“One-Pot” Aminolysis/Thiol–Maleimide End-Group Functionalization of RAFT Polymers: Identifying and Preventing Michael Addition Side Reactions

We show that many of the nucleophiles (catalysts, reducing agents, amines, thiols) present during “one-pot” aminolysis/thiol–maleimide end-group functionalization of RAFT polymers can promote side reactions that substantially reduce polymer end-group functionalization efficiencies. The nucleophilic catalyst 1,8-diazabicyclo[5.4.0]­undec-7-ene and the reducing agent tributyl­phosphine were shown to initiate anionic polymerization of <i>N</i>-methyl­maleimide (NMM) in both polar and nonpolar solvents whereas hexylamine-initiated polymerization of NMM occurred only in high-polarity solvents. Furthermore, triethylamine-catalyzed Michael reactions of the representative thiol ethyl 2-mercapto­propionate (E2MP) and NMM in polar solvents resulted in anionic maleimide polymerization when [NMM]₀ > [E2MP]₀. Base-catalyzed enolate formation on the α-carbon of thiol–maleimide adducts was also shown as an alternative initiation pathway for maleimide polymerization in polar solvents. Ultimately, optimal “one-pot” reaction conditions were identified allowing for up to 99% maleimide end-group functionalization of dithiobenzoate-terminated poly­(<i>N</i>,<i>N</i>-dimethyl­acrylamide). Much of the work described herein can also be used to ensure near-quantitative conversion of small molecule thiol–maleimide reactions while preventing previously unforeseen side reactions

Mechanistic Insights into Temperature-Dependent Trithiocarbonate Chain-End Degradation during the RAFT Polymerization of <i>N</i>‑Arylmethacrylamides

Mechanistic insights into trithiocarbonate degradation during the RAFT polymerization of <i>N</i>-aryl­methacryl­amides are reported. Previous work by our group showed significant RAFT agent degradation during the polymerization of <i>N</i>-arylmethacryloyl sulfonamides at 70 °C. Herein we report the influence of methacrylamide structure on trithiocarbonate degradation during the RAFT polymerizations of <i>N</i>-phenyl­methacryl­amide (PhMA) and <i>N</i>-benzyl­methacrylamide (BnMA) in DMF at 70 and 30 °C. UV–vis spectroscopy revealed trithiocarbonate degradation occurs exclusively after covalent addition of monomer to the RAFT agent, with 60% trithiocarbonate degradation occurring after 12 h during the polymerization of PhMA at 70 °C compared to only 3% degradation measured during the polymerization of BnMA under identical conditions. Small molecule analogues of trithiocarbonate-functional poly­(PhMA) and poly­(BnMA) were synthesized by single monomer unit insertion and the kinetics and byproducts of degradation investigated by <i>in situ</i> ¹H NMR analysis at 70 °C. Trithiocarbonate degradation was ultimately shown to occur by <i>N</i>-phenyl-promoted, N-5 nucleophilic attack on the terminal thiocarbonyl by the ultimate methacrylamide unit

Synthesis of Polyisobutylene Bottlebrush Polymers via Ring-Opening Metathesis Polymerization

Polyisobutylene (PIB)-based bottlebrush polymers were synthesized via ring-opening metathesis polymerization (ROMP) of norbornene- and oxanorbornene-terminated PIB macromonomers (MM) initiated by Grubbs third-generation catalyst ((H₂IMes₂)­(pyr)₂(Cl)₂RuCHPh) (G3). While both MMs reached greater than 97% conversion as measured by ¹H NMR, the rate of propagation of PIB norbornene was measured to be 2.9 times greater than that of PIB oxanorbornene MMs of similar molecular weight (MW). The slower rate of propagation of the oxanorbornene MM was attributed to interaction between the electron-rich oxygen bridge and the metal center of G3, which slowed but did not inhibit polymerization. Both types of MMs demonstrated controlled/“living” polymerization behavior, and brush polymers with MWs up to ∼700 kg/mol with narrow dispersity (<i>Đ</i> ≤ 1.04) were achieved

Guanidinium-Functionalized Interpolyelectrolyte Complexes Enabling RNAi in Resistant Insect Pests

