A Disulfide Intercalator Toolbox for the Site‐Directed Modification of Polypeptides

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.     

Synthesis of Disulfide Surrogate Peptides Incorporating Large‐Span Surrogate Bridges Through a Native‐Chemical‐Ligation‐Assisted Diaminodiacid Strategy

The use of synthetic bridges as surrogates for disulfide bonds has emerged as a practical strategy to obviate the poor stability of some disulfide‐containing peptides. However, peptides incorporating large‐span synthetic bridges are still beyond the reach of existing methods. Herein, we report a native chemical ligation (NCL)‐assisted diaminodiacid (DADA) strategy that enables the robust generation of disulfide surrogate peptides incorporating surrogate bridges up to 50 amino acids in length. This strategy provides access to some highly desirable but otherwise impossible‐to‐obtain disulfide surrogates of bioactive peptide. The bioactivities and structures of the synthetic disulfide surrogates were verified by voltage clamp assays, NMR, and X‐ray crystallography; and stability studies established that the disulfide replacements effectively overcame the problems of disulfide reduction and scrambling that often plague these pharmacologically important peptides.     

Comparative Study of In Situ Loaded Antibody and PEG‐Fab NIPAAM Gels

Hydrogels can potentially prolong the release of a therapeutic protein, especially to treat blinding conditions. One challenge is to ensure that the protein and hydrogel are intimately mixed by better protein entanglement within the hydrogel. N‐isopropylacrylamide (NIPAAM) gels are optimized with poly(ethylene glycol) diacrylate (PEDGA) crosslinker in the presence of either bevacizumab or PEG conjugated ranibizumab (PEG₁₀‐Fab_rani). The release profiles of the hydrogels are evaluated using an outflow model of the eye, which is previously validated for human clearance of proteins. Release kinetics of in situ loaded bevacizumab‐NIPAAM gels displays a prolonged bimodal release profile in phosphate buffered saline compared to bevacizumab loaded into a preformed NIPAAM gel. Bevacizumab release in simulated vitreous from in situ loaded gels is similar to bevacizumab control indicating that diffusion through the vitreous rather than from the gel is rate limiting. Ranibizumab is site‐specifically PEGylated by disulfide rebridging conjugation. Prolonged and continuous release is observed with the in situ loaded PEG₁₀‐Fab_rani‐NIPAAM gels compared to PEG₁₀‐Fab_rani injection (control). Compared to an unmodified protein, there is better mixing due to PEG entanglement and compatibility of PEG₁₀‐Fab_rani within the NIPAAM‐PEDGA hydrogel. These encouraging results suggest that the extended release of PEGylated proteins in the vitreous can be achieved using injectable hydrogels.     

Ynamide‐Mediated Thiopeptide Synthesis

Exploration of the full potential of thioamide substitution as a tool in the chemical biology of peptides and proteins has been hampered by insufficient synthetic strategies for the site‐specific introduction of a thioamide bond into a peptide backbone. A novel ynamide‐mediated two‐step strategy for thiopeptide bond formation with readily available monothiocarboxylic acids as thioacyl donors is described. The α‐thioacyloxyenamide intermediates formed from the ynamides and monothiocarboxylic acids can be purified, characterized, and stored. The balance between their activity and stability enables them to act as effective thioacylating reagents to afford thiopeptide bonds under mild reaction conditions. Amino acid functional groups such as OH, CONH₂, and indole NH groups need not be protected during thiopeptide synthesis. The modular nature of this strategy enables the site‐specific incorporation of a thioamide bond into peptide backbones in both solution and the solid phase.     

Release of Enzymatically Active Deubiquitinating Enzymes upon Reversible Capture by Disulfide Ubiquitin Reagents

Deubiquitinating enzymes (DUBs) catalyze the cleavage of ubiquitin from target proteins. Ubiquitin is post‐translationally attached to proteins and serves as an important regulatory signal for key cellular processes. In this study, novel activity‐based probes to study DUBs were synthesized that comprise a ubiquitin moiety and a novel disulfide warhead at the C‐terminus. These reagents can bind DUBs covalently by forming a disulfide bridge between the active‐site cysteine residue and the ubiquitin‐based probe. As disulfide bridges can be broken by the addition of a reducing agent, these novel ubiquitin reagents can be used to capture and subsequently release catalytically active DUBs, whereas existing capturing agents bind irreversibly. These novel reagents allow for the study of these enzymes in their active state under various conditions.     

