Investigating Ugi/Passerini Multicomponent Reactions for the Site-Selective Conjugation of Native Trastuzumab**

Site-selective modification of proteins has been the object of intense studies over the past decades, especially in the therapeutic field. Prominent results have been obtained with recombinant proteins, for which site-specific conjugation is made possible by the incorporation of particular amino acid residues or peptide sequences. In parallel, methods for the site-selective and site-specific conjugation of native and natural proteins are starting to thrive, allowing the controlled functionalization of various types of amino acid residues. Pursuing the efforts in this field, we planned to develop a new type of site-selective method, aiming at the simultaneous conjugation of two amino acid residues. We reasoned that this should give higher chances of developing a site-selective strategy compared to the great majority of existing methods that solely target a single residue. We opted for the Ugi four-centre three-component reaction to implement this idea, with the aim of conjugating the side-chain amine and carboxylate groups of two neighbouring lysine and aspartate/glutamate. Herein, we show that this strategy can give access to valuable antibody conjugates bearing several different payloads; furthermore, the approach limits the potential conjugation sites to only six on the model antibody trastuzumab.     

Advances in polymer and polymeric nanostructures for protein conjugation

Linear polymers have been considered the best molecular structures for the formation of efficient protein conjugates due to their biological advantages, synthetic convenience and ease of functionalization. In recent years, much attention has been dedicated to develop synthetic strategies that produce the most control over protein conjugation utilizing linear polymers as scaffolds. As a result, different conjugate models, such as semitelechelic, homotelechelic, heterotelechelic and branched or star polymer conjugates, have been obtained that take advantage of these well-controlled synthetic strategies. Development of protein conjugates using nanostructures and the formation of said nanostructures from protein–polymer bioconjugates are other areas in the protein bioconjugation field. Although several polymer–protein technologies have been developed from these discoveries, few review articles have focused on the design and function of these polymers and nanostructures. This review will highlight some recent advances in protein-linear polymer technologies that employ protein covalent conjugation and successful protein-nanostructure bioconjugates (covalent conjugation as well) that have shown great potential for biological applications.     

Efficient bioactive oligonucleotide-protein conjugation for cell-targeted cancer therapy

Oligonucleotide-protein conjugates have important applications in biomedicine. Simple and efficient methods are described for the preparation of these conjugates. Specifically, we describe a new method in which a bifunctional linker is attached to thiol-oligonucleotide to generate a reactive intermediate that is used to link to the protein. Having similar conjugation efficacy compared with the classical method in which the bifunctional linker is attached first to the protein, this new approach produces significantly more active conjugates with higher batch to batch reproducibility. In a second approach, direct conjugation is proposed using oligonucleotides carrying carboxyl groups. These methodologies have been applied to prepare nanoconjugates of an engineered nanoparticle protein carrying a T22 peptide with affinity for the CXCR4 chemokine receptor and oligomers of the antiproliferative nucleotide 2’-deoxy-5-fluorouridine in a very efficient way. The protocols have potential uses for the functionalization of proteins, amino-containing polymers or amino-lipids in order to produce complex therapeutic nucleic acid delivery systems.     

Orthogonal Cysteine Protection Enables Homogeneous Multi-Drug Antibody–Drug Conjugates

A strategy for the preparation of homogeneous antibody–drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site-specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug-linkers that have differing physiochemical properties and exert complementary anti-cancer activities. Dual-auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual-drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site-specific protein modification.     

Degradable polyesters through chain linking for packaging and biomedical applications

