Polymerization Of N-carboxyanhydrides Research Articles

Enzymatic degradation of the hydrogels based on synthetic poly(α-amino acid)s

Biodegradable hydrogels are studied as potential scaffolds for soft tissue regeneration. In this work biodegradable hydrogels were prepared from synthetic poly(α-amino acid)s, poly(AA)s. The covalently crosslinked gels were formed by radical copolymerization of methacryloylated poly(AA)s, e.g. poly[N (5)-(2-hydroxy-ethyl)-L-glutamine-ran-L-alanine-ran-N (6)-methacryloyl-L-lysine], as a multifunctional macro-monomer with a low-molecular-weight methacrylic monofunctional monomer, e.g. 2-hydroxyethyl methacrylate (HEMA). Methacryloylated copolypeptides were synthesized by polymerization of N-carboxyanhydrides of respective amino acids and subsequent side-chain modification. Due to their polypeptide backbone, synthetic poly(AA)s are cleavable in biological environment by enzyme-catalyzed hydrolysis. The feasibility of enzymatic degradation of poly(AA)s alone and the hydrogels made from them was studied using elastase, a matrix proteinase involved in tissue healing processes, as a model enzyme. Specificity of elastase for cleavage of polypeptide chains behind the L-alanine residues was reflected in faster degradation of L-alanine-containing copolymers as well as of hydrogels composed of them.

Optimization of N-carboxyanhydride (NCA) polymerization by variation of reaction temperature and pressure

Several new methods for the controlled ring-opening polymerization of N-carboxyanhydrides (NCA ROP) have been established in the recent past, all of which based on the normal amine mechanism. In this paper the optimal NAM polymerization conditions were investigated combining the high-vacuum and the low-temperature for the NCAs of γ-benzyl-L-glutamate (BLG), Ne-benzyloxycarbonyl-L-lysine (ZLL), L-alanine (Ala), β-benzyl-L-aspartate (BLA), O-benzyl-L-serine (BLS), and O-benzyl-L-threonine (BLT). The polymerizations were followed by FTIR, size exclusion chromatography (SEC) and MALDI-ToF-MS to provide information of the monomer conversion, polymer molecular weight and chain composition as a function of pressure and temperature. It was found that the studied NCAs could be divided into two groups: in the first group monomers of BLG, ZLL and Ala polymerized considerably faster when a lower pressure of 1 × 10−5 bar was applied. MALDI-ToF-MS analysis confirmed that the formation of side products for these monomers mostly started after full monomer conversion. The second group of monomers, i.e. BLA, BLS and BLT, polymerized considerably slower than the first group and no effect was observed from the lower pressure. On the other hand, the number of side reactions was significant at 20 °C, so that the polymerizations for the latter monomers should preferably be done at 0 °C. By combining both methods, multiblock polypeptides were synthesized including a tetrablock of PBLG-b-PAla-b-PZLL-b-PBLA with a polydispersity of 1.3.

Synthesis of glycosylated peptides by NCA polymerization for recognition of human T-cells

Peracetylated sugars (glucose, mannose, galactose) were attached via a thiourea linker to the e-amino group of α-BOC-lysine and α-Z-lysine. After transformation to the N-carboxyanhydride (NCA), the product was copolymerized with both PEGylated lysine-NCA and e-TFA-lysine-NCA by both tertiary amine and nickel complex catalysts. The resulting statistical copolypeptides are water-soluble after global deprotection and show an α-helical secondary structure. Fluorescein isothiocyanate (FITC) was then coupled to the free e-NH2 groups of the lysine repeating units. The galactosylated fluorescent peptides are specifically incorporated in human T lymphocytes at 37 °C, as shown by flow cytometry and fluorescence microscopy. Therefore they are potentially useful for selective staining of cells or targeted drug delivery.

Synthesis and characterization of Fe 3O 4@SiO 2@poly- l-alanine, peptide brush–magnetic microspheres through NCA chemistry for drug delivery and enrichment of BSA

A novel Fe 3O 4@SiO 2@poly- l-alanine peptide brush–magnetic microsphere (PBMMs) was synthesized from amine-functionalized Fe 3O 4 through the surface-initiated polymerization of N-carboxyanhydrides. Two materials with different peptide lengths were obtained from different amounts of N-carboxyanhydrides. These materials were characterized by Fourier transform infrared, transmission electron microscopy, large-angle powder X-ray diffraction, vibrating sample magnetometer and elemental analysis. Furthermore, the loading and release behavior of ibuprofen and the enrichment of bovine serum albumin on the two materials were investigated and it was shown that the PBMMs have a maximum uptake amount of ibuprofen of 40.3 mg g −1, and an enrichment of bovine serum albumin of 20.9 mg g −1. These materials are promising candidates for targeted drug delivery and protein enrichment.

