Self-promoted Controlled Ring-opening Polymerization via Side Chain-mediated Proton Transfer for the Synthesis of Tertiary Amine-pendant Polypeptoids.

Proton transfer is essential in virtually all biochemical processes, with enzymes facilitating this transfer by optimizing the proximity and orientation of reactants through site-specific hydrogen bonds. Proton transfer is also crucial in the rate-determining step for the ring-opening polymerization of N-carboxyanhydrides (NCAs), widely used to prepare various peptidomimetic materials. This study utilizes side chain-assisted strategy to accelerate the rate of chain propagation by using NCAs with tertiary amine pendants. This moiety enables hydrogen bond formation between the incoming NCA and the polymer amino growing end. The tertiary amine side chain of the NCA forms a proton shuttle, via a less constrained transition state, to facilitate the proton transfer process. Moreover, the tertiary amine side chains enable the precipitation of NCA monomers through in situ protonation during the monomer synthesis. This greatly facilitates the synthesis of these unreported monomers, allowing the direct controlled synthesis of tertiary amine-pendant polypeptoids. This side chain-promoted polymerization has rarely been reported. Additionally, the tertiary amine side chains, as widely used functional groups, endow the polymers with unique properties including pH- and thermo-responsiveness, tunable pKas, and siRNA transfection capability. The self-promoted synthesis, facile monomer preparation, and attractive properties make tertiary amine-pendant polypeptoids promising materials for various applications.

Research Progress in Polypeptoids Prepared by Controlled Ring-Opening Polymerizations.

Polypeptoids, structural mimics of polypeptides, have attracted considerable attention due to their biocompatibility, proteolytic stability, thermal processability, good solubility, synthetic accessibility, and structural diversity. Polypeptoids have emerged as an interesting material in both polymer science and biological field. This review primarily discusses the research progress of polypeptoids prepared by controlled ring-opening polymerizations in the past decade, including synthetic strategies of monomers, polymerizations by different initiators, postfunctionalization, fundamental properties, crystallization-driven self-assembly, and potential biological applications.

DNA origami protection and molecular interfacing through engineered sequence-defined peptoids.

DNA nanotechnology has established approaches for designing programmable and precisely controlled nanoscale architectures through specific Watson-Crick base-pairing, molecular plasticity, and intermolecular connectivity. In particular, superior control over DNA origami structures could be beneficial for biomedical applications, including biosensing, in vivo imaging, and drug and gene delivery. However, protecting DNA origami structures in complex biological fluids while preserving their structural characteristics remains a major challenge for enabling these applications. Here, we developed a class of structurally well-defined peptoids to protect DNA origamis in ionic and bioactive conditions and systematically explored the effects of peptoid architecture and sequence dependency on DNA origami stability. The applicability of this approach for drug delivery, bioimaging, and cell targeting was also demonstrated. A series of peptoids (PE1-9) with two types of architectures, termed as "brush" and "block," were built from positively charged monomers and neutral oligo-ethyleneoxy monomers, where certain designs were found to greatly enhance the stability of DNA origami. Through experimental and molecular dynamics studies, we demonstrated the role of sequence-dependent electrostatic interactions of peptoids with the DNA backbone. We showed that octahedral DNA origamis coated with peptoid (PE2) can be used as carriers for anticancer drug and protein, where the peptoid modulated the rate of drug release and prolonged protein stability against proteolytic hydrolysis. Finally, we synthesized two alkyne-modified peptoids (PE8 and PE9), conjugated with fluorophore and antibody, to make stable DNA origamis with imaging and cell-targeting capabilities. Our results demonstrate an approach toward functional and physiologically stable DNA origami for biomedical applications.

Compact Peptoid Molecular Brushes for Nanoparticle Stabilization.