RNAi-based technologies are ideal for pest control as they can provide species specificity and spare nontarget organisms. However, in some pests biological barriers prevent use of RNAi, and therefore broad application. In this study we tested the ability of a synthetic cationic polymer, poly-[<i>N</i>-(3-guanidinopropyl)­methacrylamide] (pGPMA), that mimics arginine-rich cell penetrating peptides to trigger RNAi in an insensitive animalSpodoptera frugiperda. Polymer–dsRNA interpolyelectrolyte complexes (IPECs) were found to be efficiently taken up by cells, and to drive highly efficient gene knockdown. These IPECs could also trigger target gene knockdown and moderate larval mortality when fed to <i>S. frugiperda</i> larvae. This effect was sequence specific, which is consistent with the low toxicity we found to be associated with this polymer. A method for oral delivery of dsRNA is critical to development of RNAi-based insecticides. Thus, this technology has the potential to make RNAi-based pest control useful for targeting numerous species and facilitate use of RNAi in pest management practices

RAFT Polymerization of “Splitters” and “Cryptos”: Exploiting Azole‑<i>N</i>‑carboxamides As Blocked Isocyanates for Ambient Temperature Postpolymerization Modification

A postpolymerization modification strategy based on ambient temperature nucleophilic chemical deblocking of polymer scaffolds bearing <i>N</i>-heterocycle-blocked isocyanate moieties is reported. Room temperature RAFT polymerization of three azole-<i>N</i>-carboxamide methacrylates, including 3,5-dimethylpyrazole, imidazole, and 1,2,4-triazole derivatives, afforded reactive polymer scaffolds with well-defined molecular weights and narrow dispersities (<i><i>Đ</i></i> < 1.2). Model analogues possessing the same <i>N</i>-heterocycle blocking agents with varied leaving group abilities were synthesized to determine optimal deblocking conditions. The reactivity of the azole-<i>N</i>-carboxamide moieties toward nucleophiles can be tuned simply by varying the structure of the azole blocking agents (reactivity order: pyrazole < imidazole < triazole). DBU-catalyzed reactions of thiols with imidazole- and 1,2,4-triazole-blocked isocyanate scaffolds were shown to occur rapidly and quantitatively under ambient conditions. Differences in reactivity of 1,2,4-triazole- and 3,5-dimethylpyrazole-blocked isocyanate copolymers with various nucleophiles at room temperature facilitated sequential and postpolymerization modification. This strategy advances the utility of blocked isocyanates and promotes the chemistry as a powerful postmodification tool to access multifunctional polymeric materials

Endolytic, pH-Responsive HPMA‑<i>b</i>‑(l‑Glu) Copolymers Synthesized via Sequential Aqueous RAFT and Ring-Opening Polymerizations

A facile synthetic pathway for preparing block copolymers with pH-responsive l-glutamic acid segments for membrane disruption is reported. Aqueous reversible addition–fragmentation chain transfer (<i>a</i>RAFT) polymerization was first used to prepare biocompatible, nonimmunogenic poly­[<i>N</i>-(2-hydroxypropyl)­methacrylamide]. This macro chain transfer agent (CTA) was then converted into a macroinitiator via simultaneous aminolysis and thiol–ene Michael addition using the primary amine substituted <i>N</i>-(3-aminopropyl)­methacrylamide. This macroinitiator was subsequently utilized in the ring-opening polymerization of the <i>N</i>-carboxyanhydride monomer of γ-benzyl-l-glutamate. After deprotection, the pH-dependent coil-to-helix transformations of the resulting HPMA-<i>b</i>-(l-Glu) copolymers were monitored via circular dichroism spectroscopy. HPMA segments confer water solubility and biocompatibility while the l-glutamic acid repeats provide reversible coil-to-helix transitions at endosomal pH values (∼5–6). The endolytic properties of these novel [HPMA-<i>b</i>-(l-Glu)] copolymers and their potential as modular components in drug carrier constructs was demonstrated utilizing red blood cell hemolysis and fluorescein release from POPC vesicles