How controlled and versatile is N-carboxy anhydride (NCA) polymerization at 0 °C? Effect of temperature on homo-, block- and graft (co)polymerization

The polymerization of N-carboxyanhydride (NCA) at low temperatures is controlled and allows the synthesis of a variety of well-defined polypetides.

Peptide block copolymers by N -carboxyanhydride ring-opening polymerization and atom transfer radical polymerization: The effect of amide macroinitiators

The synthesis of polypeptide-containing block copolymers combining N-carboxyanhydride (NCA) ring-opening polymerization and atom transfer radical polymerization (ATRP) was investigated. An amide initiator comprising an amine function for the NCA polymerization and an activated bromide for ATRP was used. Well-defined polypeptide macroinitiators were obtained from γ-benzyl-L-glutamate NCA, O-benzyl-serine NCA, and N-benzyloxy-L-lysine. Subsequent ATRP macroinitiation from the polypeptides resulted in higher than expected molecular weights. Analysis of the reaction products and model reactions confirmed that this is due to the high frequency of termination reactions by disproportionation in the initial phase of the ATRP, which is inherent in the amide initiator structure. In some cases selective precipitation could be applied to remove unreacted macroinitiator to yield well-defined block copolymers. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2009

A Mechanistic Study of α-(Amino acid)-N-carboxyanhydride Polymerization: Comparing Initiation and Termination Events in High-Vacuum and Traditional Polymerization Techniques

High-vacuum polymerization of α-(amino acid)-N-carboxyanhydrides (NCAs) affords polymers with controlled molecular weights and narrow polydispersities; however, a comprehensive study of the end-group composition of the resulting polypeptides has not yet been performed. This reveals crucial information, as the end-groups are indicative of both the polymerization mechanism (i.e., initiation event) and the termination pathways. To this end, poly(O-benzyl-l-tyrosine) initiated by 1,6-diaminohexane was synthesized and subsequently characterized by MALDI-TOF MS, NALDI-TOF MS, and 13C NMR spectroscopy to ascertain the end-group structure. Polymers were prepared by both high-vacuum and glovebox techniques in DMF/THF. Preparation of poly(O-benzyl-l-tyrosine) by high-vacuum techniques yielded a polymer initiated exclusively by the normal amine mechanism, and termination by reaction with DMF was observed. In contrast, polymers prepared in the glovebox were initiated by the normal amine and activated monomer mechanisms, and several termination products are evident. To our knowledge, this is the first rigorous and comparative analysis of the end-group structure, and it demonstrates the advantage of high-vacuum techniques for polymerization of NCAs for the preparation of well-defined polypeptides with end-group fidelity.

Hyperbranched polylysines: Mechanism of formation

To gain insight into the mechanism treating formation of hyperbranched polylysines through the polymerization of N ɛ-carbobenzoxylysine N-carboxyanhydride under conditions of the reductive removal of a N ɛ-carbobenzoxy group, hyperbranched polylysine has been synthesized with the use of trifluoroacetic acid as a terminator in the polymerization of N-carboxyanhydride. The structure of the polymers is studied by capillary electrophoresis, low-pressure gel-permeation chromatography, circular dichroism, and enzymatic hydrolysis with trypsin. At the first stage of synthesis, a low-molecular-mass strongly branched core of the polymer is formed. At the second stage, polylysine chains are grafted via one point onto amino groups of N-terminal lysine moieties of the low-molecular-mass core through their carboxyl ends.

