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Autophagy is an evolutionarily ancient catabolic pathway and has recently emerged as an integral part of the innate immune system. While the core machinery of autophagy is well defined, the physiological regulation of autophagy is less understood. Here, we identify a C-terminal fragment of human hemoglobin A (HBA1, amino acids 111-132) in human bone marrow as a fast-acting non-inflammatory inhibitor of autophagy initiation. It is proteolytically released from full-length HBA1 by cathepsin E, trypsin or pepsin. Biochemical characterization revealed that HBA1(111-132) has an in vitro stability of 52 min in human plasma and adopts a flexible monomeric conformation in solution. Structure-activity relationship studies revealed that the C-terminal 13 amino acids of HBA1(120-132) are sufficient to inhibit autophagy, two charged amino acids (D127, K128) mediate solubility, and two serines (S125, S132) are required for function. Successful viruses like human immunodeficiency virus 1 (HIV-1) evolved strategies to subvert autophagy for virion production. Our results show that HBA1(120-132) reduced virus yields of lab-adapted and primary HIV-1. Summarizing, our data identifies naturally occurring HBA1(111-132) as a physiological, non-inflammatory antagonist of autophagy. Optimized derivatives of HBA1(111-132) may offer perspectives to restrict autophagy-dependent viruses., (© 2024. The Author(s).)

In contrast to biological cell membranes, it is still a major challenge for synthetic membranes to efficiently separate ions and small molecules due to their similar sizes in the sub-nanometer range. Inspired by biological ion channels with their unique channel wall chemistry that facilitates ion sieving by ion-channel interactions, the first free-standing, ultrathin (10-17 nm) nanomembranes composed entirely of polydopamine (PDA) are reported here as ion and molecular sieves. These nanomembranes are obtained via an easily scalable electropolymerization strategy and provide nanochannels with various amine and phenolic hydroxyl groups that offer a favorable chemical environment for ion-channel electrostatic and hydrogen bond interactions. They exhibit remarkable selectivity for monovalent ions over multivalent ions and larger species with K + /Mg 2+ of ≈4.2, K + /[Fe(CN) 6 ] 3- of ≈10.3, and K + /Rhodamine B of ≈273.0 in a pressure-driven process, as well as cyclic reversible pH-responsive gating properties. Infrared spectra reveal hydrogen bond formation between hydrated multivalent ions and PDA, which prevents the transport of multivalent ions and facilitates high selectivity. Chemically rich, free-standing, and pH-responsive PDA nanomembranes with specific interaction sites are proposed as customizable high-performance sieves for a wide range of challenging separation requirements., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

Drug safety and efficacy due to premature release into the bloodstream and poor biodistribution remains a problem despite seminal advances in this area. To circumvent these limitations, we report drug cyclization based on dynamic covalent linkages to devise a dual lock for the small-molecule anticancer drug, camptothecin (CPT). Drug activity is "locked" within the cyclic structure by the redox responsive disulfide and pH-responsive boronic acid-salicylhydroxamate and turns on only in the presence of acidic pH, reactive oxygen species and glutathione through traceless release. Notably, the dual-responsive CPT is more active (100-fold) than the non-cleavable (permanently closed) analogue. We further include a bioorthogonal handle in the backbone for functionalization to generate cyclic-locked, cell-targeting peptide- and protein-CPTs, for targeted delivery of the drug and traceless release in triple negative metastatic breast cancer cells to inhibit cell growth at low nanomolar concentrations., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Viral infections pose a significant threat to human health, and effective antiviral strategies are urgently needed. Antiviral peptides have emerged as a promising class of therapeutic agents due to their unique properties and mechanisms of action. While effective on their own, combining antiviral peptides may allow us to enhance their potency and to prevent viral resistance. Here, we developed an orthogonal chemical strategy to prepare a heterodimeric peptide conjugate assembled on a protein-based nanoplatform. Specifically, we combined the optimized version of two peptides inhibiting HIV-1 by distinct mechanisms. Virus-inhibitory peptide (VIRIP) is a 20 amino acid fragment of α1-antitrypsin that inhibits HIV-1 by targeting the gp41 fusion peptide. Endogenous peptide inhibitor of CXCR4 (EPI-X4) is a 16-residue fragment of human serum albumin that prevents HIV-1 entry by binding to the viral CXCR4 co-receptor. Optimized forms of both peptides are assembled on supramolecular nanoplatforms through the streptavidin-biotin interaction. We show that the construct consisting of the two different peptides (SAv-VIR-102C9-EPI-X4 JM#173-C) shows increased activity against CCR5- and CXCR4-tropic HIV-1 variants. Our results are a proof of concept that peptides with different modes of action can be assembled on nanoplatforms to enhance their antiviral activity.

Advanced derivatives of the E ndogenous P eptide I nhibitor of C X CR 4 (EPI-X4) have shown therapeutic efficacy upon topical administration in animal models of asthma and dermatitis. Here, we studied the plasma stability of the EPI-X4 lead compounds WSC02 and JM#21, using mass spectrometry to monitor the chemical integrity of the peptides and a functional fluorescence-based assay to determine peptide function in a CXCR4-antibody competition assay. Although mass spectrometry revealed very rapid disappearance of both peptides in human plasma within seconds, the functional assay revealed a significantly higher half-life of 9 min for EPI-X4 WSC02 and 6 min for EPI-X4 JM#21. Further analyses demonstrated that EPI-X4 WSC02 and EPI-X4 JM#21 interact with low molecular weight plasma components and serum albumin. Albumin binding is mediated by the formation of a disulfide bridge between Cys10 in the EPI-X4 peptides and Cys34 in albumin. These covalently linked albumin-peptide complexes have a higher stability in plasma as compared with the non-bound peptides and retain the ability to bind and antagonize CXCR4. Remarkably, chemically synthesized albumin-EPI-X4 conjugates coupled by non-breakable bonds have a drastically increased plasma stability of over 2 h. Thus, covalent coupling of EPI-X4 to albumin in vitro before administration or in vivo post administration may significantly increase the pharmacokinetic properties of this new class of CXCR4 antagonists.

