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Stimuli Responsive NCAs for the Preparation of Novel Biomaterials
Get PDFPolypeptides are naturally occurring polymers that are utilized for a variety of different biological processes including structural support, catalysis, and signaling. Composed of repeating amino acid monomeric units, the structure and function of polypeptides is easily modified by the side chain group of the amino acids. Preparation of biomaterials from a variety of α-amino acids is best accomplished through ring opening polymerization of α-amino acid N- carboxyanhydrides (NCAs). Various initiation systems to prepare polymers via this methodologyare outlined in chapter 1. Although classical preparations include the use of primary amines and strong base systems, the field has been greatly expanded to include transition metals, alcohols, and thiols. Each of the systems provides a unique set of criteria for the resulting polymer, which allows the final function of polypeptide biomaterials to be matched to the optimized initiation system. The use of three distinct initiation systems for the preparation of biomaterials is covered in subsequent chapters.In chapter 2, the preparation of stimuli responsive chemically crosslinked polypeptide biomaterials is outlined. Biologically occurring non-canonical di-α-amino acids were converted into new di-N-carboxyanhydride (di-NCA) monomers in reasonable yields with high purity. Five different di-NCAs were separately copolymerized with tert-butyl-L-glutamate NCA to obtain covalently crosslinked copolypeptides capable of forming hydrogels with varying crosslinker densities. Comparison of hydrogel properties with residue structure revealed that different di-α- amino acids were not equivalent in crosslink formation. Notably, L-cystine was found to produce significantly weaker hydrogels compared to L-homocystine, L-cystathionine, and L-lanthionine, suggesting that L-cystine may be a sub-optimal choice of di-α-amino acid for preparation of copolypeptide networks. The di-α-amino acid crosslinkers also provided different chemical stability, where disulfide crosslinks were readily degraded by reduction, and thioether crosslinks were stable against reduction. This difference in response may provide a means to fine tune the reduction sensitivity of polypeptide biomaterial networks.In chapter 3, an approach to the preparation of poly(dehydroalanine) (ADH) is discussed. Examination of bulky side chain modified α-amino acid N-carboxyanhydrides based off of serine and cysteine is performed, including their ability to undergo fast living polymerization utilizing Co(PMe3)4. The lead candidate, tBu-MA Cys NCA, displayed unique properties similar to that of Mn-MA Cys NCA, which allowed for the preparation of long soluble polymer chains of a variety of architectures. Subsequent modification of poly(S-carbo-tert-butoxymethyl-L-cysteine) under mild conditions with iodomethane leads to selective and near quantitative conversion to ADH. Preliminary studies into the modification of these residues with small molecule nucleophiles are discussed.Finally, in chapter 4, the potential of α-amino acid N-thiocarboxyanhydrides for the preparation of polypeptides via transition metal mediated ring opening polymerization is examined. The preparation of poly(L-methionine) as a precursor to functionalizeable biomaterials from Met NTA is reported. Optimization of the polymerization is explored through systematic variation of polymerization conditions. Furthermore, examination of the polymerization mechanisms through the generation of a thioalloc α-amino acid amide ligand demonstrates that the presence of the carbonyl sulfide byproduct in the polymerization can lead to the formation of nickel carbonyl species, which may lead to poisoning of the initiator. This demonstrates that the end chain active metallocycle species is not stabile during this polymerization. Additional work will need to be performed to optimize the transition metal based polymerization of NTAs
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Poly(dehydroalanine) Based Polypeptides for Stimuli Responsive Biomaterials