Plant Antimicrobial Peptides Snakin‐1 and Snakin‐2: Chemical Synthesis and Insights into the Disulfide Connectivity

Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine‐rich snakin‐1 and ‐2 antimicrobial peptides by using a combination of solid‐phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40–50 % yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin‐2 compared to natural snakin‐2, we demonstrated that synthetic snakin‐2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin‐2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.     

Direct Synthesis of Polysubstituted Aldehydes via Visible‐Light Catalysis

Aldehydes are among the most versatile functional groups for synthetic chemistry. However, access to polysubstituted alkyl aldehydes is very limited and requires lengthy synthetic routes that involve multiple‐step functional‐group conversion. This paper reports a one‐step synthesis of polysubstituted aldehydes from readily available olefin substrates using visible‐light photoredox catalysis. Despite a number of competing reaction pathways, commercial styrenes react with vinyl ethers selectively in the presence of an acridinium salt photooxidant and a disulfide hydrogen‐atom‐transfer catalyst under blue LED irradiation. Alkyl aldehydes with different substitution patterns are prepared in good yields. This strategy can be applied to structurally sophisticated substrates.     

Surface Modification of Functional Nanoparticles for Controlled Drug Delivery

Abstract

Surface‐modified nanoparticles have received much attention as drug carriers. Natural and synthetic polymers are used as the materials to prepare nanoparticles and the properties of these nanoparticles originate with these polymeric materials. In particular, these nanoparticles are modified for specific objectives. The surface characteristics of (shell) nanoparticles are more important than those of the core, because the shell layer directly contacts body fluids and organs. Generally, the nanoparticles are coated with hydrophilic polymer to give long circulation and/or are conjugated with functional ligands or proteins for site‐specific delivery. In this review, the preparative methods and the applications of surface modification of polymeric functionalized nanoparticles for long‐circulation, site‐specific delivery, and oral delivery are discussed.     

Application of Fragment‐Based Screening to the Design of Inhibitors of Escherichia coli DsbA

The thiol‐disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram‐negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell‐based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.     

Application and Structural Analysis of Triazole‐Bridged Disulfide Mimetics in Cyclic Peptides

Ruthenium‐catalysed azide–alkyne cycloaddition (RuAAC) provides access to 1,5‐disubstituted 1,2,3‐triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross‐linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor‐1. NMR and X‐ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole‐bridged peptides also displayed superior half‐lives in liver S9 stability assays compared to disulfide‐bridged peptides. This work establishes a foundation for the application of 1,5‐disubstituted 1,2,3‐triazoles as disulfide mimetics.     

Chemical Synthesis of Human Insulin‐Like Peptide‐6

Human insulin‐like peptide‐6 (INSL‐6) belongs to the insulin superfamily and shares the distinctive disulfide bond configuration of human insulin. In this report we present the first chemical synthesis of INSL‐6 utilizing fluorenylmethyloxycarbonyl‐based (Fmoc) solid‐phase peptide chemistry and regioselective disulfide bond construction protocols. Due to the presence of an oxidation‐sensitive tryptophan residue, two new orthogonal synthetic methodologies were developed. The first method involved the identification of an additive to suppress the oxidation of tryptophan during iodine‐mediated S‐acetamidomethyl (Acm) deprotection and the second utilized iodine‐free, sulfoxide‐directed disulfide bond formation. The methodologies presented here offer an efficient synthetic route to INSL‐6 and will further improve synthetic access to other multiple‐disulfide‐containing peptides with oxidation‐sensitive residues.     