With the advent of chemical strategies that allow the design of smart bioconjugates, peptide- and protein-drug conjugates are emerging as highly efficient therapeutics to overcome limitations of conventional treatment, as exemplified by antibody-drug conjugates (ADCs). While targeting peptides serve similar roles as antibodies to recognize overexpressed receptors on diseased cell surfaces, peptide-drug conjugates suffer from poor stability and bioavailability due to their low molecular weights. Through a combination of a supramolecular protein-based assembly platform and a pH-responsive linker, the authors devise herein the convenient assembly of a trivalent protein-drug conjugate. The conjugate should ideally possess distinct features of ADCs such as 1) recognition sites that recognize cell receptor and are arranged on 2) distinct locations on a high molecular weight protein scaffold, 3) a stimuli-responsive linker, as well as 4) an attached payload such as a drug molecule. These AD-like conjugates target cancer cells that overexpress somatostatin receptors, can enable controlled release in the microenvironment of cancer cells through a new pH-responsive biotin linker, and exhibit stability in biological media., (© 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive linkers or conjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding and fast dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome the limitation in linker design, we reported herein dual responsive dynamic covalent peptide tags combining a pH responsive boronate ester with fast association and dissociation rates, and a redox-active disulfide with slow formation and dissociation rate. Precoordination by boronic acid-catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. Furthermore, the conjugate responds to pH changes within the physiological range as well as to redox conditions found inside cancer cells. Such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.

The development of bioconjugation chemistry has enabled the combination of various synthetic functionalities to proteins, giving rise to new classes of protein conjugates with functions well beyond what Nature can provide. Despite the progress in bioconjugation chemistry, there are no reagents developed to date where the reactivity can be tuned in a user-defined fashion to address different amino acid residues in proteins. Here, we report that 2-chloromethyl acryl reagents can serve as a simple yet versatile platform for selective protein modification at cysteine or disulfide sites by tuning their inherent electronic properties through the amide or ester linkage. Specifically, the 2-chloromethyl derivatives (acrylamide or acrylate) can be obtained via a simple and easily implemented one-pot reaction based on the coupling reaction between commercially available starting materials with different end-group functionalities (amino group or hydroxyl group). 2-Chloromethyl acrylamide reagents with an amide linkage favor selective modification at the cysteine site with fast reaction kinetics and near quantitative conversations. In contrast, 2-chloromethyl acrylate reagents bearing an ester linkage can undergo two successive Michael reactions, allowing the selective modification of disulfides bonds with high labeling efficiency and good conjugate stability., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

Site-selective protein functionalization serves as an invaluable tool for investigating protein structures and functions in complicated cellular environments and accomplishing semi-synthetic protein conjugates such as traceable therapeutics with improved features. Dual functionalization of proteins allows the incorporation of two different types of functionalities at distinct location(s), which greatly expands the features of native proteins. The attachment and crosstalk of a fluorescence donor and an acceptor dye provides fundamental insights into the folding and structural changes of proteins upon ligand binding in their native cellular environments. Moreover, the combination of drug molecules with different modes of action, imaging agents or stabilizing polymers provides new avenues to design precision protein therapeutics in a reproducible and well-characterizable fashion. This review aims to give a timely overview of the recent advancements and a future perspective of this relatively new research area. First, the chemical toolbox for dual functionalization of proteins is discussed and compared. The strengths and limitations of each strategy are summarized in order to enable readers to select the most appropriate method for their envisaged applications. Thereafter, representative applications of these dual-modified protein bioconjugates benefiting from the synergistic/additive properties of the two synthetic moieties are highlighted., (© 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Proteins have attracted increasing attention as biopharmaceutics and diagnostics due to their high specificity, biocompatibility, and biodegradability. The biopharmaceutical sector in particular is experiencing rapid growth, which has led to an increase in the production and sale of protein drugs and diagnostics over the last two decades. Since the first-generation biopharmaceutics dominated by native proteins, both recombinant and chemical technologies have evolved and transformed the outlook of this rapidly developing field. This review article presents updates on the fabrication of covalent and supramolecular fusion hybrids, as well as protein-polymer hybrids using solid-phase approaches that hold great promise for preparing protein hybrids with precise control at the macromolecular level to incorporate additional features. In addition, the applications of the resultant protein hybrids in medicine and diagnostics are highlighted where possible., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

The preparation of precise macromolecules with multiple functionalities remains a challenge in drug delivery. Here, a method to prepare stoichiometrically precise tetrafunctional streptavidin conjugates is presented with an exemplary structure combining exactly one fluorescent label, one cell targeting group, one nucleus penetrating peptide and one drug molecule.