Controlling the interfaces and interactions of colloidal nanoparticles (NPs) via tethered molecular moieties is crucial for NP applications in engineered nanomaterials, optics, catalysis, and nanomedicine. Despite a broad range of molecular types explored, there is a need for a flexible approach to rationally vary the chemistry and structure of these interfacial molecules for controlling NP stability in diverse environments, while maintaining a small size of the NP molecular shell. Here, we demonstrate that low-molecular-weight, bifunctional comb-shaped, and sequence-defined peptoids can effectively stabilize gold NPs (AuNPs). The generality of this robust functionalization strategy was also demonstrated by coating of silver, platinum, and iron oxide NPs with designed peptoids. Each peptoid (PE) is designed with varied arrangements of a multivalent AuNP-binding domain and a solvation domain consisting of oligo-ethylene glycol (EG) branches. Among designs, a peptoid (PE5) with a diblock structure is demonstrated to provide a superior nanocolloidal stability in diverse aqueous solutions while forming a compact shell (∼1.5 nm) on the AuNP surface. We demonstrate by experiments and molecular dynamics simulations that PE5-coated AuNPs (PE5/AuNPs) are stable in select organic solvents owing to the strong PE5 (amine)-Au binding and solubility of the oligo-EG motifs. At the vapor-aqueous interface, we show that PE5/AuNPs remain stable and can self-assemble into ordered 2D lattices. The NP films exhibit strong near-field plasmonic coupling when transferred to solid substrates.

Atomic-level engineering and imaging of polypeptoid crystal lattices.

Rational design of supramolecular nanomaterials fundamentally depends upon an atomic-level understanding of their structure and how it responds to chemical modifications. Here we studied a series of crystalline diblock copolypeptoids by a combination of sequence-controlled synthesis, cryogenic transmission electron microscopy, and molecular dynamics simulation. This family of amphiphilic polypeptoids formed free-floating 2-dimensional monolayer nanosheets, in which individual polymer chains and their relative orientations could be directly observed. Furthermore, bromine atom side-chain substituents in nanosheets were directly visualized by cryogenic transmission electron microscopy, revealing atomic details in position space inaccessible by conventional scattering techniques. While the polypeptoid backbone conformation was conserved across the set of molecules, the nanosheets exhibited different lattice packing geometries dependent on the aromatic side chain para substitutions. Peptoids are inherently achiral, yet we showed that sequences containing an asymmetric aromatic substitution pattern pack with alternating rows adopting opposite backbone chiralities. These atomic-level insights into peptoid nanosheet crystal structure provide guidance for the future design of bioinspired nanomaterials with more precisely controlled structures and properties.

2,5-Dimethylfuran/Acrylonitrile as Latent Monomer for Sequence-Controlled Copolymer and Sequence-Dependent Thermo-Responsivity.

Sequence control has attracted increasing attention for its ability of regulating polymer property and performance. Herein, the sequence-controlled polymer containing acrylonitrile (AN) is achieved by using 2,5-dimethylfuran/acrylonitrile adduct as a latent monomer. The temperature-dependent retro Diels-Alder reaction is engaged in controlling the release of AN during RAFT polymerization, that is, regulating the instant AN concentration via a non-invasive and in situ manner. Such control over the instant AN concentration and particularly the molar ratio of comonomer pair leads to the simultaneous change of monomer units in "living" polymeric chain, thus resulting in the sequence-controlled polymeric structures. By delicately manipulating the polymerization temperature, diverse sequence-on-demand structures of AN-containing copolymers, such as poly(AN/methyl methacrylate), poly(AN/styrene), poly(AN/butyl acrylate), poly(AN/N,N-dimethylacrylamide), and poly(AN/N-isopropylacrylamide) are created. Meanwhile, this study presents an initial attempt in tuning the thermal responsivity of poly(AN/N-isopropylacrylamide), which is closely correlated to the sequence of polymer structure. More importantly, the polymer with averagely distributed AN units results in the higher thermal sensitivity. Therefore, the synthetic strategy proposed in this work offers a promising platform for accessing the sequence-controlled copolymers containing AN structures, thus expanding the investigation on the relationship between the polymer structures and correlated properties.

