Α-amino Acid N-carboxyanhydrides Research Articles

Insight into the cross-linking of synthetic polypeptide gels prepared by ring-opening polymerization using l-cystine N-carboxyanhydride

Synthetic polypeptides have promising potential for various biomedical applications that often require a cross-linked network for stability against solvents or mechanical stress. Due to the natural occurrence and availability of the l-cystine α-amino acid, l-cystine N-carboxyanhydride (NCA) is commonly used as a cross-linker in the ring-opening (co)polymerization of α-amino acid NCAs to prepare covalently cross-linked synthetic polypeptides. In this work, we show that the l-cystine NCA tends to undergo undesired decomposition side reactions, i.e., β-elimination and formation of 2,5-diketopiperazine, which reduce the amount of the cross-linker available and make it difficult to control the final properties of the materials prepared from it. By using the analogous l-homocystine NCA instead, the decomposition side reactions can be completely avoided. We demonstrate the cross-linking performance of l-cystine NCA compared to l-homocystine NCA by studying the corresponding gels prepared by copolymerization with γ-benzyl-l-glutamate NCA.

PH- and Temperature-responsive Hydrogels Based on Tertiary Amine-modified Polypeptides for Stimuli-responsive Drug Delivery.

pH- and temperature-responsive hydrogels have attracted considerable attention due to their responsiveness to dual physiologically-relevant stimuli. In this study, we developed stimuli-responsive hydrogels based on monomethoxy poly(ethylene glycol) (mPEG)-polypeptide block copolymers containing various tertiary amine pendants (EEP-TAs). The EEP-TAs were synthesized via ring-opening copolymerization of α-amino acid N-carboxyanhydrides, and further modified post-polymerization with click chemistry. The EEP-TAs exhibited an α-helix-to-β-sheet transition when the pH was increased from 4.0 to 7.4. At elevated polymer concentrations, aqueous solutions of the EEP-TAs underwent thermo-induced sol-gel phase transitions, which were dependent on the pH. The hydrogels almost fully degraded within 3 weeks in the subcutaneous layer of mice and exhibited good histocompatibility in vivo. Additionally, doxorubicin (DOX)-loaded hydrogels exhibited pH-responsive drug release profiles in vitro, which were composed of rapid release at acidic pH and more sustained release at neutral pH. Thus, these polypeptide hydrogels hold potential as depots for environment-responsive delivery of therapeutic agents.

Rapid and column-chromatography-free peptide chain elongation via a one-flow, three-component coupling approach.

Short peptides are extremely important as drugs and building blocks for the syntheses of longer peptides. Both solid- and liquid-phase peptide syntheses suffer from a large number of synthetic steps, high cost, and/or tedious purification. Here, we developed a rapid, mild, inexpensive, and column-chromatography-free peptide chain elongation via a one-flow, three-component coupling (3CC) approach that is the first to use α-amino acid N-carboxy anhydrides (α-NCAs) both as electrophiles and nucleophiles. We demonstrated the high-yielding and column-chromatography-free syntheses of 17 tripeptides, as well as a gram-scale synthesis of a tripeptide. The total synthesis of beefy meaty peptide was achieved by repeating the 3CC approach with the addition of only one column chromatographic purification. We also demonstrated a one-flow tripeptide synthesis via in situ preparation of α-NCA starting from three readily available protected amino acids. With this study, we achieved dramatic reductions in both time and cost compared with typical solid-phase synthesis.

Precision Synthesis of Polypeptides via Living Anionic Ring-Opening Polymerization of N-Carboxyanhydrides by Tri-thiourea Catalysts.