The major route to convert lactic acid to high-molecular-weight polymers is ring-opening polymerization of lactide. We have investigated alternative synthesis routes based on oligomerization and chain linking to produce high-molecular-weight thermoplastic degradable polymers cost-effectively. Chain linking also offers new possibilities to prepare degradable polyesters for biomedical applications by extending the range of polymer properties achievable. In this paper, we briefly review different chain linking techniques used in our laboratory. Typically, lactic acid prepolymers with molecular weights of around 3,000-15,000 g x mol(-1) have been prepared by direct polycondensation. Hydroxyl terminated oligomers have been chain linked by using diisocyanate coupling agents, preferably 1,4-butane diisocyanate, forming poly(ester-urethanes). Poly(ester-amides) have been prepared by using 2,2'-bis(2-oxazoline) as coupling agent for carboxylic acid telechelic oligomers. Chain linking by end functionalization has been used in the preparation of poly(ester-anhydrides). In addition, a variety of crosslinked degradable polymers and copolymers have been synthesized through different crosslinking routes, by using methacrylic, itaconic or maleic double bonds or triethoxysilane moieties. A biodegradation test and ecotoxicological evaluation of the degradation products were carried out in addition to hydrolysis tests. Lactic acid based chain linked polymers were biodegradable and the degradation products were harmless. In hydrolysis tests, enzymatic degradation was pronounced in the chain linked poly(epsilon-caprolactone).     

Effects on peptide binding affinity for TNFα by PEGylation and conjugation to hyaluronic acid

Conjugation of cytokine-neutralizing monoclonal antibodies (mAb) to hyaluronic acid (HA) having M_w of 1.6 MDa was previously shown to be an effective strategy for localized delivery to sites of inflammation. Despite the disparity in size of the mAb and HA, the mAb–HA conjugate was found bind tumor necrosis factor-α (TNFα) as strongly as the non-conjugated antibody, suggesting conjugation to this charged polysaccharide can provide an alternative to poly(ethylene glycol) (PEG) conjugation, which has been shown to reduce binding interactions for many proteins. To explore conjugation chemistries more systematically, we report a study on a model peptide inhibitor of tumor necrosis factor-α to investigate the effects of site-specific conjugation to HA and PEG. We compared the binding affinities of a variety of WP9QY peptide–polymer conjugates for TNFα in order to examine the effects of PEG molecular weight as well as the effects of PEG versus functionalized hyaluronic acid (HA) conjugation. The results indicate that the binding affinity of the PEG conjugates decreases in comparing PEG with mass 2 k, 10 k, and 30 k, which was attributed to PEG shrouding of the peptide, while conjugation to a 66 kDa HA chain preserved peptide binding affinity. We attribute this difference to the increased solubility of HA compared to PEG, potentially due to the carboxylic acid functional groups. In addition, the results demonstrate that conjugation to HA via a short PEG linker significantly enhances the association rate k_on, which may reflect an increased peptide accessibility. By balancing both the advantages associated with the PEG conjugates and with the HA conjugates, the HA–PEG2k–WP9QY conjugate was able to improve the binding affinity of the peptide for TNFα by a factor of two. Optimization of polymer chemistry could be used to improve delivery of protein therapeutics for localized and systemic administration.     

Biological–synthetic hybrid block copolymers: Combining the best from two worlds

Although biopolymers and synthetic polymers share many common features, each of these two classes of materials is also characterized by a distinct and very specific set of advantages and disadvantages. Combining biopolymer elements with synthetic polymers into a single macromolecular conjugate is an interesting strategy for synergetically merging the properties of the individual components and overcoming some of their limitations. This article focuses on a special class of biological–synthetic hybrids that are obtained by site‐selective conjugation of a protein or peptide and a synthetic polymer. The first part of the article gives an overview of the different liquid‐phase and solid‐phase techniques that have been developed for the synthesis of well‐defined, that is, site‐selectively conjugated, synthetic polymer–protein hybrids. In the second part, the properties and potential applications of these materials are discussed. The conjugation of biological and synthetic macromolecules allows the modulation of protein binding and recognition properties and is a powerful strategy for mediating the self‐assembly of synthetic polymers. Synthetic polymer–protein hybrids are already used as medicines and show significant promise for bioanalytical applications and bioseparations. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1–17, 2005     

Metal–Organic‐Framework‐Templated Polyelectrolyte Nanocapsules for the Encapsulation and Delivery of Small‐Molecule–Polymer Conjugates

Herein, we report a strategy for exploiting nanoscale metal–organic frameworks (nano‐MOFs) as templates for the layer‐by‐layer (LbL) assembly of polyelectrolytes. Because small‐molecule drugs or imaging agents cannot be efficiently encapsulated by polyelectrolyte nanocapsules, we investigated two promising and biocompatible polymers (comb‐shaped polyethylene glycol (PEG) and hyperbranched polyglycerol‐based PEG) for the conjugation of model drugs and imaging agents, which were then encapsulated inside the nano‐MOF‐templated nanocapsules. Furthermore, we also systemically explored the release kinetics of the encapsulated conjugates, and examined how the encapsulation and/or release processes could be controlled by varying the composition and architecture of the polymers. We envision that our nano‐MOFs‐templated nanocapsules, through combining with small‐molecule–polymer conjugates, will represent a new type of delivery system that could open up new opportunities for biomedical applications.     