Get PDFFunctionalized polypeptides provide access to unique biomaterials that can be varied by the identity of the amino acid side chain; however, access to highly functionalized polypeptides is limited by the routes for sidechain modification. Modification of the polypeptide side chain requires careful post polymerization modification to ensure quantitative functionalization or the synthesis and stringent purification of functionalized monomers. The Deming group recently developed methods for the synthesis of high molecular weight poly(dehydroalanine) (ADH), which possesses reactive electrophilic groups that allows its efficient modification to functionalized polypeptides after reactions with thiol and amine nucleophiles. In this dissertation, I will detail the synthesis and characterization of various biomaterials derived from functionalized ADH polypeptides.Chapter 2 describes the synthesis and characterization of amino acid-functionalized poly(S-alkyl-rac-cysteines) polypeptides. These polypeptides possessed the ability to form coacervates with a variety of anions and ssRNA under physiological conditions. Furthermore, these coacervates could be tuned to respond to environmental stimuli, such as temperature, pH, and redox conditions to trigger coacervation formation and dissolution.Chapter 3 describes the synthesis and characterization of amphiphilic poly(L-methionine sulfoxide)x-b-poly(dehydroalanine)y, di-block copoly-peptides, MOxADHy, and their self-assembly into submicrometer-diameter unilamellar vesicles in aqueous media. The formation of vesicles was observed over an unprecedented range of copolypeptide compositions due to the unique properties and chain conformations of the ADH hydrophobic segments. These copolypeptides incorporate two distinct thiol reactive components where each segment can respond differently to a single thiol stimulus, allowing for in situ inversion of the vesicle assemblies and vesicle disruption via glutathione under intra-cellular mimetic conditions.Chapter 4 details the assembly and characterization of MOxADHy copolypeptides to form membranes that provide plasticity and selective permeability in aqueous mixtures, which allows predictable control of vesicle shape by variation of dialysis conditions. These findings expand upon vesicle shape transformation methods for biodegradable and thiol responsive polypeptide vesicles, which are amenable to development for applications in therapeutic delivery.Chapter 5 describes the synthesis and characterization of racemic mucin analogues bearing native glycans from the functionalization of ADH with un-protected thiol modified monosaccharides. Functionalization of ADH resulted in the formation of glycosylated poly(rac-cysteine) polypeptides, with no unnatural sidechain modification and high saccharide conjugation
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Functionalized Methionine Polypeptides And Their Self Assembly
Get PDFThe synthesis and modification of polypeptides from NCAs is described. Diblocks containing a hydrophilic segment of poly(Met) and a hydrophobic segment of rac-Leu were self-assembled into micelles. The poly(Met) was either oxidized to the sulfoxide or alkylated to the carboxymethylated sulfonium. Additionally, a diblock containing poly(Met) and PEG was synthesized. The poly(Met) segment was alkylated with bromo methyl boronic acid, and the polymers bound covalently and reversibly to compounds that contain diol groups
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Transition Metal-Mediated N-Alkyl Amino Acid N-Carboxyanhydride Polymerization for Synthetic Polypept(o)id-Based Biomaterials
Get PDFPolyethylene glycol (PEG) is a polymer that has been widely utilized in the pharmaceutical industry. PEG has been found to cause health risks in recent years, especially after its incorporation in COVID-19 vaccine formulations. Consequently, there is an impetus to explore other polymers that could serve as safer alternatives to PEG, such as polypeptides and polypeptoids. This dissertation develops and investigates the mechanism of transition metal-mediated polymerization of N-alkyl amino acid N-carboxyanhydrides (NNCAs) to advance the preparation of polypept(o)id-based biomaterials. Chapter 2 demonstrates the potential of polypept(o)ide (polypeptide, polypeptoid, or statistical peptide-peptoid copolymers) based biomaterials. To prepare safe and effective lipid nanoparticle (LNP) carriers that do not contain PEG, synthetic methods were developed for polypept(o)ide-lipids that could substitute PEG-lipids in LNP formulations. These polypept(o)ide-lipids, which made use of poly(L-methionine sulfoxide) as the hydrophilic component, provide the advantages of being biodegradable and nontoxic. Both lipid tail length and polypeptide chain length could be easily adjusted.
Chapter 3 elucidates the initiation requirements for transition metal-mediated NNCA polymerization. Zerovalent Ni and Co initiators were found to be able to initiate living polymerization of sarcosine NNCA. Pro NNCA, a sterically hindered NNCA, can be polymerized with particular metallacycle and ligand combinations. Contrary to what has been previously published, an N-H group on N-carboxyanhydride (NCA) monomers is not required for metallacycle-mediated polymerizations. New divalent metallacycle structures were synthesized and initiated living polymerization of NNCAs and NCAs.
