Well-Defined Homopolypeptides, Copolypeptides, and Hybrids of Poly(l-proline)

l-Proline is the only, out of 20 essential, amino acid that contains a cyclized substituted α-amino group (is formally an imino acid), which restricts its conformational shape. The synthesis of well-defined homo- and copolymers of l-proline has been plagued either by the low purity of the monomer or the inability of most initiating species to polymerize the corresponding N-carboxy anhydride (NCA) because they require a hydrogen on the 3-N position of the five-member ring of the NCA, which is missing. Herein, highly pure l-proline NCA was synthesized by using the Boc-protected, rather than the free amino acid. The protection of the amine group as well as the efficient purification method utilized resulted in the synthesis of highly pure l-proline NCA. The high purity of the monomer and the use of an amino initiator, which does not require the presence of the 3-N hydrogen, led for the first time to well-defined poly(l-proline) (PLP) homopolymers, poly(ethylene oxide)-b-poly(l-proline), and poly(l-proline)-b-poly(ethylene oxide)-b-poly(l-proline) hybrids, along with poly(γ-benzyl-l-glutamate)-b-poly(l-proline) and poly(Boc-l-lysine)-b-poly(l-proline) copolypeptides. The combined characterization (NMR, FTIR, and MS) that results for the l-proline NCA revealed its high purity. In addition, all synthesized polymers exhibit high molecular and compositional homogeneity

Complexation-Driven Mutarotation in Poly(l‑proline) Block Copolypeptides

Novel poly­(l-lysine)-<i>block</i>-poly­(l-proline) (PLL-<i>b</i>-PLP)-based materials with all PLP helical conformers, i.e., PLP II and the rare PLP I are here reported. Electrostatic supramolecular complexation of the adjacent cationic PLL with anionic molecules bearing DNA analogue H-bonding functionalities, such as deoxyguanosine monophosphate (dGMP), preserves the extended PLP II helix, and the complexed molecule is locked and held in position by orthogonal shape-persistent hydrogen-bonded dGMP ribbons and their extended π-stacking. The branched anionic surfactant dodecylbenzenesulfonic acid (DBSA) on the other hand, introduces periodicity frustration and interlayer plasticization, leading to a reversed mutarotation to the more compact PLP I helix by complexation, without external stimuli, and is here reported for the first time. We foresee that our findings can be used as a platform for novel molecularly adaptive functional materials, and could possibly give insight in many proline-related transmembrane biological functions

Hierarchical Smectic Self-Assembly of an ABC Miktoarm Star Terpolymer with a Helical Polypeptide Arm

We demonstrate the first hierarchical smectic self-assembly in miktoarm star terpolymers, using a polymer/polypeptide hybrid (macromolecular chimera) composed of two coil-like arms (polystyrene, PS, and polyisoprene, PI) and a mesogenic α-helical polypeptide arm (poly(ε-tert-butyloxycarbonyl-l-lysine), PBLL). The PBLL α-helices are packed within lamellar nanodomains which leads to an overall smectic alteration of rod- and coil-containing layers typically observed in rod−coil block copolymers. Furthermore, the coil-containing lamellae have an inner structure composed of PS and PI rectangular cylinders, leading to what we call a hierarchical smectic phase. To elucidate the role of polypeptide helices in directing the self-assembly, the ordering is studied both after thermal annealing and after quick drop-casting from chloroform solution. The possibility to combine mesogen packing, tiling patterns, and conformational control of polypeptide blocks makes self-assembled hierarchies of star-shaped macromolecular chimeras appealing for future studies

Side-Chain-Controlled Self-Assembly of Polystyrene–Polypeptide Miktoarm Star Copolymers

We show how the self-assembly of miktoarm star copolymers can be controlled by modifying the side chains of their polypeptide arms, using A₂B and A₂B₂ type polymer/polypeptide hybrids (macromolecular chimeras). Initially synthesized PS₂PBLL and PS₂PBLL₂ (PS, polystyrene; PBLL, poly­(ε-<i>tert</i>-butyloxycarbonyl-l-lysine)) miktoarms were first deprotected to PS₂PLLHCl and PS₂PLLHCl₂ miktoarms (PLLHCl, poly­(l-lysine hydrochloride)) and then complexed ionically with sodium dodecyl sulfonate (DS) to give the supramolecular complexes PS₂PLL­(DS) and PS₂(PLL­(DS))₂. The solid-state self-assemblies of these six miktoarm systems were studied by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and small- and wide-angle X-ray scattering (SAXS, WAXS). The side chains of the polypeptide arms were observed to have a large effect on the solubility, polypeptide conformation, and self-assembly of the miktoarms. Three main categories were observed: (i) lamellar self-assemblies at the block copolymer length scale with packed layers of α-helices in PS₂PBLL and PS₂PBLL₂; (ii) charge-clustered polypeptide micelles with less-defined conformations in a nonordered lattice within a PS matrix in PS₂PLLHCl and PS₂PLLHCl₂; (iii) lamellar polypeptide–surfactant self-assemblies with β-sheet conformation in PS₂PLL­(DS) and PS₂(PLL­(DS))₂ which dominate over the formation of block copolymer scale structures. Differences between the 3- and 4-arm systems illustrate how packing frustration between the coil-like PS arms and rigid polypeptide conformations can be relieved by the right number of arms, leading to differences in the extent of order