Copper‐Mediated Selenazolidine Deprotection Enables One‐Pot Chemical Synthesis of Challenging Proteins

While chemical protein synthesis has granted access to challenging proteins, the synthesis of longer proteins is often limited by low abundance or non‐strategic placement of cysteine residues, which are essential for native chemical ligations, as well as multiple purification and isolation steps. We describe the one‐pot total synthesis of human thiosulfate:glutathione sulfurtransferase (TSTD1). WT‐TSTD1 was synthesized in a C‐to‐N synthetic approach involving multiple NCL reactions, Cu^II‐mediated deprotection of selenazolidine (Sez), and chemoselective deselenization. The seleno‐analog Se‐TSTD1, in which the active site Cys is replaced with selenocysteine, was also synthesized with a kinetically controlled ligation with an N‐to‐C synthetic approach. The catalytic activity of the two proteins indicated that Se‐TSTD1 possessed only four‐fold lower activity than WT‐TSTD1, thus suggesting that selenoproteins can have physiologically comparable sulfutransferase activity to their cysteine counterparts.     

Mitochondria‐Targeting,Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles

Mitochondria are key organelles in mammalian cells whose dysfunction is linked to various diseases. Drugs targeting mitochondrial proteins provide a highly promising strategy for potential therapeutics. Methods for the delivery of small‐molecule drugs to the mitochondria are available, but these are not suitable for macromolecules, such as proteins. Herein, we report the delivery of native proteins and antibodies to the mitochondria using biodegradable silica nanoparticles (BS–NPs). The modification of the nanoparticle surface with triphenylphosphonium (TPP) and cell‐penetrating poly(disulfide)s (CPD) facilitated their rapid intracellular uptake with minimal endolysosomal trapping, providing sufficient time for effective mitochondrial localization followed by glutathione‐triggered biodegradation and of native, functional proteins into the mitochondria.     

A chemoenzymatic approach enables the site‐specific conjugation of recombinant proteins

Many biotechniques including protein microarray, drug screening, biosensors rely on the immobilization of recombinant proteins on the solid supports. It is well known that random orientation of the immobilized proteins could impair their biologic functions. Thus, it is very important to develop new site‐specific immobilization approach. In this study, we presented a chemoenzymatic approach for site‐specific conjugation of recombinant proteins onto solid support. In this strategy, the affinity tag on recombinant protein was enzymatically cleaved to expose the N‐terminal serine, which was oxidized to carry an aldehyde group and was then covalently coupled to hydrazide resin through hydrazone ligation. As this approach takes advantage of the most frequently used TEV protease, it requires no further sequence design on recombinant protein. This method was validated by site specific coupling of a synthetic peptide and a recombinant protein onto solid supports. It was found that the site specific immobilized SH2 domain is functional and could be used to enrich tyrosine phosphorylated peptides.     

Structural diversity in poly(disulfide)s

This article reports a synthetic methodology for single step preparation of telechelic poly(disulfide)s (PDS) by step‐growth polymerization between a di‐thiol and a commercially available monomer 2,2′‐dithiodipyridine in presence of a functional group appended pyridyl disulfide moiety as the “mono‐functional impurity” (MFI). Redox‐destructible well‐defined segmented PDSs with functional chain terminal, predicted and tunable degree of polymerization and narrow polydispersity index (<2.0) could be synthesized under a mild reaction condition. Using an appropriate MFI, PDS could be synthesized with trithiocarbonate chain terminals in a single step, which could be further used as macro chain‐transfer agent (CTA) for chain growth polymerization under RAFT mechanism producing ABA type tri‐block copolymer wherein the B block consists of the degradable PDS chain. By copolymerization between a hydrophobic di‐thiol monomer and a hydroxyl group appended di‐thiol monomer, PDS could be prepared with pendant hydroxyl functional group which was utilized to initiate ring opening polymerization of cyclic lactide monomers producing well‐defined degradable graft‐copolymer. The pendant hydroxyl groups were further utilized to anchor a polar carboxylic group to the degradable PDS backbone which under basic condition showed aqueous self‐assembly generating micelle‐like structure with hydrophobic guest encapsulation ability and glutathione responsive sustained release. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 194–202     

Mitochondria‐Targeting,Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles

Mitochondria are key organelles in mammalian cells whose dysfunction is linked to various diseases. Drugs targeting mitochondrial proteins provide a highly promising strategy for potential therapeutics. Methods for the delivery of small‐molecule drugs to the mitochondria are available, but these are not suitable for macromolecules, such as proteins. Herein, we report the delivery of native proteins and antibodies to the mitochondria using biodegradable silica nanoparticles (BS–NPs). The modification of the nanoparticle surface with triphenylphosphonium (TPP) and cell‐penetrating poly(disulfide)s (CPD) facilitated their rapid intracellular uptake with minimal endolysosomal trapping, providing sufficient time for effective mitochondrial localization followed by glutathione‐triggered biodegradation and of native, functional proteins into the mitochondria.     