Minimizing Crinkling of Soft Specimens Using Holey Gold Films on Molybdenum Grids for Cryogenic Electron Microscopy.

We introduce a novel composite holey gold support that prevents cryo-crinkling and reduces beam-induced motion of soft specimens, building on the previously introduced all-gold support. The composite holey gold support for high-resolution cryogenic electron microscopy of soft crystalline membranes was fabricated in two steps. In the first step, a holey gold film was transferred on top of a molybdenum grid. In the second step, a continuous thin carbon film was transferred onto the holey gold film. This support (Au/Mo grid) was used to image crystalline synthetic polymer membranes. The low thermal expansion of Mo is not only expected to avoid cryo-crinkling of the membrane when the grids are cooled to cryogenic temperatures, but it may also act to reduce whatever crinkling existed even before cooling. The Au/Mo grid exhibits excellent performance with specimens tilted to 45°. This is demonstrated by quantifying beam-induced motion and differences in local defocus values. In addition, images of specimens on the Au/Mo grids that are tilted at 45° show high-resolution information of the crystalline membranes that, after lattice-unbending, extends beyond 1.5 Å in the direction perpendicular to the tilt axis.

Amphiphilic Polypeptoids Rupture Vesicle Bilayers To Form Peptoid-Lipid Fragments Effective in Enhancing Hydrophobic Drug Delivery.

Peptoids are highly biocompatible pseudopeptidic polyglycines with designable substituents on the nitrogen atoms. The therapeutic and drug-carrying potential of these materials requires a fundamental understanding of their interactions with lipid bilayers. In this work, we use amphiphilic polypeptoids with up to 100 monomeric units where a significant fraction (26%) of the nitrogen atoms are functionalized with decyl groups (hydrophobes) that insert into the lipid bilayer through the hydrophobic effect. These hydrophobically modified polypeptoids (HMPs) insert their hydrophobes into lipid bilayers creating instabilities that lead to the rupture of vesicles. At low HMP concentrations, such rupture leads to the creation of large fragments which remarkably anchor to intact vesicles through the hydrophobic effect. At high HMP concentrations, all vesicles rupture to smaller HMP-lipid fragments of the order of 10 nm. We show that the technique for such nanoscale polymer-lipid fragments can be exploited to sustain highly hydrophobic drug species in solution. Using the kinase inhibitor, Sorafenib as a model drug, it is shown that HMP-lipid fragments containing the drug can efficiently enter a hepatocellular carcinoma cell line (Huh 7.5), indicating the use of such fragments as drug delivery nanocarriers.

Effect of processing and end groups on the crystal structure of polypeptoids studied by cryogenic electron microscopy at atomic length scales.

Cryogenic electron microscopy at atomic length scales was used to study the structure of self-assembled crystalline nanosheets obtained from a series of polypeptoids with the same chain architecture but with different end groups. While long-range order is enhanced by slowing down the self-assembly process, the dominant crystalline motif was found to be a sensitive function of both processing details and end group chemistry. In some cases, adjacent rows of polypeptoid molecules adopt anti-parallel V-shaped side chain conformations. In other cases, adjacent rows of polypeptoid molecules adopt parallel V-shaped side chain conformations. Interestingly, the unit cell is rectangular in both cases with dimensions a = 4.5 Å and c = 50 Å. In all cases, long-range order, quantified by the average number of concatenated unit cells of the same type, is more prevalent along the a direction.

Polypeptoid polymers: Synthesis, characterization, and properties.

Polypeptoids, a class of peptidomimetic polymers, have emerged at the forefront of macromolecular and supramolecular science and engineering as the technological relevance of these polymers continues to be demonstrated. The chemical and structural diversity of polypeptoids have enabled access to and adjustment of a variety of physicochemical and biological properties (eg, solubility, charge characteristics, chain conformation, HLB, thermal processability, degradability, cytotoxicity and immunogenicity). These attributes have made this synthetic polymer platform a potential candidate for various biomedical and biotechnological applications. This review will provide an overview of recent development in synthetic methods to access polypeptoid polymers with well-defined structures and highlight some of the fundamental physicochemical and biological properties of polypeptoids that are pertinent to the future development of functional materials based on polypeptoids.