The chemistry of α-amino acid N-carboxyanhydrides (NCAs) has a history of over 100 years, but precise and efficient ring-opening polymerization methods for NCAs remain highly needed to facilitate the studies of polypeptides─that is, mimics of natural proteins─in various disciplines. Moreover, the universally accepted NCA polymerization mechanisms are largely limited to the "amine" and the "activated monomer" mechanisms, and the anionic ring-opening polymerization of NCAs has so far not been invoked. Herein, we show an unprecedented anion-binding catalytic system combining tripodal tri-thiourea with sodium thiophenolate that enables the fast and selective anionic ring-opening polymerization of NCAs. This method leads to the precision construction of various polypeptides with living polymerization behavior and is evidenced by narrow molecular weight distributions (Mw/Mn < 1.2), chain extension experiments, and minimal "activated monomer" pathway. Calculations and experimental results elucidate a living anionic polymerization mechanism, and high selectivities for monomer propagation relative to other deleterious side reactions, such as the "activated monomer" pathway, are attributed to the enhanced stabilization of the propagating carbamate anion, which is enforced by an intramolecular hydrogen bond within the tri-thiourea structure.

Composition-dependent wettability of nature-inspired homo poly(amino acid) coating and its influences on bacterial adhesion

A variety of natural proteins have shown anti-adhesive functionality against the non-specific adsorption by proteins and bacteria. A deep understanding of the contribution of amino acid composition to the antifouling performance of a natural protein is crucial for engineering a protein-based coating layer. Herein, we synthesized five types of homo poly(amino acid)s via the ring-opening polymerization of α-amino acid N-carboxy anhydride (NCA) to include exclusively one type of amino acid from lysine (K), glutamic acid (E), tyrosine (Y), alanine (A), and phenylalanine (F). Homo poly(amino acid)s were covalently immobilized on the surfaces of quartz and polyurethane (PU), respectively. The amino acid composition effects on the wetting and anti-bacterial adhesion of a homo poly(amino acid) coating layer were evaluated. We found that the wettability of homo poly(amino acid) coating layers exhibited a dependence on the intrinsic properties of amino acid side-chain, such as the hydration free energy and dipole moment of side-chain. The activity of a homo poly(amino acid) coating layer against bacterial adsorption was found to be independent of the wetting property of the amino acid side-chain. Our findings are different from the conventional opinion that the hydration property of a material surface coating layer correlates to its anti-bacterial functionality, emphasizing the complexity of the amino acid composition contribution to the activity against the non-specific adsorption of bacteria for a protein-mimicking system.

Polyoxometalate-polypeptide nanoassemblies as peroxidase surrogates with antibiofilm properties.

Developing artificial metalloenzymes that possess a superior performance to their natural counterparts is an attractive concept. Polyoxometalates (POMs) are a class of anionic molecular metal-oxides with excellent redox properties and bioactivity. We have recently introduced "POMlymers" - covalently conjugated POM-peptide hybrid materials - where the polypeptide chain is obtained through a ring-opening polymerisation (ROP) of α-amino acid N-carboxyanhydrides (NCA) on an inorganic POM scaffold. Attracted by the idea of preparing artificial metalloenzymes, here we report the supramolecular self-assembly of POMlymer hybrids into nanoparticles where an optimal environment for catalysis is created. Our results demonstrate that the self-assembly of covalent POMlymers, enhances the peroxidase-like activity of the parent POM anion whereas, in contrast, the catalytic activity for nanoparticles obtained by ionic self-assembly of the same peptide and POM components practically disappears. Furthermore, POMlymer nanoparticles also present antimicrobial and antibiofilm activity against the skin bacterium Staphylococcus epidermidis; whereas, ionic POM-peptide hybrids significantly increase biofilm production and endogenous production of reactive oxygen species. In summary, we present the self-assembly of POMlymer hybrids into nanoparticles and a combination of peroxidase activity and microbiology assays that show that the POM-peptide covalent bond is essential for the stability of the self-assembled nanoparticles and therefore for their catalytic and biological activity.

A micro-flow rapid dual activation approach for urethane-protected α-amino acid N-carboxyanhydride synthesis.

