Polymerization Of N-carboxyanhydrides Research Articles

Facile synthesis of amphiphilic AB3 and A3B miktoarm PeptoMiktoStars

Amphiphilic miktoarm star copolymers have a unique internal structure and many attractive properties with respect to solution self-assembly, such as a high density of internal and peripheral functionalities, low CMC values, and high loading efficiency. However, compared to their linear analogs, the complex architecture demands asymmetric polymer arms emanating from a single core, which poses a significant synthetic challenge. Herein, we demonstrate an approach for the synthesis of polypept(o)ide-based AB3- and A3B-type miktoarm PeptoStars consisting of polypeptidic poly(γ-benzyl-l-glutamate (pGlu(OBn)) as the hydrophobic A arm and polypeptoidic polysarcosine (pSar) as the hydrophilic B arm. These structures, which are completely derived from endogenous amino acids, were realized by the core-first method using a tetrafunctional initiator and the corresponding N-carboxyanhydrides (NCAs) via a controlled, living nucleophilic ring-opening polymerization (ROP). The asymmetric architecture was achieved through an orthogonal protecting group strategy with multiple protection/deprotection steps. Characterization via 1H NMR-, 1H DOSY-spectroscopy, and size-exclusion chromatography (SEC) indicate the presence of well-formed miktoarm PeptoStars with low dispersities (Đ = 1.09–1.14), the precise control over the degree of polymerization and Poisson-like molecular weight distributions. Schwiertz et al. report on the synthesis of miktoarm star polymers based on polypept(o)ides by nucleophilic ring opening polymerization of N-carboxyanhydrides. The reported procedures allow for precise control over chain length, number of arms and end group functionality.

Recent Advances and Future Perspectives of Synthetic Polypeptides from N-Carboxyanhydrides

Synthetic polypeptides, the analogues of natural proteins, are important biomaterials that have found broad biomedical applications. Ring-opening polymerization of N-carboxyanhydrides offers a reliable way to prepare high-molecular-weight polypeptides in large scale with diverse side-chain functionalities. The past two decades have seen significant advances in the polypeptide field, with the development of various controlled polymerization methodologies, the deeper understanding on secondary structures, and the discovery of new assembly behaviors and applications. In this Perspective, we highlight several key advances in the chemical synthesis and materials application of synthetic polypeptides and discuss promising future directions in this area.

Polypeptide Nanoparticles Obtained from Emulsion Polymerization of Amino Acid N-Carboxyanhydrides.

Polypeptide nanoparticles were obtained by the miniemulsion polymerization of S-(o-nitrobenzyl)-l-cysteine (NBC) N-carboxyanhydride (NCA). Through process optimization, reaction conditions were identified that allowed the polymerization of the water sensitive NCA to yield nanoparticles of about 220 nm size. Subsequent UV-irradiation of the nanoparticle emulsions caused the in situ removal of the nitrobenzyl group and particle cross-linking through disulfide bond formation accompanied by the shrinkage of the particles.

Hierarchical Fractal Assemblies from Poly(ethylene oxide- b-lysine- b-leucine).

Poly(ethylene oxide43- b-lysine62- b-leucine72) (wherein subscripts denote the degree of polymerization) was synthesized via ring-opening polymerization of N-carboxyanhydrides using an amine-terminated poly(ethylene oxide) macroinitiator, with polypeptide blocks produced by sequential monomer addition. Infrared and circular dichroism spectroscopy indicated that the peptide blocks in this polymer formed α-helices in the solid and solution states, respectively. In the aqueous solution, this polymer self-assembled into spherical micelles with a hydrodynamic radius of approximately 90 nm at concentrations between 0.05 and 0.20% w/w and pH values between 2 and 6.5. Upon preparation of transmission electron microscopy (TEM) grids, the micelles at pH 2 underwent hierarchical assembly to produce fractal assemblies, whereas small clusters were observed for micellar solutions at pH 6.5. Cryogenic-TEM of solutions showed spherical micelles, and dynamic light scattering showed no large (∼1 μm) aggregates in the solution, which suggests that fractal formation was a result of the drying process, and that fractals were not present in the solution. This system provides a facile route to nanostructured surfaces, which can be used for applications such as modulating cell adhesion or promoting the growth of neurons.

