Functional polypeptides obtained by living ring opening polymerizations of N-carboxyanhydrides

N-Carboxyanhydride ring opening polymerization (NCA ROP) is a method to prepare polypeptides with a high degree of polymerization in large quantities. The living polymerization technique of NCA ROP gave the opportunity to synthesize many polymer architectures with well-defined blocks and copolymers with a well-controllable composition. By combining other polymerization techniques, biohybrid polymers have been prepared. Although the polypeptides prepared by NCA ROP have a random amino acid order and a polydispersity, which is uncommon for natural peptides and proteins, they still can be considered as natural polymers and still have some of the features of natural peptides. For example, they can form secondary structures and can be degraded enzymatically. This provides opportunities for biomedical applications such as drug-delivery and hydrogels for the polypeptides and the hybrid polymers prepared by NCA ROP. The goals of this thesis were to study the living ROP of the NCAs and to make use of the versatility of the polymerization technique to obtain polypeptide and hybrid polymer architectures. Finally, the functionality of the polypeptide products was investigated for biomimetic crystallization, self-organization and enzymatic degradation. In the field of NCA ROP there are several methods known for living polymerizations. These can be classified as methods were the mechanism is altered to ensure that no side reactions can occur at the reactive polypeptide chain end and methods in which the reaction conditions are optimized to obtain living polymerizations. A lower temperature and a decreased pressure have both been claimed by separate groups to give the best results. In a systematic study for several different NCA monomers the monomer conversion, molecular weight distribution and chain composition were studied for reactions performed at different temperatures and different pressures. Depending on the monomer species, different side reactions were identified; these were found to be temperature dependent. Monomer conversion studies identified two groups of monomer. The first group of the NCA monomers (¿-benzyl-L-glutamate, Ne-benzyloxycarbonyl-L-lysine and L-alanine) showed fast monomer conversion and responded to the low pressure, showing an increase in the speed of propagation at room temperature. The number of side reactions was low, so the optimal reaction conditions for this group of monomers is under high vacuum and at room temperature. The second group (ß-benzyl-L-aspartate, O-benzyl-Lserine and O-benzyl-L-threonine) showed a lower rate of monomer conversion and no beneficial effect was observed at low pressures. For this second group of monomers, the number of side reactions was also much higher. The best results for a living polymerization of this group of NCAs were obtained at 0 ºC under atmospheric pressure. Using the previously mentioned living ring opening polymerization techniques, different polypeptide architectures have been synthesized. Copolypeptides, graft copolypeptides and block copolypeptides have been synthesized. Although NCA ROP is known as a living polymerization the solubility and the formation of secondary structures can decrease the solubility of the reaction products, resulting in less well-defined block copolypeptides upon macroinitiation. Therefore, the block copolypeptides have been intensively studied to identify the optimal block order synthesis. Improved and quicker reaction conditions were found for tetrablock copolypeptides by combining the optimal solubility and reaction conditions. Biohybrid block and graft copolymers were synthesized by combining radical polymerizations with NCA ROP. Grafted structures were obtained from the free radical chain transfer reaction or thiol-ene reaction with the thiols of poly(¿-benzyl-L-glutamate-co-L-cysteine). Biohybrid block copolymers were obtained by using amine-functionalized bifunctional initiators for atomic transfer radical polymerization (ATRP) and nitroxide mediated radical polymerization (NMRP). The functionality of the copolypeptides was investigated for the biomimetic crystallization of calcium carbonate. Due to the random distribution of amino acids in copolypeptides, this enabled a facile understanding of the function of amino acid species in natural peptides in biomineralization. Fluorescein-labeled copolypeptides of L-glutamic acid, L-aspartic acid and L-alanine were prepared and used in the crystallization of calcium carbonate. The crystal morphology was highly altered by the addition of the independent copolypeptides. An elongated crystal was found for the crystallization in the presence of poly(L-aspartic acid-co-L-alanine) and a crystal with round features was found for the crystallization with poly(L-glutamic acid-co-L-alanine). The fluorescent-labeled polypeptides were incorporated in the crystals. The enzymatic degradation of the polypeptides and biohybrid block copolymers containing L-glutamic acid and L-alanine was also studied. The enzymes elastase and thermolysin were used for this study, since these are known to be selective towards L-alanine-containing peptide bonds. First, biohybrid block copolymers were prepared by using NMRP in combination with NCA ROP. In the hydrophilic polypeptide block the quantity of the L-alanine was altered to direct enzymatic degradation. The hydrophobic block was either polystyrene with a Tg of 100 ºC or poly(n-butylacrylate) with a Tg of -49 ºC. In phosphate buffer solutions these biohybrid block copolymers formed micelles and vesicles. Upon addition of the enzymes, the poly(n-butylacrylate)-containing polymers with a 50% L-alanine content in the hydrophilic block did give an enzymatic response, manifesting itself as an increased particle size and precipitation. For the polystyrene biohybrid block copolymers no response was found for the same polypeptide composition, due to the stability of the high Tg core or membrane material. Block copolypeptides of L-glutamic acid and L-alanine were prepared by living NCA ROP and were found to self-assemble into vesicles in water. A first attempt was made to make vesicles with cellmembrane recognition combined with an enzymatic release trigger for targeted delivery

