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Hydroxybenzylammonium compounds can undergo a reversible 1,4- or 1,6-elimination to afford quinone methide intermediates after release of the amine. These molecules are useful for the reversible conjugation of payloads to amines. We hypothesized that aromaticity could be used to alter the rate of reversibility as a distinct thermodynamic driving force. We describe the use of density functional theory (DFT) calculations to determine the effect of aromaticity on the rate of release of the amine from hydroxybenzylammonium compounds. Namely, the aromatic scaffold affects the dearomatization reaction to reduce the kinetic barrier and prevent the reversibility of the amine elimination. We consequently synthesized a small library of polycyclic hydroxybenzylammoniums, which resulted in a range of release half-lives from 18 minutes to 350 hours. The novel mechanistic insight provided herein significantly expands the range of release rates amenable to hydroxybenzylammonium-containing compounds. This work provides another way to affect the rate of payload release in hydroxybenzylammoniums.
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Development of bioconjugation strategies to efficiently modify biomolecules is of key importance for fundamental and translational scientific studies. Cysteine S-arylation is an approach which is becoming more popular due to generally rapid kinetics and high chemoselectivity, as well as the strong covalently bonded S-aryl linkage created in these processes. Organometallic approaches to cysteine S-arylation have been explored that feature many advantages compared to their more traditional organic counterparts. In this Viewpoint, progress in the use of Au(III) and Pd(II) oxidative addition (OA) complexes for stoichiometric cysteine S-arylation is presented and discussed. A focus is placed on understanding the rapid kinetics of these reactions under mild conditions, as well as the ability to generate biomolecular heterostructures. Potential avenues for further exploration are addressed and usefulness of these methods to the practitioner are emphasized in the discussion.
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Cisteína , Oxidación-Reducción , Paladio , Cisteína/química , Oro/química , Cinética , Compuestos Organometálicos/química , Paladio/químicaRESUMEN
Reversible conjugation of polymers to proteins is important for a variety of applications, for example to control protein activity. Light is often employed as an external trigger to allow for spatio and temporal control over release of a payload. In this report, we demonstrate preparation of photocleavable poly(polyethylene glycol) acrylate)-lysozyme (pPEGA-Lys) conjugates via ortho-nitrobenzyl linkages. The conjugates were made by both grafting-to and grafting-from in order to compare and contrast the two synthetic approaches. First, a lysine-reactive ortho-nitrobenzyl atom transfer radical polymerization (ATRP) initiator was synthesized. For the grafting-to strategy, the initiator was employed in the ATRP of PEGA, and the subsequent polymer was conjugated to the lysine residues of lysozyme. For the grafting-from strategy, lysozyme was modified first with the photocleavable initiator, and the purified macroinitiator was then subjected to polymerization conditions to synthesize the protein-polymer conjugate. The polymer was cleaved from the protein via UV light, and activity before and after polymer removal was evaluated, showing 83% recovery. This work provides evidence that reversing conjugation is successful for activity modulation for ortho-nitrobenzyl linked protein-polymer conjugates.
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Bioconjugation of polymers to proteins is a method to impart improved stability and pharmacokinetic properties to biologic systems. However, the precise effects of polymer architecture on the resulting bioconjugates are not well understood. Particularly, cyclic polymers are known to possess unique features such as a decreased hydrodynamic radius when compared to their linear counterparts of the same molecular weight, but have not yet been studied. Here, we report the first bioconjugation of a cyclic polymer, poly(ethylene glycol) (PEG), to a model protein, T4 lysozyme, containing a single engineered cysteine residue (V131C). We compare the stability and activity of this conjugate with those of a linear PEG-T4 lysozyme analogue of similar molecular weight. Furthermore, we used molecular dynamics (MD) simulations to determine the behavior of the polymer-protein conjugates in solution. We introduce cyclic polymer-protein conjugates as potential candidates for the improvement of biologic therapeutics.