Nanoparticulate delivery of irinotecan active metabolite (SN38) in murine colorectal carcinoma through conjugation to poly (2-ethyl 2-oxazoline)-b-poly (L-glutamic acid) double hydrophilic copolymer

SN-38 is the active metabolite of irinotecan, an FDA-approved chemotherapeutic agent indicated for colorectal carcinoma, which would not be clinically applicable due to its very poorly soluble and hydrolytic degradation properties. To overcome these limitations, it was proposed to conjugate SN38 to residing carboxylic acid residues in poly (2-ethyl 2-oxazoline) block poly (L-glutamic acid), inducing nano-assembly in aqueous medium. Following a series of reactions including poly (2-ethyl oxazoline) macro-initiated ring opening polymerization of N-carboxyanhydride, deprotection of benzyl group and chemical conjugation of SN38 via biodegradable ester linkage, the as-synthesized product was characterized by dynamic light scattering, ζ potential and transmission electron microscopy. The resulting particles presented about 90% loading efficiency with a mean size of 90 nm. Upon incubation with colorectal carcinoma CT26 cell line, higher association of SN-38 fluorescence and significantly more specific cytotoxicity was noticed for the SN38 conjugated particles than free drug. Therapeutic applicability of the as-synthesized product was evaluated in CT26 allograft tumor model in BALB/c mice, showing superior efficiency of the SN38 conjugated particles particularly in tumors with sizes larger than 200 mm3 than parent irinotecan and reduced mortality rate by 2.5 times. Conclusively, the poly (2-ethyl 2-oxazoline) decorated nano-conjugates of poly (L-glutamic acid) and SN38 can be regarded as a novel and potentially efficient drug delivery system for advanced colorectal carcinoma.

Synthesis of polypeptides via bioinspired polymerization of in situ purified N-carboxyanhydrides

Ribozymes synthesize proteins in a highly regulated local environment to minimize side reactions caused by various competing species. In contrast, it is challenging to prepare synthetic polypeptides from the polymerization of N-carboxyanhydrides (NCAs) in the presence of water and impurities, which induce monomer degradations and chain terminations, respectively. Inspired by natural protein synthesis, we herein report the preparation of well-defined polypeptides in the presence of competing species, by using a water/dichloromethane biphasic system with macroinitiators anchored at the interface. The impurities are extracted into the aqueous phase in situ, and the localized macroinitiators allow for NCA polymerization at a rate which outpaces water-induced side reactions. Our polymerization strategy streamlines the process from amino acids toward high molecular weight polypeptides with low dispersity by circumventing the tedious NCA purification and the demands for air-free conditions, enabling low-cost, large-scale production of polypeptides that has potential to change the paradigm of polypeptide-based biomaterials.

Proximity-Induced Cooperative Polymerization in "Hinged" Helical Polypeptides.

Cooperative interactions and transitions are among the most important strategies utilized by biological systems to regulate a variety of physical and chemical processes. We report herein an auto-accelerated, rapid cooperative polymerization of N-carboxyanhydrides (NCAs) with initiators structurally as simple as linear aliphatic diamines for the synthesis of polypeptides. The polymerization initiated by diamines proceeds via the formation of "hinged" polypeptides, which are two blocks of helical chains connected head-to-head by the diamine molecules in the polymerization solution. The reactions follow a two-stage, cooperative polymerization kinetic; the cooperative interactions between the macrodipoles of the two hinged helical polypeptides dramatically accelerate the polymerization. Compared to the NCA polymerization initiated by the hexylamine (CH3(CH2)5NH2), the chain propagation rate of the NCA polymerization is increased by more than 600 times when initiated by its diamine analogue (1,6-diaminohexane, NH2(CH2)6NH2). This proximity-induced cooperative polymerization showcases the single helix as a remarkable cooperativity-enabling motif in synthetic chemistry.

Gelating polypeptide matrices based on the difunctional N‐carboxyanhydride diaminopimelic acid cross‐linker

ABSTRACTThis work reports the synthesis of a new difunctional N‐carboxyanhydride (NCA) monomer, namely diaminopimelic acid (DAP), and its use in the one‐pot preparation of an organogelating copolypeptide. The organogel is formed in situ through ring‐opening polymerization (ROP) of DAP NCA from helical poly(ε‐carbobenzyloxy‐L‐lysine) (PZLL) blocks in a mixture of dimethylformamide/chloroform. Gelation occurs by immobilizing the solvent through core crosslinking and is stabilized through physical intermolecular conformations. After removal of the carbobenzyloxy (cbz or Z) protecting groups, the network remains intact in exceedingly high aqueous concentrations (99.5%). FTIR is used to characterize the secondary structure, revealing the conformational arrangements that contributed to these stabilized gel networks with their relative mechanical properties determined via real‐time rheological assays. DAP core crosslink of the random coil forming polypeptoid poly(sarcosine) (PSar) is also resulting in networks but is devoid of any stabilized physical interactions, thus yielding significantly weaker gels as a result. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019

PH-responsive polymeric vesicles from branched copolymers.