Synthesis of Maleimide-End Functionalized Star Polymers and Multimeric Protein-Polymer Conjugates

Protein-polymer conjugates exhibit superior properties to unmodified proteins, generating a high demand for these materials in the fields of medicine, biotechnology, and nanotechnology. Multimeric conjugates are predicted to surpass the activity of monomeric conjugates. Herein, we report a straightforward method to synthesize multimeric polymer-conjugates. Four armed poly(N-isopropylacrylamide) (pNIPAAm) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization in the presence of a tetra-functionalized trithiocarbonate chain transfer agent (CTA). The polymer molecular weight, architecture and polydispersity index (PDI) were verified by gel permeation chromatography (GPC), dynamic light scattering gel permeation chromatography (DLS-GPC), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This approach afforded well-defined polymers (PDI's < 1.06) and the ability to target various molecular weights. Maleimide functional groups were introduced at the chain ends by heating the polymers in the presence of a furan-protected azo-initiator. This allowed for site-specific conjugation of V131C T4 lysozyme to the polymers to generate multimeric protein-polymer conjugates. MALDI-TOF mass spectrometry, electrospray ionization gas-phase electrophoretic-mobility macromolecule analysis (ESI-GEMMA), gel electrophoresis, and liquid chromatography tandem mass spectrometry (LC-MS/MS) of the trypsin digests demonstrated that multimeric protein-polymer conjugates had formed. This simple strategy provides ready access to star protein-polymer conjugates for application in the fields of drug discovery, drug delivery, and nanotechnology.     

Site directed maleimide bifunctional chelators for the M(CO)3+ core (M =(99m)Tc, Re)

A series of bifunctional chelates containing a tridentate donor set for complexation of the M(CO)3+ core and a maleimide group for site-specific coupling to peptides and proteins containing free thiol groups has been prepared and their Re(CO)3+ complexes and glutathione conjugates structurally characterized. The flexibility of design allows preparation of ligands suitable for both fluorescence imaging, radioimaging and radiotherapeutic studies of proteins and peptides as well as other biopolymers using site specific conjugation.     

Novel chiral conjugated macromolecules for potential electrical and optical applications

Optically active 1,1′‐binaphthyl molecules have been used to construct novel chiral dendrimers and linear polymers. Efficient light harvesting effects of the dendrimers have been observed. They have shown enantioselective fluorescence responses in the presence of chiral amino alcohol quenchers. They are potentially useful as fluorescent sensors for the recognition of chiral organic compounds. Linear binaphthyl polymers have shown strong light emitting properties. Their colors of emission can be systematically tuned by incorporating linkers of various conjugation length. A very efficient light emitting diode has been prepared from the binaphthyl‐based conjugated polymers. Nonlinear optical chromophores have been organized in the chiral binaphthyl polymer chains to construct noncentrosymmetric and multipolar materials. These novel propeller‐like polymers have shown significant second‐order nonlinear optical effects.     

Impact of open-shell loading on mass transport and doping in conjugated radical polymers

Radical-containing polymers are an evolving class of redox-active macromolecules that have received great interest; however, most reports regarding radical polymers have focused on materials with nonconjugated backbones because their application drivers did not require this conjugation. Conversely, there has been a recent rise in the development of radical polymers for next-generation applications where imparting conjugation to the backbone of the radical polymer could be of significant benefit. To this end, we designed and synthesized a series of 3,4-propylenedioxythiophene (ProDOT)-based polymers bearing nitroxide radical pendent groups via direct arylation polymerization. Specifically, we present four radical polymers with open-shell loadings ranging from 24% to 82% of the total number of repeat units per polymer chain. The impact of open-shell loading on the electrochemical behaviors of these polymers in different electrolytes was then established using cyclic voltammetry, spectroelectrochemical analyses, and electrochemical quartz-crystal microbalance with dissipation monitoring. We demonstrate that incorporating the open-shell moieties in the ProDOT-based polymers lowers the oxidation onset potential of the conjugated backbone and increases the solvent and ion uptake significantly. Thus, this effort provides a clear picture of the mass transfer and doping mechanism of the ProDOT-based radical polymers to aid in guiding their future design.     