Amphiphilic Polypeptoids Serve as the Connective Glue to Transform Liposomes into Multilamellar Structures with Closely Spaced Bilayers.

We report the ability of hydrophobically modified polypeptoids (HMPs), which are amphiphilic pseudopeptidic macromolecules, to connect across lipid bilayers and thus form layered structures on liposomes. The HMPs are obtained by attaching hydrophobic decyl groups at random points along the polypeptoid backbone. Although native polypeptoids (with no hydrophobes) have no effect on liposomal structure, the HMPs remodel the unilamellar liposomes into structures with comparable diameters but with multiple concentric bilayers. The transition from single-bilayer to multiple-bilayer structures is revealed by small-angle neutron scattering (SANS) and cryo-transmission electron microscopy (cryo-TEM). The spacing between bilayers is found to be relatively uniform at ∼6.7 nm. We suggest that the amphiphilic nature of the HMPs explains the formation of multibilayered liposomes; i.e., the HMPs insert their hydrophobic tails into adjacent bilayers and thereby serve as the connective glue between bilayers. At higher HMP concentrations, the liposomes are entirely disrupted into much smaller micellelike structures through extensive hydrophobe insertion. Interestingly, these small structures can reattach to fresh unilamellar liposomes and self-assemble to form new two-bilayer liposomes. The two-bilayer liposomes in our study are reminiscent of two-bilayer organelles such as the nucleus in eukaryotic cells. The observations have significance in designing new nanoscale drug delivery carriers with multiple drugs on separate lipid bilayers and extending liposome circulation times with entirely biocompatible materials.

Synthesis and Characterization of Well-Defined PEGylated Polypeptoids as Protein-Resistant Polymers.

Well-defined polypeptoids bearing oligomeric ethylene glycol side chains (PNMe(OEt)nG, n = 1-3) with a controlled molecular weight (3.26-28.6 kg/mol) and narrow molecular weight distribution (polydispersity index, PDI = 1.03-1.10) have been synthesized by ring-opening polymerization of the corresponding N-carboxyanhydrides having oligomeric ethylene glycol side chains (Me(OEt)n-NCA, n = 1-3) using primary amine initiators. Kinetic studies of polymerization revealed a first-order dependence on the monomer concentration, consistent with living polymerization. The obtained PEGylated polypeptoids are highly hydrophilic with good water solubility (>200 mg/mL) and are amorphous, with a glass transition temperature in the -41.1 to +46.4 °C range that increases with increasing molecular weight and decreasing side chain length. DLS and SANS analyses revealed no appreciable adsorption of lysozyme to PNMeOEtG. PNMeOEtG having different molecular weights exhibited minimal cytotoxicity toward HEp2 cells. These combined results suggest the potential use of PEGylated polypeptoids as protein-resistant materials in biomedical and biotechnological fields.

Enhanced Hypsochromic Shifts, Quantum Yield, and π-π Interactions in a meso,ß-Heteroaryl-Fused BODIPY.

We report the synthesis and investigation of an unprecedented 8-heteroaryl-fused BODIPY 4. This compound exhibits enhanced π-π stacking in the solid state, unusually large blue-shifts in the absorbance and emission spectra, and higher fluorescence quantum yield than its unfused precursor; DFT calculations suggest a small energy gap for 4 and strong electronic communication between the 8-OPh and the BODIPY core.

Synthesis and Spectroscopic and Cellular Properties of Near-IR [a]Phenanthrene-Fused 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacenes.