This study demonstrated the rapid dual activation (10 s, 20 °C) of a combination of an α-amino acid N-carboxyanhydride and alkyl chloroformate in the synthesis of a urethane-protected α-amino acid N-carboxyanhydride in a micro-flow reactor. The key to success was the combined use of two amines that activated both substrates with proper timing. Three amines, i-Pr2NEt, Me2NBn, or N-ethylmorpholine, were used with pyridine in accordance with the steric bulkiness of a side chain in the α-amino acid N-carboxyanhydride. A variety of 16 urethane-protected α-amino acid N-carboxyanhydrides were synthesized in high yields. The role of amines was investigated based on the measurement of the time-dependent (0.5 to 10 s) decrease of α-amino acid N-carboxyanhydrides and alkyl chloroformates in the presence of amines via flash mixing technology using a micro-flow reactor. It was suggested that the in situ generated acylpyridinium cation was highly active and less prone to causing undesired decomposition compared with the acylammonium cation examined in this study. Thus, even at a very low concentration, the acylpyridinium cation facilitated the desired coupling reaction.

Poly(l-cysteine) Peptide Amphiphile Derivatives Containing Disulfide Bonds: Synthesis, Self-Assembly-Induced β-Sheet Nanostructures, pH/Reduction Dual Response, and Drug Release

Three disulfide bond-containing peptide amphiphiles (PA1-3) with different lengths of alkyl tails (PA1 for C6, PA2 for C12, and PA3 for C18) were synthesized by ring-opening polymerization of α-amino acid N-carboxyanhydride followed by post-polymerization modification. The peptide segments were composed of 3-mercaptopropionic acid-modified poly(l-cysteine) [P(Cys-SS-CH2CH2COOH)]. We characterized the chemical structure and molecular parameters by 1H NMR, 13C NMR, gel permeation chromatography, Fourier transform infrared spectroscopy, and circular dichroism spectroscopy. It is found that alkyl-P(Cys-SS-CH2CH2COOH) mainly presents a β-sheet conformation at the solid state. However, the PAs present predominant random coils at pH 7.4 in aqueous solutions. The β-sheet conformation increased dramatically when the concentration of the PA exceeded its critical micelle concentration (ca. 0.3 mg/mL for PA3), indicating the formation of self-assembly-induced β-sheet nanostructures. Elongation of the alkyl chain length or a decrease of the pH of the PA solution can promote the formation of the β-sheet conformation. The three PAs can self-assemble into spherical micelles or nanofibrous hydrogels, which can be utilized as nanocarriers for systemic drug delivery or implants for localized drug delivery, respectively. Cisplatin (CDDP) was loaded as a model medicine to examine the drug delivery potential of PA3. We found that the CDDP-loaded PA3 micelles are stable at pH 7.4, have a spherical morphology with a hydrodynamic diameter of ca. 52 nm, and accomplish pH/reduction dual-responsive release of CDDP. In addition, alkyl-P(Cys-SS-CH2CH2COOH) can self-assemble into nanofibrous hydrogels at pH 5.0-6.0 or upon the addition of certain metal ions and show excellent dynamic reversibility. Moreover, the CDDP-loaded PA3 hydrogel exhibits a sustained release profile and a nearly linear release over 48 h. In vitro cytotoxicity of PA3 also indicates its nontoxicity. Thus, our findings suggest that alkyl-P(Cys-SS-CH2CH2COOH) has great potential for both systemic and localized delivery of therapeutics.

Superfast and Water-Insensitive Polymerization on α-Amino Acid N-Carboxyanhydrides to Prepare Polypeptides Using Tetraalkylammonium Carboxylate as the Initiator.