Peptide Brush—Ordered Mesoporous Silica Nanocomposite Materials

Organic−inorganic nanocomposites are synthesized through the surface-initiated polymerization of N-carboxyanhydrides from amine-functionalized ordered mesoporous silica (OMS). Composites with high densities of grafted peptide were obtained which possess dramatically decreased porosity compared to the parent OMS material. The effect of the amine loading, pore size, pore topology, and monomer identity on the peptide brush layer obtained is investigated. Nitrogen porosimetry, thermal gravimetric analysis, elemental analysis, infrared and NMR spectroscopy, and mass spectrometry verify the formation of the peptide brushes and indicate much of the organic phase formed is in the silica mesopores. The l-Lys(Z) peptide brushes can be deprotected on the solid surface as evidenced by thermal gravimetric analysis, infrared spectroscopy, and NMR spectroscopy. l-Ala peptide brushes may also be synthesized, and polymer growth confinement is evidenced by mass spectrometry as the number of alanine units per brush appears ...

Controllable shape selectivity based on highly ordered carbonyl and methyl groups in simple β-structural polypeptide on silica

Poly( l-alanine)-grafted porous silica (Sil-Ala 22) was prepared by polymerization of N-carboxyanhydride of l-alanine initiated by 3-aminopropylated silica. The retention behaviors of the column packed with Sil-Ala 22 were investigated by using alkylbenzenes and polycyclic aromatic hydrocarbons as injection samples in liquid chromatography. The Ala 22 phase was in a rigid β-form structure and thus provided specific interaction sites, which were derived from the highly ordered carbonyl and methyl groups. These interaction sites bring unique molecular-shape discriminations: molecular-length and non-planarity selectivity, which are controllable by altering organic solvent used as a part of mobile phase.

Preparation of Polypeptide via Living Polymerization of Z-Lys-NCA Initiated by Platinum Complexes

Two novel platinum complexes, [bis(diphenylphosphino)ethane][N-((1S,2R)-2-hydroxo-1,2-diphenylethyl)-4-methyl-benzenesulfonamidato]platinum(II) [(dppe)Pt(MBS−O), 2a] and [bis(diphenylphosphino)ethane][N-((1S,2R)-2-amido-1,2-diphenylethyl)-4-methylbenzenesulfonamidato]platinum [(dppe)Pt(MBS−NH), 2b] were synthesized and structurally characterized. Experimental results show that compound 2b is an efficient initiator toward ring opening polymerization of amino acid N-carboxyanhydride (NCA) yielding polypeptides with narrow PDIs (1.07−1.19). By comparing the activity of complex 2a with that of 2b, the mechanism for polymerization of NCA initiated by an amido−sulfonamidate is proposed.

Synthesis of Sugar-Based Polypeptides

Synthetic polypeptides containing sugar moieties can find interesting applications as drug-delivery systems or as hydrogels. Only a few studies report on the successful synthesis of sugar-based polypeptides. The aim of this study is to access a range of well-defined polypeptides carrying linear gluconoyl moieties as side-groups, including homo-polypeptides and co-polypeptides with hydrophilic and hydrophobic sequences. The strategy consisted in first preparing the polypeptide via ring-opening polymerization of N-carboxyanhydrides using either an Al-Schiff's base metal complex or a primary amine. The free amino side-groups of the polylysine units are then reacted with gluconolactone, thus leading to a main polypeptide backbone and linear sugar moieties as pendant groups. Sugar moieties have also been attached to statistical and block co-polypeptides bearing hydrophilic and hydrophobic units such as L-lysine and L-leucine, respectively. The anchoring of linear gluconoyl moieties onto poly(L-lysine) and onto statistical and block co-polypeptides with lysine and leucine sequences is here reported for the first time.

Hexamethyldisilazane-Mediated Controlled Polymerization of α-Amino AcidN-Carboxyanhydrides

Ring-opening polymerizations of α-amino acid N-carboxyanhydrides (NCAs) initiated with amines typically form polypeptides with uncontrolled molecular weights and broad molecular weight distributions. However, we found that hexamethyldisilazane (HMDS)-mediated controlled NCA polymerizations gave polypeptides with predictable molecular weights and narrow molecular weight distributions. Using MS, NMR, and FT-IR, we demonstrated that the initiation step involved the cleavage of the N−Si bond of HMDS and the formation of a trimethylsilyl carbamate (TMS-CBM) terminal group. Polypeptide chains were propagated through the migration of TMS of the TMS-CBM end group to the incoming monomer and formed a new TMS-CBM terminal group. This organosilicon reagent mediated NCA polymerization offers a metal-free strategy for the convenient synthesis of homo- or block polypeptides with predictable molecular weights and narrow molecular weight distributions.

