic ligands. Selective chemical modification of proteins is at the forefront of developments in chemical biology and biopharmaceuticals. Insulin's structure has made it amenable to organic and inorganic chemical reactions. This Review provides a synthetic organic chemistry perspective on this small protein. It gives an overview of key chemical and physico-chemical aspects of the insulin molecule, with a focus on chemoselective reactions. This includes N-acylations at the N-termini or at LysB29 by pH control, introduction of protecting groups on insulin, binding of metal ions, ligands to control the nano-scale assembly of insulin, and more.