Poly(<i>S</i>‑ethylsulfonyl‑l‑cysteines) for Chemoselective Disulfide Formation

The amino acid cysteine possesses a unique role in nature due to its ability to reversibly cross-link proteins. To transfer this feature to polypeptides and control the process of disulfide formation, a protective group needs to provide stability against amines during synthesis, combined with chemoselective reactivity toward thiols. A protective group providing these unique balance of stability and reactivity toward different nucleophiles is the <i>S</i>-alkylsulfonyl group. In this work we report the polymerization of <i>S</i>-ethylsulfonyl-l-cysteine <i>N</i>-carboxyanhydride and kinetic evaluations with respect to temperature (−10, 0, and +10 °C) and monomer concentration. The polymerization degree of poly­(<i>S</i>-ethylsulfonyl-l-cysteines) can be controlled within a range of 10–30, yielding well-defined polymers with molecular weights of 6900–12 300 g/mol with dispersity indices of 1.12–1.19 as determined by GPC and MALDI–TOF analysis. The limitation of chain length is, however, not related to side reactions during ring-opening polymerization, but to physical termination during β-sheet assembly. In the case of poly­(<i>S</i>-ethylsulfonyl-l-cysteines), circular dichroism as well as FT-IR experiments confirm an antiparallel β-sheet conformation. The reaction of poly­(<i>S</i>-ethylsulfonyl-l-cysteines) with thiols is completed in less than a minute, leading quantitatively to asymmetric disulfide bond formation in the absence of side reactions. Therefore, poly­(<i>S</i>-ethylsulfonyl cysteines) are currently the only reactive cysteine derivative applicable to NCA synthesis and polymerization, which allows efficient and chemoselective disulfide formation in synthetic polypeptides, bypassing additional protective group cleavage steps

Solution Properties of Polysarcosine: From Absolute and Relative Molar Mass Determinations to Complement Activation

Polysarcosine (pSar) was one of the first polymers synthesized in a controlled living manner, but it was only recently when it was reconsidered as a promising alternative for poly­(ethylene glycol) (PEG) in biomedical applications. Despite receiving more and more attention, very little is known about the solution properties of pSar, such as coil dimensions and thermodynamic interactions. In this article, we report on these properties of pSar with degrees of polymerization 50 < <i>X</i>_n < 400 that were prepared by controlled living ring-opening polymerization. The polymers are characterized by gel permeation chromatography (GPC), MALDI-TOF mass spectrometry, dynamic and static light scattering (SLS), and viscometry. The chain stiffness of pSar in PBS in terms of the Kuhn statistical segment length, <i>l</i>_k, was estimated to <i>l</i>_k = 1.5 nm by application of the Yamakawa–Fujii wormlike chain theory to the experimentally determined hydrodynamic radii, <i>R</i>_h, thus being higher than <i>l</i>_k = 1.1 nm for PEG in PBS. Also, the second virial coefficients, <i>A</i>₂, of pSar and PEG in PBS were similar and reflect their good solubility in aqueous solution. While the universal calibration of GPC elution volumes failed for pSar in HFIP utilizing PMMA standards, it worked better in PBS buffer with PEG standards. Alternatively, an <i>R</i>_h–<i>M</i>_w relation is established in the present work, which enables the determination of molar masses of pSar by simple DLS measurements. In addition, it is demonstrated that pSar independent from its chain length (50 < <i>X</i>_n < 400) does not induce any detectable complement activation (C5a) in human serum

Revisiting Secondary Structures in NCA Polymerization: Influences on the Analysis of Protected Polylysines

Two series (degree of polymerization: 20–200) of polylysines with Z and TFA protecting groups were synthesized, and their behavior in a range of analytical methods was investigated. Gel permeation chromatography of the smaller polypeptides reveals a bimodal distribution, which is lost in larger polymers. With the help of GPC, NMR, circular dichroism (CD), and MALDI-TOF, it was demonstrated that the bimodal distribution is not due to terminated chains or other side reactions. Our results indicate that the bimodality is caused by a change in secondary structure of the growing peptide chain that occurs around a degree of polymerization of about 15. This change in secondary structure interferes strongly with the most used analysis method for polymersGPCby producing a bimodal distribution as an artifact. After deprotection, the polypeptides were found to exhibit exclusively random coil conformation, and thus a monomodal GPC elugram was obtained. The effect can be explained by a 1.6-fold increase in the hydrodynamic volume at the coil–helix transition. This work demostrates that secondary structures need to be carefully considered when performing standard analysis on polypeptidic systems