Determination of Dendrigraft Poly-l-Lysine Diffusion Coefficients by Taylor Dispersion Analysis

This work focuses on the physicochemical characterization of dendrigraft poly-L-lysines (DGLs) obtained by polymerization of N-carboxyanhydride in buffered water (pH 6.5). Diffusion coefficients (D) and hydrodynamic radii (Rh) of five successive DGL generations were determined by Taylor dispersion analysis (TDA). To our knowledge, this is the first experimental work using TDA for the characterization of dendrimer-like structures. Experimental Rh values obtained by TDA were compared to those derived from dynamic light scattering and size exclusion chromatography coupled to a triple detection (refractive index, viscosimetry, and static light scattering). Significant differences were obtained, especially for the highest generations, as a result of the inherent contribution of aggregates to the light scattering intensity. For that reason, TDA was found to be the most appropriate technique for determining the D values of these hyperbranched macromolecules. Regarding their physicochemical behavior, the experimental results confirm that DGLs are very similar to trifunctional dendrimers (exponential growth of the molar mass, almost linear variation of the hydrodynamic radius, high branching density, and maximum of the intrinsic viscosity or of the free volume fraction for generation 4).

Novel Amphiphilic Poly(ε-caprolactone)-g-poly(l-lysine) Degradable Copolymers

As part of the search of novel degradable polymers, amphiphilic and cationic poly(epsilon-caprolactone)-g-poly(l-lysine) (PCL-g-PlL) copolymers have been synthesized following a grafting "onto" or a grafting "from" method both applied to a macropolycarbanionic PCL derivative. The first approach led to PCL-g-PZlL containing 36% of epsilon-caprolactone and 64% of N-epsilon-Z-l-lysine units, by reaction of activated poly(N-epsilon-Z-l-lysine) on the macropolycarbanion derived from PCL. The second route was based on the anionic ring opening polymerization of N-carboxyanhydride of N-epsilon-benzyloxycarbonyl-l-lysine initiated by the macropolycarbanion derived from PCL and led to a similar copolymer containing 45% of of epsilon-caprolactone and 55% of N-epsilon-Z-l-lysine units. After deprotection of the lysine units, PCL-g-PlL copolymers were obtained. These copolymers are water-soluble and form nanometric micelle-like objects with mean diameters between 60 and 500 nm in distilled water depending on the synthesis route.

Preparation and Characterization of Polyion Complex Micelles with a Novel Thermosensitive Poly(2-isopropyl-2-oxazoline) Shell via the Complexation of Oppositely Charged Block Ionomers

Novel thermosensitive polyion complex (PIC) micelles were prepared in an aqueous medium based on the complexation of a pair of oppositely charged block ionomers, poly(2-isopropyl-2-oxazoline)-b-poly(amino acid)s (PiPrOx-b-PAA), containing thermosensitive PiPrOx segments. The controlled synthesis of PiPrOx-b-PAA was achieved via the ring-opening anionic polymerization of N-carboxyanhydrides (NCA) of either eta-benzyloxycarbonyl-l-lysine (Lys(Z)-NCA) or beta-benzyl-l-aspartate (BLA-NCA) with omega-amino-functionalized PiPrOx macroinitiators and the subsequent deprotection reaction under acidic or basic conditions. Gel permeation chromatography (GPC) and 1H NMR spectroscopy revealed that the syntheses of two block ionomers, poly(2-isopropyl-2-oxazoline)-b-poly(l-lysine) [PiPrOx-P(Lys)] and poly(2-isopropyl-2-oxazoline)-b-poly(aspartic acid) [PiPrOx-P(Asp)], proceeded almost quantitatively to give samples with a narrow molecular weight distribution (Mw/Mn </= 1.2). The mixing of these two oppositely charged block ionomers in an aqueous medium led to the spontaneous formation of PIC micelles, which was confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The PIC micelles were spherical particles with a narrow distribution in the range of the measured concentration (0.125-1 mg/mL) and were stable without any secondary aggregates. Furthermore, the PIC micelles had a constant cloud-point temperature (Tcp) of approximately 32 degrees C under physiological conditions regardless of the total concentration, suggesting that the concentration factor is almost negligible with respect to the Tcp of the micelles presumably because of the increased local concentration of the PiPrOx segments in the shell layer. These PIC micelles have a promising application as a size-regulated smart nanocontainer loading charged compounds as well as bearing a thermosensitive outer shell that is useful for physical affinity control.