An inverse electron demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO) holds great promise for protein modification and manipulation. Herein, we report the design and synthesis of a tetrazine-based disulfide rebridging reagent, which allows the site-selective installation of a tetrazine group into disulfide-containing peptides and proteins such as the hormone somatostatin (SST) and the antigen binding fragment (Fab) of human immunoglobulin G (IgG). The fast and efficient conjugation of the tetrazine modified proteins with three different TCO-containing substrates to form a set of bioconjugates in a site-selective manner was successfully demonstrated for the first time. Homogeneous, well-defined bioconjugates were obtained underlining the great potential of our method for fast bioconjugation in emerging protein therapeutics. The formed bioconjugates were stable against glutathione and in serum, and they maintained their secondary structure. With this work, we broaden the scope of tetrazine chemistry for site-selective protein modification to prepare well-defined SST and Fab conjugates with preserved structures and good stability under biologically relevant conditions.

Acute myeloid leukemia (AML) is characterized by relapse and treatment resistance in a major fraction of patients, underlining the need of innovative AML targeting therapies. Here we analysed the therapeutic potential of an innovative biohybrid consisting of the tumor-associated peptide somatostatin and the photosensitizer ruthenium in AML cell lines and primary AML patient samples. Selective toxicity was analyzed by using CD34 enriched cord blood cells as control. Treatment of OCI AML3, HL60 and THP1 resulted in a 92, and 99 and 97% decrease in clonogenic growth compared to the controls. Primary AML cells demonstrated a major response with a 74 to 99% reduction in clonogenicity in 5 of 6 patient samples. In contrast, treatment of CD34 + CB cells resulted in substantially less reduction in colony numbers. Subcellular localization assays of RU-SST in OCI-AML3 cells confirmed strong co-localization of RU-SST in the lysosomes compared to the other cellular organelles. Our data demonstrate that conjugation of a Ruthenium complex with somatostatin is efficiently eradicating LSC candidates of patients with AML. This indicates that receptor mediated lysosomal accumulation of photodynamic metal complexes is a highly attractive approach for targeting AML cells.

The targeted pharmacological modulation of polymorphonuclear leukocytes (PMNs) is of major medical interest. These innate immune cells play a central role in the defense against pathogenic microorganisms. However, their excessive chemotactic recruitment into tissues after traumatic injury is detrimental due to local and systemic inflammation. Rho-GTPases, being the master regulators of the actin cytoskeleton, regulate migration and chemotaxis of PMNs, are attractive pharmacological targets. Herein, supramolecular protein complexes are assembled in a "mix-and-match" approach containing the specific Rho-inhibiting clostridial C3 enzyme and three PMN-binding peptides using an avidin platform. Selective delivery of the C3 Rho-inhibitor with these complexes into the cytosol of human neutrophil-like NB-4 cells and primary human PMNs ex vivo is demonstrated, where they catalyze the adenosine diphosphate (ADP) ribosylation of Rho and induce a characteristic change in cell morphology. Notably, the complexes do not deliver C3 enzyme into human lung epithelial cells, A549 lung cancer cells, and immortalized human alveolar epithelial cells (hAELVi), demonstrating their cell type-selectivity. The supramolecular complexes represent attractive molecular tools to decipher the role of PMNs in infection and inflammation or for the development of novel therapeutic approaches for diseases that are associated with hyperactivity and reactivity of PMNs such as post-traumatic injury., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The surface of proteins is heterogeneous with sophisticated but precise hydrophobic and hydrophilic patches, which is essential for their diverse biological functions. To emulate such distinct surface patterns on macromolecules, we used rigid spherical synthetic dendrimers (polyphenylene dendrimers) to provide controlled amphiphilic surface patches with molecular precision. We identified an optimal spatial arrangement of these patches on certain dendrimers that enabled their interaction with human adenovirus 5 (Ad5). Patchy dendrimers bound to the surface of Ad5 formed a synthetic polymer corona that greatly altered various host interactions of Ad5 as well as in vivo distribution. The dendrimer corona (1) improved the ability of Ad5-derived gene transfer vectors to transduce cells deficient for the primary Ad5 cell membrane receptor and (2) modulated the binding of Ad5 to blood coagulation factor X, one of the most critical virus-host interactions in the bloodstream. It significantly enhanced the transduction efficiency of Ad5 while also protecting it from neutralization by natural antibodies and the complement system in human whole blood. Ad5 with a synthetic dendrimer corona revealed profoundly altered in vivo distribution, improved transduction of heart, and dampened vector sequestration by liver and spleen. We propose the design of bioactive polymers that bind protein surfaces solely based on their amphiphilic surface patches and protect against a naturally occurring protein corona, which is highly attractive to improve Ad5-based in vivo gene therapy applications.

Nature has evolved an optimal synthetic factory in the form of translational and posttranslational processes by which millions of proteins with defined primary sequences and 3D structures can be built. Nature's toolkit gives rise to protein building blocks, which dictates their spatial arrangement to form functional protein nanostructures that serve a myriad of functions in cells, ranging from biocatalysis, formation of structural networks, and regulation of biochemical processes, to sensing. With the advent of chemical tools for site-selective protein modifications and recombinant engineering, there is a rapid development to develop and apply synthetic methods for creating structurally defined, functional protein nanostructures for a broad range of applications in the fields of catalysis, materials and biomedical sciences. In this review, design principles and structural features for achieving and characterizing functional protein nanostructures by synthetic approaches are summarized. The synthetic customization of protein building blocks, the design and introduction of recognition units and linkers and subsequent assembly into structurally defined protein architectures are discussed herein. Key examples of these supramolecular protein nanostructures, their unique functions and resultant impact for biomedical applications are highlighted.