A new synthetic method to build aryl-fused 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPYs) is reported. The intramolecular cyclization step was completed in a short time (1-2 h) and in high yields (>90%), due to the intrinsic rigid structural conformation of the precursor BODIPY and the high reactivity of its 1,7-bromo groups. The [a]phenanthrene-fused BODIPYs 4a-c were characterized by NMR spectroscopy, HRMS, DFT calculations, and, in the case of 4a, by X-ray crystallography. Spectroscopic studies show that 4a-c strongly absorb and emit in the NIR spectral region, in the range 642-701 nm. In addition, BODIPYs 4b and 4c exhibit no toxicity in the light or dark in HEp2 cells and accumulate intracellularly in a time-dependent manner, mainly in the cell endoplasmic reticulum. These results suggest the potential use of [a]phenanthrene-fused BODIPYs as NIR bioimaging probes.

Synthesis and Spectroscopic Investigation of a Series of Push-Pull Boron Dipyrromethenes (BODIPYs).

A series of push-pull BODIPYs bearing multiple electron-donating and electron-acceptor groups were synthesized regioselectively from 2,3,5,6,8-pentachloro-BODIPY, and characterized by NMR spectroscopy, HRMS, and X-ray crystallography. The influence of the push-pull substituents on the spectroscopic and electrochemical properties of BODIPYs was investigated. Bathochromic shifts were observed for both absorbance (up to 37 nm) and emission (up to 60 nm) in different solvents upon introduction of the push-pull moieties. DFT calculations, consistent with the spectroscopic and cyclic voltammetry studies, show decreased HOMO-LUMO energy gaps upon the installation of the push-pull moieties. BODIPY 7 bearing thienyl groups on the 2 and 6 positions showed the largest λmax for both absorption (635-653 nm) and emission (706-707 nm), but also the lowest fluorescence quantum yields. All BODIPYs were nontoxic in the dark (IC50 > 200 µM) and showed low phototoxicity (IC50 > 100 µM, 1.5 J/cm2) toward human HEp2 cells. Despite the relatively low fluorescence quantum yields, the push-pull BODIPYS were effective for cell imaging, readily accumulating within cells and localizing mainly in the ER and Golgi. Our structure-property studies can guide future design of functionalized BODIPYs for various applications, including bioimaging and in dye-sensitized solar cells.

Unusual molecular mechanism behind the thermal response of polypeptoids in aqueous solutions.

Poly(α-peptoid)s, a structural isomer to polypeptides, have recently attracted a significant amount of scientific attention. However, the molecular mechanism behind the thermal response of this class of polymers is unknown. Here, the thermal response of two polypeptoids in aqueous solutions was studied by different methodologies, including dynamic light scattering, IR spectroscopies, NMR, etc. Our studies focused on two polypeptoids with identical alkyl side chain compositions, but different architecture; i.e., cyclic and linear. Aqueous solutions of the cyclic and linear polymers present phase transition temperatures at 43 °C and 47 °C, respectively, that have an anomalous dependence on the polymer morphology as expected from macromolecules having very similar solvent interactions, but different conformational entropy. The atypical trend in the phase transition temperature is found to be caused by the initiator required in the synthesis which favors the formation of soluble dimers in the cyclic polymer. Our experimental findings also demonstrate that the phase transition, irrespective of the morphology, is governed by the polymer backbone conformation which depends on the composition and structure of the alkyl side chains. This proposed mechanism is novel and different from the commonly assumed mechanism for thermo-responsive polymers in which the hydration of the polymer cause by a coil to globule transition is the determining factor. Moreover, the proposed mechanism is likely to be general since it can explain not only the experimental findings of this work, but also observations of the thermal response and conformation of other studied polypeptoids in water. Finally, our mechanism gives a molecular framework for the rational designed of polypeptoids with tailored phase transition temperatures.

Synthesis and regioselective functionalization of perhalogenated BODIPYs.

Three perhalogenated BODIPYs (1b-3b), bearing chloro and bromo groups at all carbon positions, were synthesized and characterized. The reactivity of BODIPY 3b was investigated under Stille cross-coupling reactions, and single crystal X-ray analysis was used to confirm the regioselectivity of the reactions. Further substitution at the boron atom produced nona-functionalized BODIPYs 7a,b, which show 676 and 739 nm emissions with 91 and 100 nm Stokes shifts, respectively.