Separation of living and dead polymers in synthetic polypeptide mixtures by nonaqueous capillary electrophoresis using differences in ionization states

The complexity in the mechanisms of polymerization of N-carboxyanhydrides requires the development of new analytical techniques able to separate mixtures of synthetic polypeptides. This work focuses on the separation of poly(N(epsilon)-trifluoroacetyl-L-lysine) (PTLL) mixtures by nonaqueous capillary electrophoresis (CE). The main goal of this work was to find electrophoretic conditions that permit the separation and the quantification of the dead polymer families that were previously identified in the samples. The influence of the pH of the electrolyte on the selectivity of the separation was carefully investigated. The mechanisms of separation of the PTTLs are discussed as a function of their ionization state. The separations obtained on a noncovalently coated capillary were compared with those obtained on a fused-silica capillary. Finally, using two different electrolytes, it is possible to quantify the three families of PTLLs, namely, the living PTLLs, the dead PTLLs with N-formyl end group and the dead PTLLs with a carboxylic end group. These results confirm the importance of CE for the separation of synthetic organic polymers in nonaqueous electrolytes.

Self-assembling of biocompatible BAB amphiphilic triblock copolymers PLL(Z)–PEG–PLL(Z) in aqueous medium

A novel biocompatible BAB amphiphilic triblock copolymers which consist of poly(ethylene glycol) (PEG) and poly( ε-benzyloxycarbonyl l-Lysine) (PLL(Z)) were synthesized by anion ring opening polymerization of N-carboxyanhydride of ε-benzyloxycarbonyl- l-Lysine ( l-Lys(z)-NCA) using α-amino- ω-amino-poly(ethylene glycol) as initiator in DMF. The block copolymers were characterized by IR, 1H-NMR, GPC, DSC. The results showed that the block copolymers were of narrow molecular distribution and well defined structure. The self-assembled behaviors of block copolymers in aqueous medium were investigated. The effects of various factors, such as cosolvents, initial concentration, temperature, annealing time and times of frozen-thaw cycle etc., on the aggregate morphologies were investigated by transmission electron microscopy (TEM). Different morphological aggregates such as sphere, rod and vesicle etc. could be obtained at controlled conditions, at the same time, a novel helical aggregate was observed. These regular nanometer structures have potential applications in biomedicine due to the biocompatibility of two blocks.

Molecular-length and chiral discriminations by β-structural poly( l-alanine) on silica

Poly( l-alanine)-grafted porous silica (Sil-Ala 22) was prepared by polymerization of N-carboxyanhydride of l-alanine initiated by 3-aminopropylated silica. Its selective interaction with aromatic guest molecules was evaluated by the retention time in liquid chromatography using the column packed with Sil-Ala 22. Sil-Ala 22 showed a specific selective retention with discriminating molecular shapes, such as molecular length, linearity and planarity. This selectivity can be explained by a multiple π–π interaction with the carbonyl groups one dimensionally-aligned on the rigid β-form structure in the peptide main chain. Chiral separation with Sil-Ala 22 was also described.

Adsorption immobilization of Candida rugosa lipases on polypropylene hollow fiber microfiltration membranes modified by hydrophobic polypeptides

Poly(γ-ethyl- l-glutamate) (PELG) and poly(γ-stearyl- l-glutamate) (PSLG), two polypeptides with short and long hydrophobic side chains, respectively, were tethered on the polypropylene hollow fiber microfiltration membrane (PPHFMM) surface through the ring opening polymerization of N-carboxyanhydride of γ-ethyl- l-glutamate (γ-stearyl- l-glutamate) initiated by amino groups. Lipases from Candida rugosa were immobilized on these membranes by adsorption. Results on the basis of the enzyme adsorption capacity, activity and thermal stability were compared with those of the nascent PPHFMM. It was found that, as for the modified PPHFMM, the adsorption capacities of lipase are lower than that of the nascent ones, but the activity retention of the immobilized enzymes increases from 57% to 72% and to 62%, respectively for the PSLG-modified and PELG-modified PPHFMM. In addition, the experimental results of thermal stability show that the residual activity of the immobilized lipases at 50 °C for 2 h is respectively 64% for the PELG-modified PPHFMM and 58% for the PSLG-modified PPHFMM, which are higher than that of the nascent ones.