A new type of branched copolymer, poly(l-lactide)2-b-poly(l-glutamic acid) (PLLA2–PLGA), based on polypeptide PLGA is synthesized by the ring-opening polymerization (ROP) of N-carboxyanhydride of γ-benzyl-l-glutamate (BLG–NCA) with amino-terminated PLLA2–NH2 and subsequent deprotection. The branched copolymer is characterized by 1H NMR, FTIR and GPC measurements. The self-assembly of the copolymers in aqueous media has been systematically discussed. A pyrene probe has been used to demonstrate the aggregated formation of PLLA2–PLGA in solution by measuring the critical micelle concentration (cmc). The morphology and size of the micelles have further been studied by transmission electron microscopy (TEM), dynamic light scattering (DLS) and field emission scanning electron microscopy (ESEM). We demonstrated that the Rh of the vesicle is depending on solution pH and salt concentration. The vesicles show good stability with remained shapes and sizes during the lyophilizing process. These vesicles have great potential in the application of drug delivery.

Synthesis of water soluble and multi-responsive selenopolypeptides via ring-opening polymerization of N-carboxyanhydrides.

We report here the synthesis of water soluble selenopolypeptides via the ring-opening polymerization of N-carboxyanhydrides. The oligoethylene glycol-bearing selenopolypeptides are thermally responsive in aqueous solutions with tunable lower critical solution temperatures. The polymers can also undergo rapid and reversible helix-coil transitions upon responding to the added redox cycle.

Lithium hexamethyldisilazide initiated superfast ring opening polymerization of alpha-amino acid N-carboxyanhydrides

Polypeptides have broad applications and can be prepared via ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCAs). Conventional initiators, such as primary amines, give slow NCA polymerization, which requires multiple days to reach completion and can result in substantial side reactions, especially for very reactive NCAs. Moreover, current NCA polymerizations are very sensitive to moisture and must typically be conducted in a glove box. Here we show that lithium hexamethyldisilazide (LiHMDS) initiates an extremely rapid NCA polymerization process that is completed within minutes or hours and can be conducted in an open vessel. Polypeptides with variable chain length (DP = 20–1294) and narrow molecular weight distribution (Mw/Mn = 1.08–1.28) were readily prepared with this approach. Mechanistic studies support an anionic ring opening polymerization mechanism. This living NCA polymerization method allowed rapid synthesis of polypeptide libraries for high-throughput functional screening.

α-Amino acid N-thiocarboxyanhydrides: A novel synthetic approach toward poly(α-amino acid)s

Poly(α-amino acid)s, containing amino acid repeat units, are remarkable biomaterials with similar structures to protein, which are mainly synthesized by solid phase peptide synthesis, ring opening polymerization of N-carboxyanhydrides, polymerization of activated urethane derivatives of α-amino acids and so on. α-Amino acid N-thiocarboxyanhydride (NTA) and N-substituted glycine N-thiocarboxyanhydride (NNTA) have recently emerged as a subject of scientific interest due to their easy synthesis and purification in open air, long shelf-life and well controlled polymerization to produce poly(α-amino acid)s. In this feature article, we review the history and the recent development on the design of NTA monomers, and summarize the recent advances in poly(α-amino acid)s synthesis through ring-opening polymerization of NTA and NNTA by using different initiators and reaction mediums. We also highlight the applications of poly(α-amino acid) materials resulting from NTA polymerization.

Rapid and Mild Synthesis of Amino Acid N-Carboxy Anhydrides: Basic-to-Acidic Flash Switching in a Microflow Reactor.

Polymerization of N-carboxy anhydrides (NCAs) is the primary process used to prepare polypeptides. The synthesis of various pure NCAs is key to the efficient synthesis of polypeptides. The only practical method that can be used to synthesize NCAs requires harsh acidic conditions that make acid-labile substrates unusable and results in an undesired ring opening of NCAs. Basic-to-acidic flash switching and subsequent flash dilution technology in a microflow reactor was used to demonstrate the synthesis of NCAs. It is both rapid (0.1 s) and mild (20 °C) and includes substrates containing acid-labile functional groups. The basic-to-acidic flash switching enabled both an acceleration of the desired NCA formation and avoided the undesired ring opening of NCAs. The flash dilution precluded the undesired decomposition of acid-labile functional groups. The developed process allowed the synthesis of various NCAs which cannot be readily synthesized using conventional batch methods.