Chapter 4 investigates the propagation mechanism behind transition metal-mediated NNCA polymerization. It was hypothesized that these polymerizations proceed via reversible hydrogen (H) transfer reactions from ?-C-H bonds that NNCAs possess. Transition metal-mediated statistical copolymerizations of NNCAs with isotopically labeled N-D NCAs and NMR experiments on oligomerizations using N-D NCAs were conducted, but H/D transfer was not observed. Weak organic acids enhanced the rate of transition metal-mediated NCA polymerizations, but no H/D transfer was observed when deuterated acid was added. Based on these mechanistic studies, the carbon bound to the metal in these metallacyclic initiators is not protonated off and chain propagation more likely proceeds through a different process
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Diblock Polypeptide Hydrogel Synthesis and Biomedical Applications in Central Nervous System
Get PDFPeptides and proteins are present throughout the living world, where nature's ability to produce these macromolecules with precise geometry, length, and sequence specificity results in biomaterials that can perform tasks as varied as catalyzing complex biochemical reactions and serving as the structural framework of many living organisms. These unique features make the preparation of synthetic peptides and polypeptides an area with the potential for great rewards. Well-defined block copolypeptides, developed in our lab at UCLA, are novel synthetic biomaterials, whose defined conformations and multitude of possible block compositions allow for the precise design of polymers which can self-assemble into complex supramolecular structures such as vesicles, micelles or hydrogels.Amphiphilic diblock copolypeptide hydrogels (DCHs) are synthetic polypeptide based materials with many features that make them attractive as scaffolds and depots for central nervous system (CNS) applications. We have developed DCH as depots that can be safely and easily injected into specific sites in CNS tissues to deliver potentially therapeutic molecules. My current work showed that DCH depots could provide prolonged release of bioactive growth factors that influence local neurons in predictable ways and form gradients that are effective up to 5 mm away from depots in mouse CNS. We also demonstrated the facile and predictable tunability of DCH to achieve a wide range of loading capacities and release profiles of hydrophobic molecules while retaining CNS compatible physical properties.Recently, I have developed modifications to our DCH that make them non-ionic and thermoresponsive. These new DCH are viscous liquids at room temperature but quickly form stiff hydrogels when warmed to 37 °C and provide the possibility to protect, support and regulate the differentiation of neural stem cells (NSC) that can be grafted into sites of CNS injury, stroke or degenerative disease. Together with our colleagues in Dr. Sofroniew's lab from the UCLA Neuroscience department, we showed that NSC grafted in growth factor loaded DCH have better survivability, controlled differentiation and greater integration with the host tissues
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Introducing Multifunctionality into Polypeptide Vesicles for Biomedical Applications
Get PDFThe delivery of naked drugs, DNA, RNA and proteins within living organisms is a challenging endeavor where renal clearance, liver accumulation, solubility issues, enzymatic and proteolytic degradation may reduce the effectiveness of the drug. Researchers are developing drug carriers such as liposomes, micelles, emulsions and vesicles to overcome these obstacles. Such carriers are used to encapsulate drugs and protect them from degradation, and more importantly to protect the patient from toxic side effects. Polypeptide vesicles are of interest because they are made up of long chains of amino acids and may be advantageous for in vivo applications since they can degrade to non-toxic metabolites. Natural and unnatural amino acids can be used as building blocks allowing a variety of functionality and tuning of physical properties. Polypeptides are also advantageous in that they can form secondary structures (i.e., alpha-helices, beta-sheets) stabilized by hydrogen bonding, which help to direct their self-assembly. Our group had developed polypeptide vesicles containing polyarginine hydrophilic segments of the general structure: poly(L-arginine)60-block-poly(L-leucine)20, R60L20. The R60L20 vesicles were able to encapsulate Texas Red labeled dextran and were taken up by T84, HeLa, and HULEC-5A cell lines, indicating that polyarginine segments are useful for intracellular delivery. While these polypeptide vesicles (R60L20) have shown promise for intracellular delivery there are issues that remain to be addressed, such as cytotoxicity and cargo release. In my research, I have focused on addressing these issues by optimizing the hydrophobic segment and introducing multifunctionality into polypeptide vesicles, creating improved drug delivery vehicle candidates. In order to optimize vesicle self-assembly and the ability to obtain diameters in the nanoscale range, the hydrophobic domain length and composition was varied. Fine-tuning the length of the poly(L-leucine) domain to 20 residues led to stable vesicular assemblies that had reduced cytotoxicity. To reduce the rigidity of the vesicle membrane a statistical copolypeptide was incorporated in the hydrophobic domain to disrupt the crystallinity of the poly(L-leucine)20. The incorporation of L-alanine and L-phenylalanine residues allowed vesicle diameters to be manipulated below 200 nanometers with a 1 to 1 ratio of L-leucine to L-phenylalanine resulting in narrow polydispersities. Replacing the cationically charged hydrophilic domains with neutral segments led to reduced cytotoxicity of block copolypeptide vesicles. It was found that incorporating neutrally charged segments, containing