Programmable Assembly of Peptide Amphiphile via Noncovalent-to-Covalent Bond Conversion

Controlling the number of monomers in a supramolecular polymer has been a great challenge in programmable self-assembly of organic molecules. One approach has been to make use of frustrated growth of the supramolecular assembly by tuning the balance of attractive and repulsive intermolecular forces. We report here on the use of covalent bond formation among monomers, compensating for intermolecular electrostatic repulsion, as a mechanism to control the length of a supramolecular nanofiber formed by self-assembly of peptide amphiphiles. Circular dichroism spectroscopy in combination with dynamic light scattering, size-exclusion chromatography, and transmittance electron microscope analyses revealed that hydrogen bonds between peptides were reinforced by covalent bond formation, enabling the fiber elongation. To examine these materials for their potential biomedical applications, cytotoxicity of nanofibers against C2C12 premyoblast cells was tested. We demonstrated that cell viability increased with an increase in fiber length, presumably because of the suppressed disruption of cell membranes by the fiber end-caps

Combining Orthogonal Reactive Groups in Block Copolymers for Functional Nanoparticle Synthesis in a Single Step

We report on the synthesis of polysarcosine-<i>block</i>-poly­(<i>S</i>-alkylsulfonyl)-l-cysteine block copolymers, which combine three orthogonal addressable groups enabling site-specific conversion of all reactive entities in a single step. The polymers are readily obtained by ring-opening polymerization (ROP) of corresponding α-amino acid <i>N</i>-carboxyanhydrides (NCAs) combining azide and amine chain ends, with a thiol-reactive <i>S</i>-alkylsulfonyl cysteine. Functional group interconversion of chain ends using strain-promoted azide–alkyne cycloaddition (SPAAC) and activated ester chemistry with NHS- and DBCO-containing fluorescent dyes could be readily performed without affecting the cross-linking reaction between thiols and <i>S</i>-alkylsulfonyl protective groups. Eventually, all three functionalities can be combined in the formation of multifunctional disulfide core cross-linked nanoparticles bearing spatially separated functionalities. The simultaneous attachment of dyes in core and corona during the formation of core-cross-linked nanostructures with controlled morphology is confirmed by fluorescence cross-correlation spectroscopy (FCCS)

Multidentate Polysarcosine-Based Ligands for Water-Soluble Quantum Dots

We describe the synthesis of heterotelechelic polysarcosine polymers and their use as multidentate ligands in the preparation of stable water-soluble quantum dots (QDs). Orthogonally functionalized polysarcosine with amine and dibenzo­cyclooctyl (DBCO) end groups is obtained by ring-opening polymerization of <i>N</i>-methyl­glycine <i>N</i>-carboxy­anhydride with DBCO amine as initiator. In a first postpolymerization modification step, the future biological activity of the polymeric ligands is adjusted by modification of the amine terminus. Then, in a second postpolymerization modification step, azide functionalized di- and tridentate anchor compounds are introduced to the DBCO terminus of the polysarcosine via strain-promoted azide–alkyne cycloaddition (SPAAC). Through the separate synthesis of the anchor compounds, it is possible to ensure reproducible introduction of a well-defined number of multiple anchor groups to all polymers studied. Finally, the obtained multidentate polymeric ligands are successfully used in the ligand exchange procedures to yield stable, water-soluble QDs. As polysarcosine-based ligands can provide biocompatibility, prevent nonspecific interactions, and simultaneously enable specific targeting, the systems presented here are promising candidates to provide QDs well suitable for <i>ex vivo</i> analytics or bioimaging

HPMA Copolymers as Surfactants in the Preparation of Biocompatible Nanoparticles for Biomedical Application

In this work we describe the application of amphiphilic <i>N</i>-(2-hydroxypropyl)­methacrylamide (HPMA)-based copolymers as polymeric surfactants in miniemulsion techniques. HPMA-based copolymers with different ratios of HPMA (hydrophilic) to laurylmethacrylate (LMA; hydrophobic) units were synthesized by RAFT polymerization and postpolymerization modification. The amphiphilic polymers can act as detergents in both the miniemulsion polymerization of styrene and the miniemulsion process in combination with solvent evaporation, which was applied to polystyrene and polylactide. Under optimized conditions, monodisperse colloids can be prepared. The most promising results could be obtained by using the block copolymer with a ratio of 90/10. Preliminary cell uptake studies showed that polymer-stabilized nanoparticles have only minor unspecific cellular internalization in HeLa cells. Furthermore, cytotoxicity assays showed no particle-attributed toxicity. In addition, the copolymer-stabilized particles preserved the shape and size in human blood serum as demonstrated by dynamic light scattering