Block Copolypeptides Prepared by N-Carboxyanhydride Ring-Opening Polymerization

N-Carboxyanhydride ring-opening polymerization (NCA ROP) is a synthetically straightforward methodology to generate homopolypeptides. Extensive control over the polymerization permits the production of highly monodisperse synthetic polypeptides to a targeted molecular weight in the absence of unfavorable side reactions. Sequential NCA ROP permits the creation of block copolypeptides composed of individual polypeptide blocks boasting different functionalities, secondary structures, and desirable chemical properties. Consequently, a plethora of novel materials have been generated that have found wide-range applicability. This review offers an insight into contemporary synthetic approaches toward NCA ROP before highlighting a number of block copolypeptide architectures generate

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 -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

Block Copolypeptides Prepared by N-Carboxyanhydride Ring-Opening Polymerization

N-Carboxyanhydride ring-opening polymerization (NCA ROP) is a synthetically straightforward methodology to generate homopolypeptides. Extensive control over the polymerization permits the production of highly monodisperse synthetic polypeptides to a targeted molecular weight in the absence of unfavorable side reactions. Sequential NCA ROP permits the creation of block copolypeptides composed of individual polypeptide blocks boasting different functionalities, secondary structures, and desirable chemical properties. Consequently, a plethora of novel materials have been generated that have found wide-range applicability. This review offers an insight into contemporary synthetic approaches toward NCA ROP before highlighting a number of block copolypeptide architectures generate

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 ¿-benzyl-L-glutamate (BLG), Ne-benzyloxycarbonyl-L-lysine (ZLL), L-alanine (Ala), ß-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

Thiol chemistry on well-defined synthetic polypeptides

Well-defined cysteine-containing synthetic polypeptides were synthesised and the versatility of various chemical reactions on these thiol groups was investigated

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 ¿-benzyl-L-glutamate (BLG), Ne-benzyloxycarbonyl-L-lysine (ZLL), L-alanine (Ala), ß-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

Hydrolytically stable bioactive synthetic glycopeptide homo- and copolymers by combination of NCA polymerization and click reaction

The synthesis of poly(dl-propargylglycine) and poly(¿-benzyl-l-glutamate-co-dl-propargylglycine) was performed by NCA polymerization at 0 °C to yield well-defined polypeptides with polydispersity indices below 1.3. FTIR results confirm ß-sheet and a-helical conformation of the homopolymer and copolymer, respectively. The subsequent glycosylation was achieved by Huisgen [3 + 2] cylcoaddition ("click" reaction) with azide-functional galactose. FTIR, NMR, SEC, and MALDI-ToF analyses verify the successful glycosylation and suggest a high efficiency of the click reaction. The homoglycopeptide was found to be water-soluble and to form aggregates in water above a critical concentration of 0.079 mg/mL. Selective lectin binding experiments confirmed that the glycopeptides can be used in biorecognotion applications. Moreover, the selective hydrolysis of the benzyl ester groups in the copolymer was achieved without loss of the galactose

Selective enzymatic degradation of self-assembled particles from amphiphilic block copolymers obtained by the combination of N-carboxyanhydride and nitroxide-mediated polymerization

Combining controlled radical polymerizations and a controlled polypeptide synthetic technique, such as N-carboxyanhydride (NCA) ring-opening polymerization, enables the generation of well-defined block copolymers to be easily accessible. Here we combine NCA polymerization with the nitroxide-mediated radical polymerization of poly(n-butyl acrylate) (PBA) and polystyrene (PS), using a TIPNO and SG1-based bifunctional initiator to create a hybrid block copolymer. The polypeptide block consists of (block) copolymers of poly(l-glutamic acid) embedded with various quantities of l-alanine. The formed superstructures (vesicles and micelles) of the block copolymers possessed varying degrees of enzyme responsiveness when exposed to elastase and thermolysin, resulting in controlled enzymatic degradation dictated by the polypeptide composition. The PBA containing block copolymers possessing 50% l-alanine in the polypeptide block showed a high degradation response compared to polymers containing lower l-alanine quantities. The particles stabilized by copolypeptides with l-alanine near the hydrophobic block showed full degradation within 4 days. Particles containing polystyrene blocks revealed no appreciable degradation under the same conditions, highlighting the specificity of the system and the importance of synthetic polymer selection. However, when the degradation temperature was increased to 70 °C, degradation could be achieved due to the higher block copolymer exchange between the particle and the solution. A number of novel biohybrid structures are disclosed that show promise as enzyme-responsive materials with potential use as payload release vehicles, following their controlled degradation by specific, target, enzymes