Straightforward access to linear and cyclic polypeptides

Ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCAs) is a powerful synthetic methodology for generating well-defined functional polypeptides. However, conventional procedures require a compromise between obtaining controlled microstructures and employing the optimized polymerization conditions. Specifically, a versatile method to access sequenced cyclic polypeptides remains challenging due to the difficulty in site-specific cyclization. Here we describe a general and straightforward method for the synthesis of both linear and cyclic polypeptides using organocatalytic living polymerization of NCAs. The use of an air-stable organocatalyst, imidazolium hydrogen carbonate, allows for the rapid and controlled polymerization of a variety of NCAs, leading to high conversion within a few minutes under mild conditions. Linear and cyclic block copolypeptides are also accessible simply by controlling the type of initiators and the order of addition of NCA monomers.

Advances and Biomedical Applications of Polypeptide Hydrogels Derived from α-Amino Acid N-Carboxyanhydride (NCA) Polymerizations.

Polypeptide hydrogels, having the ability to mimic certain properties of natural, native extracellular matrix components, are being actively designed and described for various applications in the construction of tissue engineering scaffolds, living cell encapsulation, and drug delivery systems. Compared to conventional hydrogels, polypeptide hydrogels possess biocompatibility, biodegradability, bioactivity, functional diversity, and structural advantage based on the unique secondary structures (α-helix and β-sheet). Furthermore, the progresses in functional N-carboxyanhydride polymerization combined with advanced orthogonal conjugation techniques significantly promote the development of the polypeptide materials. This progress report focuses on the recent advances in designing and engineering polypeptide hydrogels obtained from ring opening polymerization, highlighting the precise manipulation of their properties for biomedical applications.

Synthesis and characterization of well-defined poly(2-deoxy-2-methacrylamido-d-glucose) and its biopotential block copolymers via RAFT and ROP polymerization

Well-defined homopolymers of 2-deoxy-2-methacrylamido-d-glucose (MAG) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization using 4-cyanopentanoic acid-4-dithiobenzoate as chain transfer agent (CTA) and 2,2′-azobisisobutyronitrile (AIBN) as initiator. The effect of polymerization conditions such as molar ratio of RAFT agent to initiator and monomer to RAFT agent on conversion and molecular mass characteristics of prepared polymers was estimated. Kinetics of the polymerization under various reaction conditions was studied as a function of time and conversion using in situ1H NMR spectroscopy. The living character of polymerization was demonstrated by carriyng out the further chain extension experiments using isolated PMAG-CTA as a macroRAFT agent. For that, the PMAG with higher molecular weight, as well as block copolymer with acrylic acid were synthesized and characterized using 1H NMR and SEC analysis. A novel amphiphilic copolymer representing a hybrid of glycopolymer and polypeptide fragments was synthesized using RAFT and ring-opening polymerization (ROP). In this case, dithiobenzoate end-group of PMAG was transformed via simultaneous reduction and in situ thiol capping reaction by cysteamine hydrochloride into amino functionality suitable for polymerization of N-carboxyanhydrides (NCA). Two block copolymers of MAG with poly-l-phenylalanine (PPhe) differed with hydrophobic block length were synthesized, characterized and used for preparation of particles via copolymer self-assembly. The hydrodynamic diameter, morphology and cytotoxicity of polymer particles based on PMAG-b-PPhe were evaluated using DLS, TEM and MTT-assay, respectively. Moreover, the encapsulation of model compound (fluorescent dye rhodamine 6G) inside PMAG-b-PPhe micelles, as well as its release, were also tested.

Nanoparticulate polyelectrolyte complexes of thermally sensitive poly(l-lysine)-based copolymers and DNA

The present contribution describes the synthesis and feasibility of using a series of hybrid copolymers, designed and evaluated as stimuli-sensitive, polypeptide-based gene-delivery vectors. The copolymers of the type (PNIPAm)77-g-(PEG)9-b-(PLL)z were prepared by a combination of free radical copolymerization and ring-opening polymerization of N-carboxyanhydride of protected l-lysine with successive protective groups cleavage. They comprised fixed composition of thermally responsive poly(N-isopropylacrylamide) (PNIPAm) to which hydrophilic, non-ionic (poly(ethylene glycol), PEG) chains were grafted and a polycationic (poly(l-lysine), PLL) block with increasing from 10 to 65 degrees of polymerization. The abrupt heating of dilute aqueous solutions above the transition temperature resulted in formation of nanosized particles, which exhibited strong ability to condense DNA into well-defined polyplex particles. The latter displayed exceptional colloidal stability at room temperature. Preliminary in vitro studies showed that both polyplexes and the pure copolymers of the lowest degree of polymerization of the polypeptide block were devoid of intrinsic cytotoxicity. The findings indicate that the (PNIPAm-g-PEG)-b-PLL copolymers can be considered prospective as gene-delivery platforms.