Dynamic covalent chemistry is a versatile and powerful tool that integrates both stable chemical bonds and stimulus responsiveness into the construction of smart biotherapeutics. With minimalistic molecular design, a dynamic covalent protein assembly that incorporates selective targeting and intracellular release upon pH stimulus is presented. The construct comprises an active enzymatic protein core (cytochrome c) self-assembled with cancer cell targeting motifs (somatostatin) through boronic acid/salicylhydroxamate chemistry. The bioorthogonal assembly takes place rapidly under neutral aqueous conditions while the release of the protein is initiated under acidic conditions found within cellular vesicles during uptake. By demonstrating that these modular components act in synergy, we show the broad applicability of such chemical strategies to advance the frontier of modern nanomedicine., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Protein-based polymeric polyelectrolytes are emerging as alternative synthetic nanoparticles owing to their biodegradability and biocompatibility. However, potential in vivo toxicity remains a significant challenge. Herein an array of protein polyelectrolytes generated from cationic human serum albumin (cHSA) and polyethylene glycol (PEG) are synthesized via synthetic customization as antimicrobials for the treatment of systemic infections. By varying PEG molecular weight and chain length, in vitro hemolytic activity can be fine-tuned without significantly affecting antimicrobial potency. The optimal hybrid material, PEG (2000) 18 -cHSA, with potent antimicrobial character, low hemolytic activity, and in vitro biofilm disruptive properties is identified. Surface plasmon resonance (SPR) evaluation demonstrates significantly higher binding activity of the protein nanoparticles to bacteria cell wall components and microfluidic live-cell imaging indicates that the nanoparticles act through a membranolytic mechanism. Given their low susceptibility to drug resistance and potent activity against resistant bacteria strains, these findings establish the PEGylated albumin nanoparticles as a potent weaponry against drug resistance and biofilm-related infection., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Once materials come into contact with a biological fluid containing proteins, proteins are generally-whether desired or not-attracted by the material's surface and adsorb onto it. The aim of this Review is to give an overview of the most commonly used characterization methods employed to gain a better understanding of the adsorption processes on either planar or curved surfaces. We continue to illustrate the benefit of combining different methods to different surface geometries of the material. The thus obtained insight ideally paves the way for engineering functional materials that interact with proteins in a predetermined manner., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The development of small protein tags that exhibit bioorthogonality, bond stability, and reversibility, as well as biocompatibility, holds great promise for applications in cellular environments enabling controlled drug delivery or for the construction of dynamic protein complexes in biological environments. Herein, we report the first application of dynamic covalent chemistry both for purification and for reversible assembly of protein conjugates using interactions of boronic acid with diols and salicylhydroxamates. Incorporation of the boronic acid (BA) tag was performed in a site-selective fashion by applying disulfide rebridging strategy. As an example, a model protein enzyme (lysozyme) was modified with the BA tag and purified using carbohydrate-based column chromatography. Subsequent dynamic covalent "click-like" bioconjugation with a salicylhydroxamate modified fluorescent dye (BODIPY FL) was accomplished while retaining its original enzymatic activity.

A facile chemical approach integrating supramolecular chemistry, site-selective protein chemistry, and molecular biology is described to engineer synthetic multidomain protein therapeutics that sensitize cancer cells selectively to significantly enhance antitumor efficacy of existing chemotherapeutics. The desired bioactive entities are assembled via supramolecular interactions at the nanoscale into structurally ordered multiprotein complexes comprising a) multiple copies of the chemically modified cyclic peptide hormone somatostatin for selective targeting and internalization into human A549 lung cancer cells expressing SST-2 receptors and b) a new cysteine mutant of the C3bot1 (C3) enzyme from Clostridium botulinum , a Rho protein inhibitor that affects and influences intracellular Rho-mediated processes like endothelial cell migration and blood vessel formation. The multidomain protein complex, SST3-Avi-C3, retargets C3 enzyme into non-small cell lung A549 cancer cells and exhibits exceptional tumor inhibition at a concentration ≈100-fold lower than the clinically approved antibody bevacizumab (Avastin) in vivo. Notably, SST3-Avi-C3 increases tumor sensitivity to a conventional chemotherapeutic (doxorubicin) in vivo. These findings show that the integrated approach holds vast promise to expand the current repertoire of multidomain protein complexes and can pave the way to important new developments in the area of targeted and combination cancer therapy.

The attachment of two different functionalities in a site-selective fashion represents a great challenge in protein chemistry. We report site specific dual functionalizations of peptides and proteins capitalizing on reactivity differences of cysteines in their free (thiol) and protected, oxidized (disulfide) forms. The dual functionalization of interleukin 2 and EYFP proceeded with no loss of bioactivity in a stepwise fashion applying maleimide and disulfide rebridging allyl-sulfone groups. In order to ensure broader applicability of the functionalization strategy, a novel, short peptide sequence that introduces a disulfide bridge was designed and site-selective dual labeling in the presence of biogenic groups was successfully demonstrated.