disordered chain conformations, provides the optimal conditions for obtaining minimally toxic vesicles with the ability be extruded to sizes below 200 nanometers in diameter. Glycosylated block copolypeptides not only provided a neutral non-toxic vesicle suspension, but also provide a method for incorporating biofunctionality, with the ability to bind to lectins. Recent advances in the purification of alpha-amino acid N-carboxyanhydrides (NCAs) led to the use of L-methionine NCA, which has not been polymerized incorporated into block copolypeptides before. The unique sulfur chemistry of methionine provided a quick alternative to introducing new functionalities into polypeptide vesicles. Oxidation of poly(L-methionine) segments provided polypeptide vesicles with the ability to release its cargo within an environment containing either reducing chemicals or reductase enzymes found in human, animal and plant cells
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Functional Polypeptides for Biomedical Applications
Get PDFSynthetic polypeptides have shown great promise as materials for biotechnology and medicine, with applications in tissue engineering, drug delivery, and as therapeutics. Despite significant advances in the preparation of well-defined polypeptides and the development of self-assembling materials, a need remains for a broader scope of polypeptides with functionalities that mimic the complexity and function of post-translationally modified natural proteins. The display of functionalities that have therapeutic effects, target materials to specific tissues, passivate the immune response, or have stimuli responsive behavior, are highly desirable yet generally require complex and inefficient synthetic approaches. This dissertation reports several distinct advances in the preparation of a wide variety of highly functional polypeptide materials. Progress in both the polymerization of functionalized NCA monomers and the post-polymerization modification of polypeptides is described. A new method of NCA purification was developed, and has allowed access to diverse monomers with functionalities previously unattainable due to impurities that impeded polymerization. This purification technique was applied to the synthesis of glycosylated NCAs, which yielded the first living polymerizations of glycosylated NCAs and gave access to glycopolypeptides with unique properties and conformations. The display of sugar functionalities from synthetic polymers is an area of great interest due to the many attractive properties imparted upon the parent material, such as non-ionic water solubility, biological targeting, and shielding of the polypeptide from proteases. These glycopolypeptides were used to explore the effect of conformation on self-assembly and ligand binding, and to develop vesicles with potential medical applications in targeted drug delivery. In addition, a new conjugation technique for facile post-polymerization modification of polypeptides is described. This conjugation utilizes the unique chemistry of the natural amino acid methionine to allow chemoselective introduction of a wide variety of functional groups via alkylation. We investigated the stability of various alkylated methionines to thiolysis, and developed a simple method to attach and later remove different groups. Finally, work performed during an NSF-IGERT funded internship at HRL Laboratories Inc. is described. Surface modification of carbon foam anodes for microbial fuel cell applications was explored, and the use of thermogravimetric analysis as technique to evaluate biofilm formation was developed
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Modification of poly(L-Homoallylglycine) for the Synthesis of Functional Polypeptides
Get PDFSynthetic polypeptides have demonstrated great potential for a variety of biomaterial applications due to their ability to self-assemble into ordered structures. The ability to design polypeptides with a variety of functionalities is necessary for tailoring the solubility, conformational properties, and environmental responses of polypeptide-based materials. Post polymerization modification of reactive residues has emerged as a desirable method for the design of functional polypeptides due to straightforward monomer synthesis and purification and the potential to synthesize a variety of functional polypeptides from a single reactive polypeptide precursor. This dissertation describes the development and subsequent modification of poly(L-homoallylglycine), a soluble, α-helical alkene bearing polypeptide. L-Homoallylglycine N-carboxyanhydride monomers were synthesized and used to prepare poly(L-homoallylglycine) polypeptides with controllable lengths of up to 245 residues. These polypeptides were modified under mild conditions via UV initiated thiol-ene chemistry to give a variety of α-helical water soluble thioether containing polypeptides. These derivativeswere able to undergo a conformational change from α-helix to random coil upon thioether oxidation or alkylation. Incorporation of L-homoallylglycine residues into block copolypeptides with L-methionine residues allowed for the synthesis of block copolypeptides with separate ordered segments of sulfoxide residues and sulfonium residues. The thiol-ene reactivity of poly(L-homoallylglycine) was then utilized to synthesize polypeptides containing N-methylaminooxy functionality. The solubility, conformation, and reactivity of these poly(L-Homoallylglycine) derived polypeptides was compared to more hydrophilic N-methylaminooxy polypeptides synthesized via a functional monomer approach. The unique reactivity of N-methylaminooxy groups with non-protected reducing sugars facilitated the straightforward synthesis of glycopolypeptides which possessed good aqueous solubility and were stable at pH 7.4 for one week. This post polymerization modification technique shows promise as a potential strategy for the synthesis of proteoglycan mimics. The thiol-ene chemistry of poly(L-homoallylglycine) was proven to be highly versatile, but the resulting thiol-ene conjugates often have limited aqueous solubility due to their long hydrophobic side chains. Therefore, poly(L-homoallylglycine) was oxidized to an epoxide bearing polypeptide, poly(5,6-epoxy-L-norleucine) and modified with thiols under basic conditions to synthesize a variety of β-hydroxy thioether containing polypeptides with considerably higher aqueous solubility than previously synthesized thiol-ene conjugates. Diethylene glycol thiol modified poly(5,6-epoxy-L-norleucine) derivatives displayed lower critical solution temperature properties in water that could be modulated by varying polypeptide concentration, polypeptide composition, and could be switched off through oxidation of their thioether groups