Preparation of biodegradable and thermoresponsive enzyme–polymer conjugates with controllable bioactivity via RAFT polymerization

The preparation of biodegradable and thermoresponsive enzyme–polymer bioconjugates with controllable enzymatic activity via reversible addition−fragmentation chain transfer (RAFT) polymerization and amidation conjugation reaction is presented. A new 2-mercaptothiazoline ester functionalized RAFT agent with intra-disulfide linkage was synthesized and used as chain transfer agent (CTA) to generate a biocompatible homopolymer, poly(ethyleneglycol) acrylate (polyPEG-A) and a thermoresponsive copolymer of poly(ethyleneglycol) acrylate with di(ethyleneglycol)ethyl ether acrylate [poly(PEG-A-co-DEG-A)]. These biodegradable and thermoresponsive polymers were then conjugated to the surface of glucose oxidase (GOx) under mild condition to afford the biodegradable and thermoresponsive enzyme–polymer conjugates. Cleavage of the polymer chains from the GOx surface obviously recovered the enzymatic activity. The thermoresponsive test of GOx-poly(PEG-A-co-DEG-A) revealed that the bioconjugate exhibited regular enzymatic activity fluctuation upon the temperature change below or above the lower critical solution temperature (LCST). The as-prepared enzyme–polymer conjugates were also characterized using ¹H NMR, UV–vis spectroscopy, polyacrylamide gel electrophoresis (PAGE) and biocatalytic activity tests. These smart enzyme–polymer conjugates would envision promising applications in biotechnology and biomedicine.     

阴离子印迹聚合物的制备及其应用研究进展

离子印迹聚合物是利用印迹技术对模板离子进行印迹、聚合进而得到能够选择性吸附该离子的一种特殊聚合物.目前的报道大多是阳离子印迹聚合物,因为阴离子模板结构更复杂多样且电荷尺寸比更小,致使阴离子印迹聚合物的发展相对滞后.为能更有效地指导阴离子印迹聚合物的制备,本文概述了阴离子印迹聚合物的发展现状,介绍了与不同阴离子有相互作用...     

Poly(ethyleneimine)/arginine–glycine–aspartic acid conjugates prepared with N‐succinimidyl 3‐(2‐pyridyldithio)propionate: An investigation of peptide coupling and conjugate stability

Poly(ethylene imine) (PEI), a highly cationic polymer, is being used for deoxyribonucleic acid (DNA) complexation and delivery into cells. To enhance the cellular uptake of polymer/DNA complexes, arginine–glycine–aspartic acid (RGD) peptides have been conjugated to PEI with N‐succinimidyl 3‐(2‐pyridyldithio)propionate (SPDP). This coupling scheme creates a disulfide‐linked conjugate, the stability of which in the presence of thiols is uncertain. We have investigated the conjugation of an RGD peptide, glycine–arginine–glycine–aspartic acid–serine–proline–cysteine (GRGDSPC), to PEI with SPDP and subsequently assessed the stability of the conjugates in the presence of two thiol compounds, mercaptoethanol and cysteine. SPDP effectively controls the extent of GRGDSPC substitution on PEI. The conjugates, however, are readily cleaved in the presence of the thiols; the cleavage is rapid (～50% cleavage in 2–4 h) and inversely related to the degree of peptide substitution on the polymers. The peptide coupling is stable in the absence of thiols, and its cleavage is strongly dependent on the pH of the medium but not on the ionic strength of the medium. We conclude that RGD peptides coupled to PEI are labile in the presence of physiological concentrations of thiols, and this should be taken into account when such polymer–peptide conjugates are used for DNA delivery. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 6143–6156, 2004     