In osteoporosis, bone structure can be improved by the introduction of therapeutic molecules inhibiting bone resorption by osteoclasts. Here, biocompatible hydrogels represent an excellent option for the delivery of pharmacologically active molecules to the bone tissue because of their biodegradability, injectability, and manifold functionalization capacity. The present study reports the preparation of a multifunctional hybrid hydrogel from chemically modified human serum albumin and rationally designed DNA building blocks. The hybrid hydrogel combines advantageous characteristics, including rapid gelation through DNA hybridization under physiological conditions and a self-healing and injectable nature with the possibility of specific loading and spatiotemporally controlled release of active proteins, making it an advanced biomaterial for the local treatment of bone diseases, for example, osteoporosis. The hydrogels are loaded with a recombinant Rho-inhibiting C3 toxin, C2IN-C3lim-G205C. This toxin selectively targets osteoclasts and inhibits Rho-signaling and, thereby, actin-dependent processes in these cells. Application of C2IN-C3lim-G205C toxin-loaded hydrogels effectively reduces osteoclast formation and resorption activity in vitro, as demonstrated by tartrate-resistant acid phosphatase staining and the pit resorption assay. Simultaneously, osteoblast activity, viability, and proliferation are unaffected, thus making C2IN-C3lim-G205C toxin-loaded hybrid hydrogels an attractive pharmacological system for spatial and selective modulation of osteoclast functions to reduce bone resorption., (© 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The synthetic transformation of polypeptides with molecular accuracy holds great promise for providing functional and structural diversity beyond the proteome. Consequently, the last decade has seen an exponential growth of site-directed chemistry to install additional features into peptides and proteins even inside living cells. The disulfide rebridging strategy has emerged as a powerful tool for site-selective modifications since most proteins contain disulfide bonds. In this Review, we present the chemical design, advantages and limitations of the disulfide rebridging reagents, while summarizing their relevance for synthetic customization of functional protein bioconjugates, as well as the resultant impact and advancement for biomedical applications., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nature has provided a highly optimized toolbox in bacterial endotoxins with precise functions dictated by their clear structural division. Inspired by this streamlined design, a supramolecular approach capitalizing on the strong biomolecular (streptavidin (SA))-biotin interactions is reported herein to prepare two multipartite fusion constructs, which involves the generation 2.0 (D2) or generation 3.0 (D3) polyamidoamine-dendronized transporter proteins (dendronized streptavidin (D3SA) and dendronized human serum albumin (D2HSA)) non-covalently fused to the C3bot1 enzyme from Clostridium botulinum, a potent and specific Rho-inhibitor. The fusion constructs, D3SA-C3 and D2HSA-C3, represent the first examples of dendronized protein transporters that are fused to the C3 enzyme, and it is successfully demonstrated that the C3 Rho-inhibitor is delivered into the cytosol of mammalian cells as determined from the characteristic C3-mediated changes in cell morphology and confocal microscopy. The design circumvents the low uptake of the C3 enzyme by eukaryotic cells and holds great promise for reprogramming the properties of toxin enzymes using a supramolecular approach to broaden their therapeutic applications., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Water-soluble allyl sulfones provide convenient site-specific disulfide rebridging of native proteins and cyclic peptides. The site-selective functionalization of (a) the peptide hormone somatostatin, (b) the interchain disulfide of bovine insulin and (c) functionalization of the proteins GFP and lysozyme with allyl sulfones proceeds in aqueous solution. Allyl sulfones offer three functionalizable sites that react with thiol containing molecules in a step-wise fashion. Dual labeling of proteins and cyclic peptides is achieved i.e. the attachment of a chromophore and an affinity tag in a single reaction step, which is of great significance for the construction of precise multifunctional peptide and protein conjugates.

We present a versatile approach for the synthesis of cyclic peptide amphiphiles of the hormone somatostatin (SST) with tunable lipophilic tails to program bioactive nanoarchitectures. A novel bis-alkylation reagent is synthesized that facilitates the functionalization of SST with a thiol anchor. Different hydrophobic moieties are introduced inspired by a biomimetic palmitoylation approach which opens access to cyclic peptide amphiphiles that display rich self-organization and cell membrane interactions., (Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The design and synthesis of a polyphenylene dendrimer (PPD 3) with discrete binding sites for lipophilic guest molecules and characteristic surface patterns is presented. Its semi-rigidity in combination with a precise positioning of hydrophilic and hydrophobic groups at the periphery yields a refined architecture with lipophilic binding pockets that accommodate defined numbers of biologically relevant guest molecules such as fatty acids or the drug doxorubicin. The size, architecture, and surface textures allow to even penetrate brain endothelial cells that are a major component of the extremely tight blood-brain barrier. In addition, low to no toxicity is observed in in vivo studies using zebrafish embryos. The unique PPD scaffold allows the precise placement of functional groups in a given environment and offers a universal platform for designing drug transporters that closely mimic many features of proteins., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The development of protein-polymer hybrids emerged several decades ago with the vision that their synergistic combination will offer macromolecular hybrids with manifold features to succeed as the next generation therapeutics. From the first generation of protein-polymer therapeutics represented by PEGylated proteins, the field has since advanced, reinforced by the progress in contemporary chemical techniques for designing polymeric scaffolds and protein engineering. Novel polymerization techniques that offer multifunctional strategies as well as a greater understanding of proteins and their biological behavior have both proven to be exceptional tools in the construction of these hybrid materials. In this review, we seek to summarize and highlight the recent progress in these semi-synthetic protein hybrids in terms of their preparation, design, resulting bioarchitectures and bioactivities for their intended bio-applications.