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Simulación de Dinámica Molecular , Muramidasa , Polietilenglicoles , Polietilenglicoles/química , Muramidasa/química , Bacteriófago T4/enzimologíaRESUMEN
Glucagon is a peptide hormone that acts via receptor-mediated signaling predominantly in the liver to raise glucose levels by hepatic glycogen breakdown or conversion of noncarbohydrate, 3 carbon precursors to glucose by gluconeogenesis. Glucagon is administered to reverse severe hypoglycemia, a clinical complication associated with type 1 diabetes. However, due to low stability and solubility at neutral pH, there are limitations in the current formulations of glucagon. Trehalose methacrylate-based nanoparticles were utilized as the stabilizing and solubilizing moiety in the system reported herein. Glucagon was site-selectively modified to contain a cysteine at amino acid number 24 to covalently attach to the methacrylate-based polymer containing pyridyl disulfide side chains. PEG2000 dithiol was employed as the crosslinker to form uniform nanoparticles. Glucagon nanogels were monitored in Dulbecco's phosphate-buffered saline (DPBS) pH 7.4 at various temperatures to determine its long-term stability in solution. Glucagon nanogels were stable up to at least 5 months by size uniformity when stored at -20 °C and 4 °C, up to 5 days at 25 °C, and less than 12 hours at 37 °C. When glucagon stability was studied by either HPLC or thioflavin T assays, the glucagon was intact for at least 5 months at -20 °C and 4 °C within the nanoparticles at -20 °C and 4 °C and up to 2 days at 25 °C. Additionally, the glucagon nanogels were studied for toxicity and efficacy using various assays in vitro. The findings indicate that the nanogels were nontoxic to fibroblast cells and nonhemolytic to red blood cells. The glucagon in the nanogels was as active as glucagon alone. These results demonstrate the utility of trehalose nanogels towards a glucagon formulation with improved stability and solubility in aqueous solutions, particularly useful for storage at cold temperatures.
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Through mechanistic work and rational design, we have developed the fastest organometallic abiotic Cys bioconjugation. As a result, the developed organometallic Au(III) bioconjugation reagents enable selective labeling of Cys moieties down to picomolar concentrations and allow for the rapid construction of complex heterostructures from peptides, proteins, and oligonucleotides. This work showcases how organometallic chemistry can be interfaced with biomolecules and lead to a range of reactivities that are largely unmatched by classical organic chemistry tools.
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Cisteína , Oro , Cisteína/química , Oro/química , Péptidos/química , Compuestos Orgánicos de Oro/química , Compuestos Orgánicos de Oro/síntesis química , Estructura MolecularRESUMEN
Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.
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Escherichia coli , Estabilidad Proteica , Proteoma , Proteoma/metabolismo , Proteoma/química , Humanos , Escherichia coli/metabolismo , Temperatura , Betaína/química , Betaína/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Trehalosa/química , Trehalosa/metabolismo , Proteómica/métodos , Prolina/química , Prolina/metabolismo , Glucosa/química , Glucosa/metabolismo , Glicerol/química , Glicerol/metabolismo , MetilaminasRESUMEN
Herein, we describe the synthesis of bench-stable organometallic Au(III) terminated polymer reagents. These reagents mediate the chemoselective S-arylation of thiol-containing small molecules and polymers to yield functionalized mono-telechelic polymers and diblock copolymers, respectively. These transformations proceed rapidly within minutes and produce conjugates in quantitative conversion, making this strategy a robust addition to the polymer functionalization toolbox.
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Recombinant human growth hormone (rhGH) and GH receptor antagonists (GHAs) are used clinically to treat a range of disorders associated with GH deficiency or hypersecretion, respectively. However, these biotherapeutics can be difficult and expensive to manufacture with multiple challenges from recombinant protein generation through to the development of long-acting formulations required to improve the circulating half-life of the drug. In this review, we summarize methodologies and approaches used for making and purifying recombinant GH and GHA proteins, and strategies to improve pharmacokinetic and pharmacodynamic properties, including PEGylation and fusion proteins. Therapeutics that are in clinical use or are currently under development are also discussed.
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Hormona de Crecimiento Humana , Receptores de Somatotropina , Humanos , Hormona de Crecimiento Humana/genética , Hormona de Crecimiento Humana/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Receptores de Somatotropina/agonistas , Receptores de Somatotropina/antagonistas & inhibidoresRESUMEN
One of the primary global health concerns is the increase in antimicrobial resistance. Polymer chemistry enables the preparation of macromolecules with hydrophobic and cationic side chains that kill bacteria by destabilizing their membranes. In the current study, macromolecules are prepared by radical copolymerization of caffeine methacrylate as the hydrophobic monomer and cationic- or zwitterionic-methacrylate monomers. The synthesized copolymers bearing tert-butyl-protected carboxybetaine as cationic side chains showed antibacterial activity toward Gram-positive bacteria (S. aureus) and Gram-negative bacteria (E. coli). By tuning the hydrophobic content, we prepared copolymers with optimal antibacterial activity against S. aureus, including methicillin-resistant clinical isolates. Moreover, the caffeine-cationic copolymers presented good biocompatibility in a mouse embryonic fibroblast cell line, NIH 3T3, and hemocompatibility with erythrocytes even at high hydrophobic monomer content (30-50%). Therefore, incorporating caffeine and introducing tert-butyl-protected carboxybetaine as a quaternary cation in polymers could be a novel strategy to combat bacteria.