Cylindrical Brush Polymers with Polysarcosine Side Chains: A Novel Biocompatible Carrier for Biomedical Applications

Cylindrical brush polymers constitute promising polymeric drug delivery systems (nanoDDS). Because of the densely grafted side chains such structures may intrinsically exhibit little protein adsorption (“stealth” effect) while providing a large number of functional groups accessible for bioconjugation reactions. Polysarcosine (PSar) is a highly water-soluble, nonionic and nonimmunogenic polypeptoid based on the endogenous amino acid sarcosine (<i>N</i>-methyl glycine). Here we report on the synthesis, characterization and biocompatibility of cylindrical brush polymers with either polysarcosine side chains or poly-l-lysine-<i>b</i>-polysarcosine side chains. The latter leads to block copolypept­(o)­id based core–shell cylindrical brushes with a cationic poly-l-lysine (PLL) core and a neutral polysarcosine corona. The cylindrical brush polymers were prepared by ring-opening polymerization of the respective N-carboxyanhydrides (NCA) from a macroinitiator chain. Preliminary experiments on complex formation with siRNA demonstrate that a core–shell cylindrical brush polymer may complex on average up to 270 RNA molecules amounting to a high loading efficiency of N⁺/P^– = 1.1. No bridging between cylindrical brushes leading to larger aggregates is observed. In vitro studies on the silencing of the expression of ApoB100, which is abundantly expressed in AML-12 hepatocytes, induced by siRNA-cylindrical core–shell brush complexes showed high efficiency, leading to a knock-down efficiency of ApoB100 mRNA of 70%

Cooperative Catechol-Functionalized Polypept(o)ide Brushes and Ag Nanoparticles for Combination of Protein Resistance and Antimicrobial Activity on Metal Oxide Surfaces

Prevention of biofouling and microbial contamination of implanted biomedical devices is essential to maintain their functionality and biocompatibility. For this purpose, polypept­(o)­ide block copolymers have been developed, in which a protein-resistant polysarcosine (pSar) block is combined with a dopamine-modified poly­(glutamic acid) block for surface coating and silver nanoparticles (Ag NPs) formation. In the development of a novel, versatile, and biocompatible antibacterial surface coating, block lengths pSar were varied to derive structure–property relationships. Notably, the catechol moiety performs two important tasks in parallel; primarily it acts as an efficient anchoring group to metal oxide surfaces, while it furthermore induces the formation of Ag NPs. Attributing to the dual function of catechol moieties, antifouling pSar brush and antimicrobial Ag NPs can not only adhere stably on metal oxide surfaces, but also display passive antifouling and active antimicrobial activity, showing good biocompatibility simultaneously. The developed strategy seems to provide a promising platform for functional modification of biomaterials surface to preserve their performance while reducing the risk of bacterial infections

Coordinative Binding of Polymers to Metal–Organic Framework Nanoparticles for Control of Interactions at the Biointerface

Metal–organic framework nanoparticles (MOF NPs) are of growing interest in diagnostic and therapeutic applications, and due to their hybrid nature, they display enhanced properties compared to more established nanomaterials. The effective application of MOF NPs, however, is often hampered by limited control of their surface chemistry and understanding of their interactions at the biointerface. Using a surface coating approach, we found that coordinative polymer binding to Zr-fum NPs is a convenient way for peripheral surface functionalization. Different polymers with biomedical relevance were assessed for the ability to bind to the MOF surface. Carboxylic acid and amine containing polymers turned out to be potent surface coatings and a modulator replacement reaction was identified as the underlying mechanism. The strong binding of polycarboxylates was then used to shield the MOF surface with a double amphiphilic polyglutamate–polysarcosine block copolymer, which resulted in an exceptional high colloidal stability of the nanoparticles. The effect of polymer coating on interactions at the biointerface was tested with regard to cellular association and protein binding, which has, to the best of our knowledge, never been discussed in literature for functionalized MOF NPs. We conclude that the applied approach enables a high degree of chemical surface confinement, which could be used as a universal strategy for MOF NP functionalization. In this way, the physicochemical properties of MOF NPs could be tuned, which allows for control over their behavior in biological systems