Efficient Gene Delivery Mediated by a Helical Polypeptide: Controlling the Membrane Activity via Multivalency and Light-Assisted Photochemical Internalization (PCI).

The development of robust and nontoxic membrane-penetrating materials is highly demanded for nonviral gene delivery. Herein, a photosensitizer (PS)-embedded, star-shaped helical polypeptide was developed, which combines the advantages of multivalency-enhanced intracellular DNA uptake and light-strengthened endosomal escape to enable highly efficient gene delivery with low toxicity. 5,10,15,20-Tetrakis-(4-aminophenyl) porphyrin as a selected PS initiated ring-opening polymerization of N-carboxyanhydride and yielded a star-shaped helical polypeptide after side-chain functionalization with guanidine groups. The star polypeptide afforded a notably higher transfection efficiency and lower cytotoxicity than those of its linear analogue. Light irradiation caused almost complete (∼90%) endosomal release of the DNA cargo via the photochemical internalization (PCI) mechanism and further led to a 6-8-fold increment of the transfection efficiency in HeLa, B16F10, and RAW 264.7 cells, outperforming commercial reagent 25k PEI by up to 3 orders of magnitude. Because the PS and DNA cargoes were compartmentalized distantly in the core and polypeptide layers, respectively, the generated reactive oxygen species caused minimal damage to DNA molecules to preserve their transfection potency. Such multivalency- and PCI-potentiated gene delivery efficiency was also demonstrated in vivo in melanoma-bearing mice. This study thus provides a promising strategy to overcome the multiple membrane barriers against nonviral gene delivery.

Highly Effective Antibacterial Vesicles Based on Peptide-Mimetic Alternating Copolymers for Bone Repair

It is an important challenge for bone repair to effectively deliver growth factors and at the same time to prevent and cure inflammation without obvious pathogen resistance. We designed a kind of antibacterial peptide-mimetic alternating copolymers (PMACs) to effectively inhibit and kill both Gram-positive and Gram-negative bacteria. The minimum inhibition concentrations (MICs) of the PMACs against E. coli and S. aureus are 8.0 μg/mL, which are much lower than that of antibacterial peptides synthesized by other methods such as widely used ring-opening polymerization of N-carboxyanhydride. Furthermore, the PMACs can self-assemble into polymer vesicles (polymersomes) in pure water with low cytotoxicity (IC50 > 1000 μg/mL), which can encapsulate growth factors in aqueous solution and release them during long-term antibacterial process for facilitating bone repair. We also find that the alternating structure is essential for the excellent antibacterial activity. The in vivo tests in rabbits confirmed that the growth-factor-encapsulated antibacterial vesicles have better bone repair ability compared with control groups without antibacterial vesicles. Overall, we have provided a novel method for designing PMAC-based highly effective intrinsically antibacterial vesicles that may have promising biomedical applications in the future.

Exploring Tyrosine-Triazolinedione (TAD) Reactions for the Selective Conjugation and Cross-Linking of N-Carboxyanhydride (NCA) Derived Synthetic Copolypeptides.

Highly efficient functionalization and cross-linking of polypeptides is achieved via tyrosine-triazolinedione (TAD) conjugation chemistry. The feasibility of the reaction is demonstrated by the reaction of 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) with tyrosine containing block copolymer poly(ethylene glycol)-Tyr4 as well as a statistical copolymer of tyrosine and lysine (poly(Lys40 -st-Tyr10 )) prepared form N-carboxyanhydride polymerization. Selective reaction of PTAD with the tyrosine units is obtained and verified by size exclusion chromatography and NMR spectroscopy. Moreover, two monofunctional and two difunctional TAD molecules are synthesized. It is found that their stability in the aqueous reaction media significantly varied. Under optimized reaction conditions selective functionalization and cross-linking, yielding polypeptide hydrogels, can be achieved. TAD-mediated conjugation can offer an interesting addition in the toolbox of selective (click-like) polypeptide conjugation methodologies as it does not require functional non-natural amino acids.