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Enzyme nanogels (ENGs) offer a convenient method to protect therapeutic proteins from in vivo stressors. Current methodologies to prepare ENGs rely on either covalent modification of surface residues or the noncovalent assembly of monomers at the protein surface. In this study, we report a new method for the preparation of noncovalent ENGs that utilizes a heterobifunctional, photocleavable monomer as a hybrid approach. Initial covalent modification with this monomer established a polymerizable handle at the protein surface, followed by radical polymerization with poly(ethylene glycol) methacrylate monomer and ethylene glycol dimethacrylate crosslinker in solution. Final photoirradiation cleaved the linkage between the polymer and protein to afford the noncovalent ENGs. The enzyme phenylalanine ammonia lyase (PAL) was utilized as a model protein yielding well-defined nanogels 80 nm in size by dynamic light scattering (DLS) and 76 nm by atomic force microscopy. The stability of PAL after exposure to trypsin or low pH was assessed and was found to be more stable in the noncovalent nanogel compared to PAL alone. This approach may be useful for the stabilization of active enzymes.
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Hydrophilic amendments can enhance soil moisture content, which, in turn, can improve crop health under drought conditions. Understanding how different hydrogels interact with specific crops is necessary for optimal application. The soil conditioning abilities of a trehalose hydrogel and polyacrylate-based hydrogel were evaluated for tomatoes (Solanum lycopersicum) subjected to drought. Tomato plants were transplanted into individual pots with soil that contained trehalose hydrogel (0.4 wt%), polyacrylate-based hydrogel (0.4 wt%), or no hydrogel and subjected to a well-watered treatment or to pronounced soil drought, with or without rewatering. The health of tomato plants was monitored by measuring leaf total chlorophyll (a + b) concentration, leaf water potential (Ψleaf), stomatal conductance (g s) and relative growth rate (RGR). The polyacrylate-based hydrogel, but not the trehalose hydrogel, improved tomato plant function under drought conditions, as indicated by improved g s and RGR relative to the well-watered control. However, when subjected to a second drought, neither hydrogel was effective, and neither prolonged survival. The more hydrophilic polyacrylate-based hydrogel demonstrated promise in improving the growth of tomato plants under drought when included as a soil amendment at 0.4 wt%. This research is important for understanding the effects of these hydrogels as soil conditioners in drought prone systems.
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Insulin, the oldest U.S. Food and Drug Administration (FDA)-approved recombinant protein and a World Health Organization (WHO) essential medicine for treating diabetes globally, faces challenges due to its storage instability. One approach to stabilize insulin is the addition of poly(trehalose methacrylate) (pTrMA) as an excipient. The polymer increases the stability of the peptide to heat and mechanical agitation and has a low viscosity suitable for injection and pumps. However, the safety and stabilizing mechanism of pTrMA is not yet known and is required to understand the potential suitability of pTrMA as an insulin excipient. Herein is reported the immune response, biodistribution, and insulin plasma lifetime in mice, as well as investigation into insulin stabilization. pTrMA alone or formulated with ovalbumin did not elicit an antibody response over 3 weeks in mice, and there was no observable cytokine production in response to pTrMA. Micropositron emission tomography/microcomputer tomography of 64Cu-labeled pTrMA showed excretion of 78-79% ID/cc within 24 h and minimal liver accumulation at 6-8% ID/cc when studied out to 120 h. Further, the plasma lifetime of insulin in mice was not altered by added pTrMA. Formulating insulin with 2 mol equiv of pTrMA improved the stability of insulin to standard storage conditions: 46 weeks at 4 °C yielded 87.0% intact insulin with pTrMA present as compared to 7.8% intact insulin without the polymer. The mechanism by which pTrMA-stabilized insulin was revealed to be a combination of inhibiting deamidation of amino acid residues and preventing fibrillation, followed by aggregation of inactive and immunogenic amyloids all without complexing insulin into its hexameric state, which could delay the onset of insulin activity. Based on the data reported here, we suggest that pTrMA stabilizes insulin as an excipient without adverse effects in vivo and is promising to investigate further for the safe formulation of insulin.