A disulfide intercalator toolbox was developed for site-specific attachment of a broad variety of functional groups to proteins or peptides under mild, physiological conditions. The peptide hormone somatostatin (SST) served as model compound for intercalation into the available disulfide functionalization schemes starting from the intercalator or the reactive SST precursor before or after bioconjugation. A tetrazole-SST derivative was obtained that undergoes photoinduced cycloaddition in mammalian cells, which was monitored by live-cell imaging., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CONSPECTUS: Chemical programming of macromolecular structures to instill a set of defined chemical properties designed to behave in a sequential and precise manner is a characteristic vision for creating next generation nanomaterials. In this context, biopolymers such as proteins and nucleic acids provide an attractive platform for the integration of complex chemical design due to their sequence specificity and geometric definition, which allows accurate translation of chemical functionalities to biological activity. Coupled with the advent of amino acid specific modification techniques, "programmable" areas of a protein chain become exclusively available for any synthetic customization. We envision that chemically reprogrammed hybrid proteins will bridge the vital link to overcome the limitations of synthetic and biological materials, providing a unique strategy for tailoring precision therapeutics. In this Account, we present our work toward the chemical design of protein- derived hybrid polymers and their supramolecular responsiveness, while summarizing their impact and the advancement in biomedicine. Proteins, in their native form, represent the central framework of all biological processes and are an unrivaled class of macromolecular drugs with immense specificity. Nonetheless, the route of administration of protein therapeutics is often vastly different from Nature's biosynthesis. Therefore, it is imperative to chemically reprogram these biopolymers to direct their entry and activity toward the designated target. As a consequence of the innate structural regularity of proteins, we show that supramolecular interactions facilitated by stimulus responsive chemistry can be intricately designed as a powerful tool to customize their functions, stability, activity profiles, and transportation capabilities. From another perspective, a protein in its denatured, unfolded form serves as a monodispersed, biodegradable polymer scaffold decorated with functional side chains available for grafting with molecules of interest. Additionally, we are equipped with analytical tools to map the fingerprint of the protein chain, directly elucidating the structure at the molecular level. Contrary to conventional polymers, these biopolymers facilitate a more systematic avenue to investigate engineered macromolecules, with greater detail and accuracy. In this regard, we focus on denaturing serum albumin, an abundant blood protein, and exploit its peptidic array of functionalities to program supramolecular architectures for bioimaging, drug and gene delivery. Ultimately, we seek to assimilate the evolutionary advantage of these protein based biopolymers with the limitless versatility of synthetic chemistry to merge the best of both worlds.

A modular approach for the precise assembly of multi-component hydrogels consisting of protein and DNA building blocks is described for the first time. Multi-arm DNA is designed for crosslinking and stepwise, non-covalent assembly of active proteins inside the hydrogel.

The modulation of protein uptake and activity in response to physiological changes forms an integral part of smart protein therapeutics. We describe herein the self-assembly of a pH-responsive dendrimer shell onto the surface of active enzymes (trypsin, papain, DNase I) as a supramolecular protecting group to form a hybrid dendrimer-enzyme complex. The attachment is based on the interaction between boronic acid and salicyl hydroxamate, thus allowing the macromolecular assembly to respond to changes in pH between 5.0 and 7.4 in a highly reversible fashion. Catalytic activity is efficiently blocked in the presence of the dendrimer shell but is quantitatively restored upon shell degradation under acidic conditions. Unlike the native proteases, the hybrid constructs are shown to be efficiently taken up by A549 cells and colocalized in the acidic compartments. The programmed intracellular release of the proteases induced cytotoxicity, thereby uncovering a new avenue for precision biotherapeutics., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the ideal case, a precise synthesis yields molecules with a constitutional as well as a conformational perfectness. Such a case of precision is demonstrated by the synthesis of semi-rigid amphiphilic polyphenylene dendrimers (PPDs). Polar sulfonate groups are precisely placed on their periphery in such a manner that patches of polar and non-polar regions are created. Key structural features are the semi-rigid framework and shape-persistent nature of PPDs since the limited flexibility introduces a nano-phase-separated amphiphilic rim of the dendrimer. This results in both attractive and repulsive interactions with a given solvent. Frustrated solvent structures then lead to a remarkable solubility in solvents of different polarity such as toluene, methanol, and water or their mixtures. Water solubility combined with defined surface structuring and variable hydrophobicity of PPDs that resemble the delicate surface textures of proteins are important prerequisites for their biological and medical applications based upon cellular internalization., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1-like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine cells decreases. Conversely, knockdown of Nel expression by transposon-mediated introduction of RNA interference constructs results in decrease in RGC number and increase in the number of displaced amacrine cells. Modifications of Nel expression levels do not appear to affect proliferation of retinal progenitor cells, but they significantly alter the progression rate of RGC differentiation from the central retina to the periphery. Furthermore, Nel protects RGCs from apoptosis during retinal development. These results indicate that Nel positively regulates RGC production by promoting their differentiation and survival during development.

Versatile nanocarrier systems facilitating uptake of exogenous proteins are highly alluring in evaluating these proteins for therapeutic applications. The self-assembly of an efficient nano-sized protein transporter consisting of three different entities is presented: A streptavidin protein core functioning as an adapter, second generation polyamidoamine dendrons for facilitating cell uptake as well as two different therapeutic proteins (tumor suppressor p53 or pro-apoptotic cytochrome c as cargo). Well-defined dendrons containing a biotin core are prepared and display no cytotoxic behavior upon conjugation to streptavidin. The integration of biotinylated human recombinant p53 (B-p53) into the three component system allows excellent internalization into HeLa, A549 and SaOS osteosarcoma cells monitored via confocal microscopy, immunoblot analysis and co-localization studies. In addition, the conjugation of B-p53 to dendronized streptavidin preserves its specific DNA-binding in vitro, and its delivery into SaOS cells impairs cell viability with concomitant activation of caspases 3 and 7. The versatility of this system is further exhibited by the significant enhancement of the pro-apoptotic effects of internalized cytochrome c which is analyzed by flow cytometry and cell viability assays. These results demonstrate that the "bio-click" self-assembly of biotinylated dendrons and proteins on a streptavidin adapter yields a stable supramolecular complex. This efficient bionanotransporter provides an attractive platform for mediating the delivery of functional proteins of interest into living mammalian cells in a facile and rapid way., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A facile, noncovalent solid-phase immobilization platform is described to assemble Janus-like supramolecular fusion proteins that are responsive to external stimuli. A chemically postmodified transporter protein, DHSA, is fused with (imino)biotinylated cargo proteins via an avidin adaptor with a high degree of spatial control. Notably, the derived heterofusion proteins are able to cross cellular membranes, dissociate at acidic pH due to the iminobiotin linker and preserve the enzymatic activity of the cargo proteins β-galactosidase and the enzymatic subunit of Clostridium botulinum C2 toxin. The mix-and-match strategy described herein opens unique opportunities to access macromolecular architectures of high structural definition and biological activity, thus complementing protein ligation and recombinant protein expression techniques.