Guanidine-Containing Methacrylamide (Co)polymers via <i>a</i>RAFT: Toward a Cell-Penetrating Peptide Mimic

We report the synthesis and controlled radical homopolymerization and block copolymerization of 3-guanidinopropyl methacrylamide (GPMA) utilizing aqueous reversible addition–fragmentation chain transfer (<i>a</i>RAFT) polymerization. The resulting homopolymer and block copolymer with <i>N</i>-(2-hydroxypropyl) methacrylamide (HPMA) were prepared to mimic the behavior of cell-penetrating peptides (CPPs) and poly­(arginine) (>6 units), which have been shown to cross cell membranes. The homopolymerization mediated by 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl)­pentanoic acid (CEP) in aqueous buffer exhibited pseudo-first-order kinetics and linear growth of molecular weight with conversion. Retention of the “living” thiocarbonylthio ω-end group was demonstrated through successful chain extension of the GPMA macroCTA yielding GPMA₃₇-<i>b</i>-GPMA₆₁ (<i>M</i>_w/<i>M</i>_n = 1.05). Block copolymers of GPMA with the nonimmunogenic, biocompatible HPMA were synthesized yielding HPMA₂₇₁-<i>b</i>-GPMA₁₃ (<i>M</i>_w/<i>M</i>_n = 1.15). Notably, intracellular uptake was confirmed by fluorescence microscopy, confocal laser scanning microscopy, and flow cytometry experiments after incubation for 2.5 h with KB cells at 4 °C and at 37 °C utilizing FITC-labeled, GPMA-containing copolymers. The observed facility of cellular uptake and the structural control afforded by <i>a</i>RAFT polymerization suggest significant potential for these synthetic (co)­polymers as drug delivery vehicles in targeted therapies

Aqueous RAFT Synthesis of Glycopolymers for Determination of Saccharide Structure and Concentration Effects on Amyloid β Aggregation

GM1 ganglioside is known to promote amyloid-β (Aβ) peptide aggregation in Alzheimer’s disease. The roles of the individual saccharides and their distribution in this process are not understood. Acrylamide-based glycomonomers with either β-d-glucose or β-d-galactose pendant groups were synthesized to mimic the stereochemistry of saccharides present in GM1 and characterized via ¹H NMR and electrospray ionization mass spectrometry. Glycopolymers of different molecular weights were synthesized by aqueous reversible addition–fragmentation chain transfer (aRAFT) polymerization and characterized by NMR and GPC. The polymers were used as models to investigate the effects of molecular weight and saccharide unit type on Aβ aggregation via thioflavin-T fluorescence and PAGE. High molecular weight (∼350 DP) glucose-containing glycopolymers had a profound effect on Aβ aggregation, promoting formation of soluble oligomers of Aβ and limiting fibril production, while the other glycopolymers and negative control had little effect on the Aβ propagation process

Antimicrobial Peptide Mimicking Primary Amine and Guanidine Containing Methacrylamide Copolymers Prepared by Raft Polymerization

Naturally occurring antimicrobial peptides (AMPs) display the ability to eliminate a wide variety of bacteria, without toxicity to the host eukaryotic cells. Synthetic polymers containing moieties mimicking lysine and arginine components found in AMPs have been reported to show effectiveness against specific bacteria, with the mechanism of activity purported to depend on the nature of the amino acid mimic. In an attempt to incorporate the antimicrobial activity of both amino acids into a single water-soluble copolymer, a series of copolymers containing lysine mimicking aminopropyl methacrylamide (APMA) and arginine mimicking guanadinopropyl methacrylamide (GPMA) were prepared via aqueous RAFT polymerization. Copolymers were prepared with varying ratios of the comonomers, with degree of polymerization of 35–40 and narrow molecular weight distribution to simulate naturally occurring AMPs. Antimicrobial activity was determined against Gram-negative and Gram-positive bacteria under conditions with varying salt concentration. Toxicity to mammalian cells was assessed by hemolysis of red blood cells and MTT assays of MCF-7 cells. Antimicrobial activity was observed for APMA homopolymer and copolymers with low concentrations of GPMA against all bacteria tested, with low toxicity toward mammalian cells