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Excipientes , Trehalosa , Animales , Estabilidad de Medicamentos , Excipientes/química , Insulina/química , Metacrilatos , Ratones , Polímeros/química , Distribución Tisular , Tomografía Computarizada por Rayos X , Trehalosa/químicaRESUMEN
Bioconjugation techniques for biomolecule-polymer conjugation are numerous; however, slow kinetics and steric challenges generally necessitate excess reagents or long reaction times. Organometallic transformations are known to circumvent these issues; yet, harsh reaction conditions, incompatibility in aqueous media, and substrate promiscuity often limit their use in a biological context. The work reported herein demonstrates a facile and benign organometallic Au(III) S-arylation approach that enables the synthesis of poly(ethylene glycol) monomethyl ether (mPEG)-protein conjugates with high efficiency. Isolable and bench-stable 2, 5, and 10 kDa mPEG-Au(III) reagents were synthesized via oxidative addition into terminal aryl iodide substituents installed on mPEG substrates with a (Me-DalPhos)Au(I)Cl precursor. Reaction of the isolable mPEG-Au(III) oxidative addition complexes with a cysteine thiol on a biomolecule resulted in facile and selective cysteine arylation chemistry, forging covalent S-aryl linkages and affording the mPEG-biomolecule conjugates. Notably, low polymer reagent loadings were used to achieve near quantitative conversion at room temperature in 1 min due to the rapid kinetics and high chemoselectivity of this Au-based bioconjugation approach. Therefore, this work represents an important addition to the protein-polymer conjugation chemical toolbox.
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Cisteína , Polietilenglicoles , Cisteína/química , Indicadores y Reactivos , Oxidación-Reducción , Polietilenglicoles/química , Proteínas/químicaRESUMEN
Trehalose is a naturally occurring, nonreducing disaccharide that is widely used in the biopharmaceutical, food, and cosmetic industries due to its stabilizing and cryoprotective properties. Over the years, scientists have developed methodologies to synthesize linear polymers with trehalose units either in the polymer backbone or as pendant groups. These macromolecules provide unique properties and characteristics, which often outperform trehalose itself. Additionally, numerous reports have focused on the synthesis and formulation of materials based on trehalose, such as nanoparticles, hydrogels, and thermoset networks. Among many applications, these polymers and materials have been used as protein stabilizers, as gene delivery systems, and to prevent amyloid aggregate formation. In this Perspective, recent developments in the synthesis and application of trehalose-based linear polymers, hydrogels, and nanomaterials are discussed, with a focus on utilization in the biomedical field.
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Poly(styrenyl acetal trehalose) (pSAT), composed of trehalose side chains linked to a polystyrene backbone via acetals, stabilizes a variety of proteins and enzymes against fluctuations in temperature. A promising application of pSAT is conjugation of the polymer to therapeutic proteins to reduce renal clearance. To explore this possibility, the safety of the polymer was first studied. Investigation of acute toxicity of pSAT in mice showed that there were no adverse effects of the polymer at a high (10 mg/kg) concentration. The immune response (antipolymer antibody and cytokine production) in mice was also studied. No significant antipolymer IgG was detected for pSAT, and only a transient and low level of IgM was elicited. pSAT was also safe in terms of cytokine response. The polymer was then conjugated to a granulocyte colony stimulating factor (GCSF), a therapeutic protein that is approved by the Federal Drug Administration, in order to study the biodistribution of a pSAT conjugate. A site-selective, two-step synthesis approach was developed for efficient conjugate preparation for the biodistribution study resulting in 90% conjugation efficiency. The organ distribution of GCSF-pSAT was measured by positron emission tomography and compared to controls GCSF and GCSF-poly(ethylene glycol), which confirmed that the trehalose polymer conjugate improved the in vivo half-life of the protein by reducing renal clearance. These findings suggest that trehalose styrenyl polymers are promising for use in therapeutic protein-polymer conjugates for reduced renal clearance of the biomolecule.
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Acetales , Trehalosa , Animales , Factor Estimulante de Colonias de Granulocitos , Ratones , Polímeros/química , Proteínas/química , Distribución Tisular , Trehalosa/químicaRESUMEN
To improve the efficacy of antibody drug conjugates (ADCs), there has been significant focus on increasing the drug-to-antibody ratio (DAR) in order to deliver more payload. However, due to the hydrophobicity of many cytotoxics, highly-loaded conjugates often have lower physicochemical stability and poorer pharmacokinetic outcomes, requiring the development of new hydrophilic linkers. Herein, we report a platform for the preparation of functional, sequence-defined polymers for conjugation to antibodies. We demonstrate the successful synthesis of novel diazido macrocyclic sulfate monomers of varied size ranging from 4 to 7 ethylene glycol repeat units. These monomers were then successively ring-opened to produce sequence-defined polymers that contained either 4 or 6 azides for post-synthesis functionalization. Given the hydrophilic ethylene glycol backbone and chemically defined nature of the polymers, we envisioned this as a useful strategy in the preparation of highly-loaded ADCs. To demonstrate this, we prepared a model polymer-fluorophore scaffold composed of 4 coumarin molecules and conjugated it to Herceptin. We fully characterized the conjugate via mass spectrometry, which yielded a polymer-to-antibody ratio of 6.6, translating to a total of 26 fluorophores conjugated to the antibody at the inter-chain disulfides. We believe this technology to not only be a meaningful contribution to the field of sequence-defined polymers and conjugates, but also as a general and tunable platform for drug delivery.