The native transportation protein serum albumin represents an attractive nano-sized transporter for drug delivery applications due to its beneficial safety profile. Existing albumin-based drug delivery systems are often limited by their low drug loading capacity as well as noticeable drug leakage into the blood circulation. Therefore, a unique albumin-derived core-shell doxorubicin (DOX) delivery system based on the protein denaturing-backfolding strategy was developed. 28 DOX molecules were covalently conjugated to the albumin polypeptide backbone via an acid sensitive hydrazone linker. Polycationic and pegylated human serum albumin formed two non-toxic and enzymatically degradable protection shells around the encapsulated DOX molecules. This core-shell delivery system possesses notable advantages, including a high drug loading capacity critical for low administration doses, a two-step drug release mechanism based on pH and the presence of proteases, an attractive biocompatibility and narrow size distribution inherited from the albumin backbone, as well as fast cellular uptake and masking of epitopes due to a high degree of pegylation. The IC50 of these nanoscopic onion-type micelles was found in the low nanomolar range for Hela cells as well as leukemia cell lines. In vivo data indicate its attractive potential as anti-leukemia treatment suggesting its promising profile as nanomedicine drug delivery system., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The synthesis of biohybrid materials with tailored functional properties represents a topic of emerging interest. Combining proteins as natural, macromolecular building blocks, and synthetic polymers opens access to giant brush-like biopolymers of high structural definition. The properties of these precision polypeptide copolymers can be tailored through various chemical modifications along their polypeptide backbone, which expands the repertoire of known protein-based materials to address biomedical applications. In this article, the synthetic strategies for the design of precision biopolymers from proteins through amino acid specific conjugation reagents are highlighted and the different functionalization strategies, their characterization, and applications are discussed., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe the synthesis of a core-shell biohybrid consisting of a human serum albumin (HSA) core that serves as a reservoir for lipophilic molecules and a cationized shell region consisting of ethynyl-G2.0-PAMAM or ethynyl-G3.0-PAMAM dendrons. The binding capacity of lipophilic guests was quantified applying electron paramagnetic resonance (EPR) spectroscopy, and five to six out of seven pockets were still available compared with HSA. The attachment of ethynyl-G2.0-PAMAM dendrons to HSA yielded a nontoxic core-shell macromolecule that was clearly uptaken by A549 human epithelial cells due to the presence of the dendritic PAMAM shell. Significantly higher loading of doxorubicin was observed for dendronized G2-DHSA compared with the native protein due to the availability of binding pockets of the HSA core, and interaction with the dendritic shell. Dendronized G2-DHSA-doxorubicin displayed significant cytotoxicity resulting from high drug loading and high stability under different conditions, thus demonstrating its great potential as a transporter for drug molecules.

Multifunctional peptide-polymer hybrid materials have been applied as efficient and biocompatible quantum-dot coating materials. Significant pH responsiveness (e.g., an influence of the pH on the quantum yields of the peptide-polymer/QDs) was found and is attributed to conformational rearrangements of the peptide backbone.

Nel is a glycoprotein containing five chordin-like and six epidermal growth factor-like domains and is strongly expressed in the nervous system. In this study, we have examined expression patterns and in vitro functions of Nel in the chicken retinotectal system. We have found that in the developing tectum, expression of Nel is localized in specific laminae that retinal axons normally do not enter, including the border between the retinorecipient and non-retinorecipient laminae. Nel-binding activity is detected on retinal axons both in vivo and in vitro, suggesting that retinal axons express a receptor for Nel. In vitro, Nel inhibits retinal axon outgrowth and induces growth cone collapse and axon retraction. These results indicate that Nel acts as an inhibitory guidance cue for retinal axons, and suggest its roles in the establishment of the lamina-specificity in the retinotectal projection.

Disrupted-in-Schizophrenia-1 (DISC1), identified by positional cloning of a balanced translocation (1;11) with the breakpoint in intron 8 of a large Scottish pedigree, is associated with a range of neuropsychiatric disorders including schizophrenia. To model this mutation in mice, we have generated Disc1(tr) transgenic mice expressing 2 copies of truncated Disc1 encoding the first 8 exons using a bacterial artificial chromosome (BAC). With this partial simulation of the human situation, we have discovered a range of phenotypes including a series of novel features not previously reported. Disc1(tr) transgenic mice display enlarged lateral ventricles, reduced cerebral cortex, partial agenesis of the corpus callosum, and thinning of layers II/III with reduced neural proliferation at midneurogenesis. Parvalbumin GABAergic neurons are reduced in the hippocampus and medial prefrontal cortex, and displaced in the dorsolateral frontal cortex. In culture, transgenic neurons grow fewer and shorter neurites. Behaviorally, transgenic mice exhibit increased immobility and reduced vocalization in depression-related tests, and impairment in conditioning of latent inhibition. These abnormalities in Disc1(tr) transgenic mice are consistent with findings in severe schizophrenia.