Rethinking Cysteine Protective Groups: S‐Alkylsulfonyl‐l‐Cysteines for Chemoselective Disulfide Formation

The ability to reversibly cross‐link proteins and peptides grants the amino acid cysteine its unique role in nature as well as in peptide chemistry. We report a novel class of S‐alkylsulfonyl‐l ‐cysteines and N‐carboxy anhydrides (NCA) thereof for peptide synthesis. The S‐alkylsulfonyl group is stable against amines and thus enables its use under Fmoc chemistry conditions and the controlled polymerization of the corresponding NCAs yielding well‐defined homo‐ as well as block co‐polymers. Yet, thiols react immediately with the S‐alkylsulfonyl group forming asymmetric disulfides. Therefore, we introduce the first reactive cysteine derivative for efficient and chemoselective disulfide formation in synthetic polypeptides, thus bypassing additional protective group cleavage steps.     

Total Synthesis of Human Hepcidin through Regioselective Disulfide‐Bond Formation by using the Safety‐Catch Cysteine Protecting Group 4,4′‐Dimethylsulfinylbenzhydryl

A safety‐catch cysteine protecting group, S‐4,4′‐dimethylsulfinylbenzhydryl (Msbh), was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine‐rich peptides. The directed regioselective synthesis of human hepcidin, which contains four disulfide bonds, was undertaken and led to a high‐resolution NMR structure under more physiologically relevant conditions than previously. Conversely, hepcidin synthesized with the formerly assigned vicinal disulfide‐bond connectivity displayed significant conformational heterogeneity under similar conditions. The two synthetic forms of human hepcidin induced ferroportin internalization with apparent EC₅₀ values of 2.0 (native fold, 1 ) and 4.4 nM (non‐native fold, 2 ), with 2 undergoing isomerization to 1 in the presence of ferroportin expressing cells.     

p‐Azidophenylarsenoxide: An Arsenical “Bait” for the In Situ Capture and Identification of Cellular Arsenic‐Binding Proteins

Identification of arsenic‐binding proteins is important for understanding arsenic health effects and for developing arsenic‐based therapeutics. We report here a strategy for the capture and identification of arsenic‐binding proteins in living cells. We designed an azide‐labeled arsenical, p‐azidophenylarsenoxide (PAzPAO), to serve bio‐orthogonal functions: the trivalent arsenical group binds to cellular proteins in situ, and the azide group facilitates click chemistry with dibenzylcyclooctyne. The selective and efficient capture of arsenic‐binding proteins enables subsequent enrichment and identification by shotgun proteomics. Applications of the technique are demonstrated using the A549 human lung carcinoma cells and two in vitro model systems. The technique enables the capture and identification of 48 arsenic‐binding proteins in A549 cells incubated with PAzPAO. Among the identified proteins are a series of antioxidant proteins (e.g., thioredoxin, peroxiredoxin, peroxide reductase, glutathione reductase, and protein disulfide isomerase) and glyceraldehyde‐3‐phosphate dehydrogenase. Identification of these functional proteins, along with studies of arsenic binding and enzymatic inhibition, points to these proteins as potential molecular targets that play important roles in arsenic‐induced health effects and in cancer treatment.     

Electro‐Oxidative S−H/S−H Cross‐Coupling with Hydrogen Evolution: Facile Access to Unsymmetrical Disulfides

Sulfur is an essential element because it exists widely in proteins. The disulfide bond is an important moiety in many different types of significant organic molecules. A new approach for oxidant‐ and catalyst‐free S?H/S?H cross‐coupling, with hydrogen evolution, to construct unsymmetrical disulfides was developed. Under the conditions of an undivided cell at room temperature, a series of unsymmetrical disulfides were prepared with up to 87 % yield from the direct coupling of an aryl mercaptan and alkyl mercaptan. Gram‐scale synthesis also highlights the synthetic utility of this electrochemical strategy.