Stepwise Polychlorination of 8-Chloro-BODIPY and Regioselective Functionalization of 2,3,5,6,8-Pentachloro-BODIPY.

An effective, stepwise methodology for polychlorination of BODIPY using trichloroisocyanuric acid (TCCA) in acetic acid was developed. In this way, selectively substituted di-, tri-, tetra-, and pentachloro-BODIPYs 2-5 were prepared. The pentachloro-BODIPY is shown to undergo regioselective Pd(0)-catalyzed Stille and Suzuki coupling reactions, first at the 8-position followed by the 3,5- and then the 2,6-positions; nucleophilic substitution reactions occur first at the 8- followed by the 3,5-positions, while the 2,6 are unreactive.

Effect of Micellar Morphology on the Temperature-Induced Structural Evolution of ABC Polypeptoid Triblock Terpolymers into Two-Compartment Hydrogel Network.

We investigated the temperature-dependent structural evolution of thermoreversible triblock terpolypeptoid hydrogels, namely poly(N-allyl glycine)-b-poly(N-methyl glycine)-b-poly(N-decyl glycine) (AMD), using small-angle neutron scattering (SANS) with contrast matching in conjunction with X-ray scattering and cryogenic transmission electron microscopy (cryo-TEM) techniques. At room temperature, A100M101D10 triblock terpolypeptoids self-assemble into core-corona-type spherical micelles in aqueous solution. Upon heating above the critical gelation temperature (T gel), SANS analysis revealed the formation of a two-compartment hydrogel network comprising distinct micellar cores composed of dehydrated A blocks and hydrophobic D blocks. At T ≳ T gel, the temperature-dependent dehydration of A block further leads to the gradual rearrangement of both A and D domains, forming well-ordered micellar network at higher temperatures. For AMD polymers with either longer D block or shorter A block, such as A101M111D21 and A43M92D9, elongated nonspherical micelles with a crystalline D core were observed at T < T gel. Although these enlarged crystalline micelles still undergo a sharp sol-to-gel transition upon heating, the higher aggregation number of chains results in the immediate association of the micelles into ordered aggregates at the initial stage, followed by a disruption of the spatial ordering as the temperature further increases. On the other hand, fiber-like structures were also observed for AMD with longer A block, such as A153M127D10, due to the crystallization of A domains. This also influences the assembly pathway of the two-compartment network. Our findings emphasize the critical impact of initial micellar morphology on the structural evolution of AMD hydrogels during the sol-to-gel transition, providing valuable insights for the rational design of thermoresponsive hydrogels with tunable network structures at the nanometer scale.

Atomic-Scale Corrugations in Crystalline Polypeptoid Nanosheets Revealed by Three-Dimensional Cryogenic Electron Microscopy.

Amphiphilic molecules that can crystallize often form molecularly thin nanosheets in aqueous solutions. The possibility of atomic-scale corrugations in these structures has not yet been recognized. We have studied the self-assembly of amphiphilic polypeptoids, a family of bio-inspired polymers that can self-assemble into various crystalline nanostructures. Atomic-scale structure of the crystals in these systems has been inferred using both X-ray diffraction and electron microscopy. Here we use cryogenic electron microscopy to determine the in-plane and out-of-plane structures of a crystalline nanosheet. Data were collected as a function of tilt angle and analyzed using a hybrid single-particle crystallographic approach. The analysis reveals that adjacent rows of peptoid chains, which are separated by 4.5 Å in the plane of the nanosheet, are offset by 6 Å in the direction perpendicular to the plane of the nanosheet. These atomic-scale corrugations lead to a doubling of the unit cell dimension from 4.5 to 9 Å. Our work provides an alternative interpretation for the observed Å X-ray diffraction peak often reported in polypeptoid crystals.