The reaction of the 1,2,3,5-dithiadiazolyls (4-R-C(6)H(4)CN(2)S(2))(2) (R = Me, 2a; Cl, 2b; OMe, 2c; and CF3, 2d) and (3-NC-5-tBu-C(6)H(3)CN(2)S(2))(2) (2e) with [CpCr(CO)(3)](2) (Cp = eta(5)-C(5)H(5)) (1) at ambient temperature respectively yielded the complexes CpCr(CO)(2)(eta(2)-S(2)N(2)CC(6)H(4)R) (R = 4-Me, 3a; 4-Cl, 3b; 4-OMe, 3c; and 4-CF(3), 3d) and CpCr(CO)(2)(eta(2)-S(2)N(2)CC(6)H(3)-3-(CN)-5-(tBu)) (3e) in 35-72% yields. The complexes 3c and 3d were also synthesized via a salt metathesis method from the reaction of NaCpCr(CO)(3) (1B) and the 1,2,3,5-dithiadiazolium chlorides 4-R-C(60H(4)CN(2)S(2)Cl (R = OMe, 8c; CF(3), 8d) with much lower yields of 6 and 20%, respectively. The complexes were characterized spectroscopically and also by single-crystal X-ray diffraction analysis. Cyclic voltammetry experiments were conducted on 3a-e, EPR spectra were obtained of one-electron-reduced forms of 3a-e, and variable temperature 1H NMR studies were carried out on complex 3d. Hybrid DFT calculations were performed on the model system [CpCr(CO)(2)S(2)N(2)CH] and comparisons are made with the reported CpCr(CO)(2)(pi-allyl) complexes.

The reaction of [Cp*RuCl2]2 (1; Cp* = eta5-C5Me5) with tetraalkyldithiuram disulfides (R2NC(S)SS(S)CNR2, R = Me, Et), isopropylxanthic disulfide ([iPrOC(S)S]2), and bis(thiophosphoryl) disulfide ([(iPrO)2P(S)S]2) led to the isolation of dark-red crystalline solids of Cp*RuIVCl2(eta2-dithiolate) complexes [dithiolate = S2CNR2, DTCR (2a, R = Me; 2b, R = Et), S2COiPr (3), and S2P(iPrO)2 (4)]. Dichlorido substitution in 2 and 3 with DTCEt and S2COiPr anions yielded RuIV derivatives containing bis(DTC) and mixed DTC-dithiocarbonate ligands. These are the first organoruthenium complexes of such ligands. The reaction of monophosphines with 2a resulted in monochlorido substitution, whereas the analogous reaction with 3 resulted in displacement of both chlorido ligands and reduction of the metal center to RuII. Reduction at Ru was also observed in the reaction of 2a with [CpCr(CO)3]2. Of these complexes, only 2 and 3 are air-stable in the solid state for an extended period. All of the complexes have been spectrally characterized, and selected compounds are also crystallographically characterized.

The title heterocyclic radicals coordinate to either 17e CpCr(CO)3 or 15e CpCr(CO)2 moieties as one-electron or as three-electron donors, respectively; in the former the bonding is via the perpendicular p orbital of the sulfur atom, while in the latter bonding is via p(pi) orbitals on both sulfur and nitrogen.

The interactions of the benzothiazolate complex, CpCr(CO)(2)(SCSN(C(6)H(4))) (2), and the tetrazole thiolate complex, CpCr(CO)(3)(eta(1)-SCN(4)Ph) (3), with controlled amounts of Me(3)OBF(4) and (MeO)(2)SO(2), respectively, produced the corresponding mu(3)-oxo trinuclear thionate-bridged complexes, [Cp(3)Cr(3)(mu(2)-OH)(mu(3)-O)(mu(2)-eta(2)-SCSN(C(6)H(4)))(2)](5)BF(4) (45%) and [Cp(3)Cr(3)(mu(2)-OH)(mu(3)-O)(mu(2)-eta(2)-SCN(4)Ph)(2)](9)(MeOSO(3)) (53%), together with their respective free dimethylated thiolate ligands, [MeSCSNMe(C(6)H(4))](4)BF(4) and (Me(2)SCN(4)Ph)(8)MeOSO(3). The reaction of 3 with Me(3)OBF(4) resulted in the isolation of a binuclear complex, [Cp(2)Cr(2)(mu-OH)(mu-eta(2)-SCN(4)Ph)(2)](7)BF(4) (43%), and (8)BF(4) (27%). The reaction of the thiopyridine complex, CpCr(CO)(2)(SPy) (4), with I(2) also produced a similar mu(3)-oxo complex 10 (31%), together with CpCrI(2)(THF) (11) and the disulfide (SPy)(2). Similar reactions with 2 and 3 and I(2) yielded species 5 and 7, together with 11 and disulfides derived from their respective ligands. Cyclic voltammograms recorded in solutions of 5 and 9 indicated that the compounds could be reduced and oxidized at very similar potentials. An EPR spectrum characteristic of a compound with axial symmetry was obtained for 9 at 7 K. Single-crystal X-ray diffraction analyses confirmed that species 7 is dinuclear, whereas 5 and 9 are structural trinuclear analogues, each containing a mu(3)-oxo central core.