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Synthesis of Biomimetic Phosphorus-Containing Polypeptides
Get PDFImmune response has historically been a major issue in the drug delivery field. In order to combat this problem, drug carriers have been designed to avoid the body's innate defenses. Following decades of research, it was found that an ideal drug delivery vehicle should enter the body, evade the immune system, stay in circulation long enough to seek out affected cells, and release the payload selectively. Furthermore, the vehicle should be comprised of materials that are non-toxic and biodegradable. While a number of potentially viable materials have been produced, there has arguably not been one which encompasses all these properties. Using phosphocholine-containing vesicles or micelles as drug delivery vehicles is one potential solution. Phospholipids, the major components of biological membranes, have also been explored for drug delivery purposes; however, their use has not been successful due to the low stability of phospholipid vesicles. Thus, biomimetic polymers with pendant phosphocholine groups were used to increase drug delivery vehicle stability. Much like other polymers, polypeptides may serve as a superior alternative due to the biodegradability of their backbone. Polypeptide synthesis has been thoroughly explored in the previous decades; however no reliable method for the preparation of phosphorus- and phosphatidylcholine-containing polypeptides has been published by the inception of this work. Herein we discuss methods for preparation of phosphonate and phosphatidylcholine containing polypeptides. Synthesis of these materials builds on robust methods often employed in solution phase DNA synthesis. Protecting group strategies as well as the failures leading up to the polymers' successful synthesis are described. The polymers are synthesized via Co(PMe3)4 initiated polymerization of NCAs and are incorporated into diblock copolypeptides. The resultant products display interesting solution and calcium-binging properties
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Diblock Copolypeptide Hydrogels for Drug Delivery and Stem Cell Grafting
Get PDFGrafting of stem cells into the central nervous system (CNS) for regeneration of damaged or degrading neural tissue has shown promise. Unfortunately, this approach is hampered by poor grafted cell survivability, uncontrollable differentiation, and limited integration with host tissue. Many of these problems have been eliminated by using a cell delivery vehicle, such as a hydrogel, which mimics extra cellular matrix (ECM). ECM is densely populated with proteins and proteoglycans that provide a scaffold to support cellular interactions and has therefore driven many researchers to develop novel protein and polysaccharide based hydrogels to replicate this environment. This dissertation encompasses the synthesis and characterization of novel L-methionine (Met) based diblock copolypeptide hydrogels (DCH) for use in drug delivery and stem cell grafting in the central nervous system (CNS). Met is a naturally occurring amino acid that has rich biochemistry and can be either alkylated or oxidized to form cationic sulfonium or non-ionic sulfoxide functionalities, respectively. We took advantage of Met reactivity to synthesize a library of chemically diverse DCH using ring opening polymerizations of N-carboxyanhydrides (NCA) monomers. These DCH were found to have tunable physical properties and underwent sheer-thinning when large amounts of strain were applied, which is an important feature for non-invasive injectable hydrogels. This DCH library consisted of cationic Met sulfonium based hydrogels (DCHMM) and non-ionic Met sulfoxide based hydrogels (DCHMO). In vitro encapsulation of neural stem/progenitor cells (NSPC) within DCHMO gave comparable cell viability to culture media alone, and cell culture studies show minimal cell attachment to these scaffolds, which preserved NSPC stemness and multipotency compared to other materials. NSPC in DCHMO injected into uninjured forebrain remained localized to the grafted deposit and, after 4 weeks, exhibited an immature astroglial phenotype that integrated with host neural tissue and acted as cellular substrates that supported growth of host-derived axons. ECM is filled with complex proteoglycans which are involved in many biological functions and provide structural and physical cellular support, all of which are desirable properties for regenerative medicine biomaterials. By utilizing polypeptides possessing N-methylaminooxy side-chain functionality, the direct functionalization of reducing saccharides to give neoglycopolypeptides was accomplished in high yields. Different side chain functionalities generated tunable chain conformation, hydrophobicity, and charge. These polypeptides were found to be stable at pH 7 for 1 week. This approach will prove useful for easy conjugation of complex saccharides and will further advance the function of future hydrogels toward regenerative medicine applications
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