Polymer-Based Therapeutics

Polymeric materials have been applied in therapeutic applications, such as drug delivery and tissue regeneration, for decades owing to their biocompatibility and suitable mechanical properties. In addition, select polymer-drug conjugates have been used as bioactive pharmaceuticals owing to their increased drug efficacy, solubility, and target specificity compared with small-molecule drugs. Increased synthetic control of polymer properties has permitted the production of polymer assemblies for the targeted and controlled delivery of drugs, and polymeric sequestrants take advantage of their lack of solubility for the sequestration of target molecules in vivo. In more recent studies reviewed in greater detail here, the properties of polymers that distinguish them from small-molecule drugs, such as their high molecular weight and their ability to display multiple pendant moieties, have been specifically exploited for activating cellular targets or inhibiting the binding of pathogens. The elucidation of relevant structure-function relationships in investigations of this kind has relied on the combination of living polymerization methods with chemical conjugation methods, and protein engineering methods have shown increasing potential in the manipulation of architectural features of such polymer therapeutics. Garnering a detailed understanding of the various mechanisms by which multivalent polymers engage biological targets is certain to expand the role of polymers as therapeutics, by enabling highly specific activities of designed polymers in the biological environment.     

Structural characterization of protein–polymer conjugates for biomedical applications with small-angle scattering

Protein–polymer conjugates, typically consisting of one or more polymers covalently attached to a protein, are an increasingly common component in biotechnology. Polymers can increase circulation time, alter immune responses, and influence the self-assembly of proteins to which they are attached. To understand and take full advantage of the benefits that protein–polymer conjugates provide, there is a strong need for structural characterization of both the conjugates and their self-assembled structures. Although X-ray crystallography is suitable for determining protein structure, protein–polymer conjugates do not generally crystallize, requiring the use of alternative techniques. Small-angle scattering, with neutrons in particular, is one such technique. In this article, we review recent work in the area of protein–polymer conjugates and highlight the important role that structure plays. We then highlight shape-dependent and shape-independent approaches for structural characterization of protein–polymer conjugates and future directions in small-angle scattering interpretation. We conclude by introducing a new model that we suggest may be useful in the future to acquire more detailed structural properties.     

Selective covalent conjugation of phosphorothioate DNA oligonucleotides with streptavidin

Protein-DNA conjugates have found numerous applications in the field of diagnostics and nanobiotechnology, however, their intrinsic susceptibility to DNA degradation by nucleases represents a major obstacle for many applications. We here report the selective covalent conjugation of the protein streptavidin (STV) with phosphorothioate oligonucleotides (psDNA) containing a terminal alkylthiolgroup as the chemically addressable linking unit, using a heterobifunctional NHS-/maleimide crosslinker. The psDNA-STV conjugates were synthesized in about 10% isolated yields. We demonstrate that the terminal alkylthiol group selectively reacts with the maleimide while the backbone sulfur atoms are not engaged in chemical conjugation. The novel psDNA-STV conjugates retain their binding capabilities for both biotinylated macromolecules and the complementary nucleic acid. Moreover, the psDNA-STV conjugate retained its binding capacity for complementary oligomers even after a nuclease digestion step, which effectively degrades deoxyribonucleotide oligomers and thus the binding capability of regular DNA-STV conjugates. The psDNA-STV therefore hold particular promise for applications e.g. in proteome research and novel biosensing devices, where interfering endogenous nucleic acids need to be removed from analytes by nuclease digestion.     

Feasibility of polymer-drug conjugates for non-cancer applications

Polymer-drug conjugates have been intensely studied in the context of improving cancer chemotherapy and yet the only polymer-drug conjugate on the market (Movantik^®) has a different therapeutic application (relieving opioid-induced constipation). In parallel, a number of studies have recently been published proposing the use of this approach for treating diseases other than cancer. In this commentary, we analyse the many and very diverse applications that have been proposed for polymer-drug conjugates (ranging from inflammation to cardiovascular diseases) and the rationales underpinning them. We also highlight key design features to be considered when applying polymer-drug conjugates to these new therapeutic areas.