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Traceless self-immolative linkers are widely used for the reversible modification of proteins and peptides. This article describes a new class of traceless linkers based on ortho- or para-hydroxybenzylamines. The introduction of electron-donating substituents on the aromatic core stabilizes the quinone methide intermediate, thus providing a platform for payload release that can be modulated. To determine the extent to which the electronics affect the rate of release, we prepared a small library of hydroxybenzylamine linkers with varied electronics in the aromatic core, resulting in half-lives ranging from 20 to 144 h. Optimization of the linker design was carried out with mechanistic insights from density functional theory (DFT) and the in silico design of an intramolecular trapping agent through the use of DFT and intramolecular distortion energy calculations. This resulted in the development of a faster self-immolative linker with a half-life of 4.6 h. To demonstrate their effectiveness as traceless linkers for bioconjugation, reversible protein-polyethylene glycol conjugates with a model protein lysozyme were prepared, which had reduced protein activity but recovered ≥94% activity upon traceless release of the polymer. This new class of linkers with tunable release rates expands the traceless linkers toolbox for a variety of bioconjugation applications.
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Polietilenglicoles , Polímeros , Polímeros/química , ProteínasRESUMEN
Antibodies and antigen binding fragments (FABs) are widely used as therapeutics and conjugated polymers can enhance the properties of these important biomolecules. However, limitations to the selectivity and stability of current conjugation methodologies can inhibit the exploration of new antibody-polymer conjugates. Herein, we describe a new strategy for the synthesis of these conjugates that forms a stable thioether bond and can be directly incorporated into an atom transfer radical polymerization (ATRP) initiator. Specifically, a bis-sulfone alkyl bromide initiator was synthesized and utilized in the activators generated by electron transfer (AGET) ATRP of ethylene glycol methacrylate and trehalose methacrylate to form the respective polymers. The trehalose polymer was then irreversibly inserted into the disulfide bonds of Herceptin and Herceptin FAB after mild reduction to form the conjugates with quantitative conversions as verified by Western Blot and mass spectrometry after cleavage of the polymer. The binding of the Herceptin and Herceptin Fab conjugates to the receptor was investigated by indirect ELISA (enzyme-linked immunosorbent assay) and the EC50's were 0.90 and 2.74 nM, respectively, compared to Herceptin (0.26 nM) and the Fab (0.56 nM). The conjugates were subjected to heating studies at a constant 75 °C, the temperature determined in a heat ramp to be the threshold of stability for the antibody and FAB; the trehalose polymer was found to considerably increase the thermal stability of both Herceptin and Herceptin Fab. This work provides a new way to prepare polymer-antibody/Fab conjugates utilizing bis-sulfone end groups installed by atom transfer radical polymerization of the functionalized initiators and a way to stabilize these important molecules by conjugation to trehalose polymers.
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Instability to storage and shipping conditions and injection administration remain major challenges in treating chronic conditions with biopharmaceuticals. Herein, formulations of poly(trehalose methacrylate) (pTrMA) were successfully optimized to stabilize insulin without appreciably increasing viscosity. Polymers were synthesized (2,400 - 29,200 Da), and added to insulin at different concentrations. pTrMA maintained >95% intact insulin against 250 rpm at 37 °C for 3 hours with at least 10 mol. eq. of 5.0 kDa, 7.5 mol. eq. of 9.4 kDa, 5 mol. eq. of 12.8 kDa, 1 mol. eq. of 19.8 kDa, and 0.5 mol. eq. of 29.2 kDa polymers, compared to 13.1% of insulin alone. The lowest pTrMA concentration formulations were more viscous than insulin alone, but the highest viscosity, U-600 with 10 mol. eq. of 5 kDa pTrMA, was only 1.43 cP at 25 °C. This data demonstrates that pTrMA is a promising low viscosity additive to stabilize the diabetes therapeutic insulin.