We design the tetraalkylammonium carboxylate-initiated superfast polymerization on α-amino acid N-carboxyanhydrides (NCA) for efficient synthesis of polypeptides. Carboxylates, as a new class of initiator for NCA polymerization, can initiate the superfast NCA polymerization without the need of extra catalysts and the polymerization can be operated in open vessels at ambient condition without the use of glove box. Tetraalkylammonium carboxylate-initiated polymerization on NCA easily affords block copolymers with at least 15 blocks. Moreover, this method avoids tedious purification steps and enables direct polymerization on crude NCAs in aqueous environments to prepare polypeptides and one-pot synthesis of polypeptide nanoparticles. These advantages and the mild polymerization condition of tetraalkylammonium carboxylate-initiated NCA polymerization imply its great potential in functional exploration and application of polypeptides.

Tunable, biodegradable grafting-from glycopolypeptide bottlebrush polymers

The cellular glycocalyx and extracellular matrix are rich in glycoproteins and proteoglycans that play essential physical and biochemical roles in all life. Synthetic mimics of these natural bottlebrush polymers have wide applications in biomedicine, yet preparation has been challenged by their high grafting and glycosylation densities. Using one-pot dual-catalysis polymerization of glycan-bearing α-amino acid N-carboxyanhydrides, we report grafting-from glycopolypeptide brushes. The materials are chemically and conformationally tunable where backbone and sidechain lengths were precisely altered, grafting density modulated up to 100%, and glycan density and identity tuned by monomer feed ratios. The glycobrushes are composed entirely of sugars and amino acids, are non-toxic to cells, and are degradable by natural proteases. Inspired by native lipid-anchored proteoglycans, cholesterol-modified glycobrushes were displayed on the surface of live human cells. Our materials overcome long-standing challenges in glycobrush polymer synthesis and offer new opportunities to examine glycan presentation and multivalency from chemically defined scaffolds.

Polypeptide-based materials prepared by ring-opening polymerisation of anionic-based α-amino acid N-carboxyanhydrides: A platform for delivery of bioactive-compounds

Polymerisation of α-amino acid N-carboxyanhydrides (NCAs) is one of the most common techniques to prepare synthetic polypeptides. Of special interest are the NCAs derived from α-amino acids, L-aspartic acid and L-glutamic acid, since most investigations have been focused on their use to synthesise multiblock copolypeptides or hybrid synthetic polypeptide-polymers to design excipients suitable for delivery of bioactive compounds. This perspective highlights advantages of using L-aspartic acid and L-glutamic over other natural α-amino acids in that their pendant carboxyl group serves as a reactive handle for coupling a variety of reactive groups, and because the resulting polypeptides have the ability to adopt secondary structures. In addition, recent progress in the ring-opening polymerisation of NCAs will be discussed. Throughout, we provide representative examples that shed light on the NCAs polymerisation process, and we finally share our perspectives concerning the practical use of anionic α-amino acids as building blocks for future investigations.

Synthesis of α-Amino Acid N-Carboxyanhydrides

A simple phosgene- and halogen-free method for synthesizing α-amino acid N-carboxyanhydrides (NCAs) is described. The reaction between Boc-protected α-amino acids and T3P reagent gave the corresponding NCA derivatives in good yield and purity with no detectable epimerization. The process is safe, is easy-to-operate, and does not require any specific installation. It generates nontoxic, easy to remove byproducts. It can apply to the preparation of NCAs for the on-demand on-site production of either little or large quantities.

Two-tailed tadpole-shaped synthetic polymer polypeptide bioconjugate nanomicelles for enhanced chemo-photothermal therapy

A series of poly(N-isopropylacrylamide)30-SS-block-(cyclic-poly(L-lysine)n)-block-SS-poly(N-isopropylacrylamide)30 (n = 10, 20, 30) synthetic polymer polypeptide bio-conjugates have been synthesized for enhanced chemo-photothermal therapy. A combination of reversible addition-fragmentation chain transfer polymerization, click cycloaddition reaction, and ring-opening polymerization of α-amino acid N-carboxyanhydrides is utilized in the synthesis of a tadpole-shaped block copolymer. All the copolymers are self-assembled to nanoparticles in aqueous solutions, which are stabilized using a terephthalaldehyde crosslinker with successfully encapsulating doxorubicin (DOX) and IR780 dye. The polymer nanomicelles exhibited excellent drug loading capacity and efficiency. The in vitro drug release profiles demonstrate that the maximum release of DOX is achieved in pH 5.0 at 42 °C, in the presence of a glutathione intracellular mimic environment. IR780 dye significantly induces photothermal cytotoxicity to HT29 colon cancer lines under near-infrared laser irradiation. The new two-tailed tadpole-shaped synthetic polymer polypeptide bioconjugates exhibiting potential chemo-photothermal therapeutic effect thus appear to be a promising platform for anticancer therapy.

Density Functional Theory Studies on the Synthesis of Poly(α-Amino Acid)s Via the Amine-Mediated Ring Opening Polymerizations of N-Carboxyanhydrides and N-Thiocarboxyanhydrides.

To synthesize well-defined poly (α-amino acid)s (PAAs), ring opening polymerizations (ROP) of cyclic monomers of α-amino acid N-carboxyanhydrides (NCAs) and N-thiocarboxyanhydrides (NTAs) are most widely used. In this mini-review, we summarize the mechanism details of the monomer preparation and ROP. The present study used density functional theory calculations to reveal the mechanisms together with experimental phenomena in the past decades. Detailed discussion includes normal amine mechanism and the selectivity of the initiators bearing various nucleophilic groups.

On‐POM Ring‐Opening Polymerisation of N‐Carboxyanhydrides

The ring-opening polymerisation of α-amino acid N-carboxyanhydrides (NCAs) offers a simple and scalable route to polypeptides with predicted and narrow molecular weight distributions. Here we show how polyoxometalates (POMs)-redox-active molecular metal-oxide anions-can serve as inorganic scaffold initiators for such NCA polymerisations. This "On-POM polymerisation" strategy serves as an innovative platform to design hybrid materials with additive or synergistic properties stemming from the inorganic and polypeptide component parts. We have used this synthetic approach to synthesise a library of bactericidal poly(lysine)-POM hybrid derivatives that can be used to prevent biofilm formation. This versatile "On-POM polymerisation" method provides a flexible synthetic approach for combining inorganic scaffolds with amino acids, and the potential to tailor and improve the specificity and performance of hybrid antimicrobial materials.

α-Amino acid N-carboxyanhydride (NCA)-derived synthetic polypeptides for nucleic acids delivery.

In recent years, gene therapy has come into the spotlight for the prevention and treatment of a wide range of diseases. Polypeptides have been widely used in mediating nucleic acid delivery, due to their versatilities in chemical structures, desired biodegradability, and low cytotoxicity. Chemistry plays an essential role in the development of innovative polypeptides to address the challenges of producing efficient and safe gene vectors. In this Review, we mainly focused on the latest chemical advances in the design and preparation of polypeptide-based nucleic acid delivery vehicles. We first discussed the synthetic approach of polypeptides via ring-opening polymerization (ROP) of N-carboxyanhydrides (NCAs), and introduced the various types of polypeptide-based gene delivery systems. The extracellular and intracellular barriers against nucleic acid delivery were then outlined, followed by detailed review on the recent advances in polypeptide-based delivery systems that can overcome these barriers to enable in vitro and in vivo gene transfection. Finally, we concluded this review with perspectives in this field.

Nano Aggregate Formation Through Self-Assembly of Poly(L-lysine)-Block-Poly(γ-benzyl-L-glutamate)-Graft-Poly(ethylene glycol) Copolymer.

Polymeric nano aggregates based on poly(L-lysine)-block-PBLG-graft-poly(ethylene glycol) (PLL-b-PBLG-g-PEG) copolymers were prepared by a dialysis method in deionized water. PLL-b-PBLG-g-PEG copolymers with different degrees of PEG substitution were synthesized by combining the ring-opening polymerization of α-amino acid N-carboxyanhydrides and ester exchange reactions. Proton nuclear magnetic resonance spectroscopy and gel permeation chromatography were employed to confirm the polymer structures and molecular weights. Transmission electron microscopy and dynamic light scattering were used to observe the self-assembly behavior. The experimental results revealed that the volume content of deionized water in the mixture significantly affects the morphology and size of the aggregates formed by PLL-b-PBLG-g-PEG copolymers. Spherical, spindle-like, needle-like, and daisy-like aggregates were observed with an increase in the volume content of deionized water in the mixture.

One-Shot Synthesis of Peptide Amphiphiles with Applications in Directed Graphenic Assembly.

High molecular weight, synthetic block copolypeptides that self-assemble are in high demand for biomedical applications. The current standard method for synthesis of block copolypeptides is the controlled ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydride (NCA) monomers, where block architectures can be created by sequential NCA monomer addition. Recently, researchers have focused on developing reaction conditions and initiation systems that make NCA ROP more convenient, particularly for interdisciplinary labs without designated polypeptide facilities. In an effort to further simplify and increase the convenience of polypeptide synthesis, we developed a one-shot copolymerization strategy that allows access to block copolypeptides by capitalizing on the inherently faster reactivity of NCA monomers, compared to NTA (N-thiocarboxyanhydride) monomers. For the first time, we combine an NCA and NTA monomer in one reaction to kinetically promote block copolypeptide formation, providing a convenient alternative to sequential monomer addition. The controlled nature of this copolymerization technique is supported by a molecular weight that is modulated by the concentration of the initiator and low dispersities. We used this one-shot copolymerization to synthesize p(lysine)-b-p(leucine), a known peptide amphiphile (PA). Our one-shot PAs are antimicrobial and can spontaneously form ordered, micron-scale assemblies. Covalent conjugation of one-shot PAs to a graphenic backbone results in a functional graphenic material (FGM) with a self-assembled morphology, paving the way for creation of sophisticated FGM scaffolds with polypeptide-templated, hierarchical order. Overall, we demonstrate that this novel, one-shot copolymerization strategy produces functional copolypeptides with macroscopic sequence control.

Nitrile N-oxide-terminated poly(γ-benzyl l-glutamate) (PBLG): synthesis and catalyst-free grafting onto polybutadiene (PBD) and natural rubber (NR)

A nitrile N-oxide-functionalized polypeptide was synthesized by a two-step method involving the combination of ring-opening polymerization of an α-amino acid N-carboxyanhydride monomer using a primary amine initiator containing a nitroalkane group and a subsequent terminal conversion. The catalyst-free grafting reaction of the formed functionalized peptide with rubbers containing double bonds was achieved with high efficiency. A nitrile N-oxide-functionalized polypeptide was synthesized by a two-step method involving the combination of ring-opening polymerization of an amino acid N-carboxyanhydride (NCA) monomer using a primary amine initiator containing a nitroalkane group and a subsequent terminal conversion. The formed peptide was fully characterized by NMR, FT-IR, and MALDI-TOF MS measurements. The catalyst-free grafting reaction of the formed functionalized peptide with rubbers containing double bonds was achieved with high efficiency via an environmentally benign process. These results surely contributed to the development of novel peptide-grafted polymers.

Controlled synthesis of polypeptides

Polypeptides are one kind of promising biodegradable and biocompatible biomedical polymers with the structural units of various α-amino acids. Polypeptides were first polymerized by the ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCAs) by Leuchs and Hermann in 1906. In the past decades, several effective strategies, including the selection of initiators, the adjustment of reaction conditions, and the introduction of catalysts, have been reported to improve the controllability of the ROP of various α-amino acid NCAs to synthesize different polypeptides with precise chemical structures and low polydispersity indexes. In this Review, the strategies, mechanisms, challenges, and opportunities for controlled synthesis of polypeptides by the ROP of different α-amino acid NCAs have been declared.