Extended Self-Assembled Long Periodicity and Zig-Zag Domains from Helix–Helix Diblock Copolymer Poly(γ-benzyl‑l‑glutamate)-<i>block</i>-poly(<i>O</i>‑benzyl‑l‑hydroxyproline)

We describe the synthesis and self-assembly of particularly high periodicity of diblock copolymers composed of poly­(benzyl-l-hydroxyproline) (PBLHyP) and poly­(γ-benzyl-l-glutamate) (PBLG), that is, two polypeptide blocks with dissimilar helical structures. The robust helicity of the PBLHyP block is driven by steric constraints of the repeat units, while PBLG forms α-helices driven by hydrogen bonding, allowing defects and deformations. Herein, high-molecular-weight diblock copolypeptides of PBLG-b-PBLHyP with three different volume fractions of the PBLHyP-blocks are discussed. For shorter PBLHyP blocks, hexagonal packing of PBLHyP helices is observed, while by increasing the length of the PBLHyP block, keeping at a similar PBLG block length, the packing is distorted. Zig-zag lamellar structures were obtained due to the mismatch in the packing periodicities of the PBLG and PBLHyP helices. The frustration that takes place at the interface leads the PBLHyP to tilt to match the PBLG periodicity. The zig-zag morphology is reported for the first time for high-molecular-weight helix–helix (rod–rod) copolypeptides, and the self-assembled periodicity is uncommonly large

Architecturally Induced Multiresponsive Vesicles from Well-Defined Polypeptides. Formation of Gene Vehicles

A series of novel, partially labeled amphiphilic triblock copolypeptides, PLL-b-PBLG-d7-b-PLL, has been synthesized, where PLL and PBLG-d7 are poly(L-lysine hydrochloride) and poly(γ-benzyl-d7-L-glutamate), respectively. The synthetic approach involved the sequential ring-opening polymerization (ROP) of γ-benzyl-L-glutamate and ε-Boc-L-lysine N-carboxy anhydrides by a diamino initiator using high-vacuum techniques, followed by the selective deprotection of the Boc groups. Combined characterization results showed that the copolypeptides exhibit high degrees of molecular and compositional homogeneity. The synthesized copolypeptides had similar molecular weights, while the composition of the middle block ranged between 19 and 74% with respect to the monomeric units. Due to the macromolecular architecture of the copolypeptide and the rigid nature of the middle block, the formation of monolayers was favored, and, surprisingly, vesicles were formed in water at neutral pH over the entire compositional range. The vesicular structures were extensively characterized by static and dynamic light scattering, small-angle neutron scattering, atomic force microscopy, cryo-transmission electron microscopy, scanning electron microscopy, UV and Fourier transform infrared spectroscopy, and circular dichroism. In contrast to other vesicular structures derived from conventional polymers, the formed polypeptidic vesicles possess the unique feature of being stimuli-responsive to pH and temperature. When the copolypeptides were mixed with plasmid DNA (pDNA), large vesicular structures were also formed. The molecular characterization of the vectors was performed with most of the methods mentioned above, and indicated that the pDNA is both partially condensed on the PLL phase and partially encapsulated inside the vesicle. Consequently, the synthesized vectors combine the advantages of the polylysine−DNA systems to condense large amounts of genes, as well as those of the liposome−DNA systems to better protect the encapsulated DNA. These vectors are expected to present better gene transfection efficiency to the cell nucleus

Double Smectic Self-Assembly in Block Copolypeptide Complexes

We show double smectic-like self-assemblies in the solid state involving alternating layers of different polypeptide α-helices. We employed rod–coil poly­(γ-benzyl l-glutamate)-block-poly­(l-lysine) (PBLG-b-PLL) as the polymeric scaffold, where the PLL amino residues were ionically complexed to di-n-butyl phosphate (diC4P), di­(2-ethylhexyl) phosphate (diC2/6P), di­(2-octyldodecyl) phosphate (diC8/12P), or di-n-dodecyl phosphate (diC12P), forming PBLG-b-PLL­(diC4P), PBLG-b-PLL­(diC2/6P), PBLG-b-PLL­(diC8/12P), and PBLG-b-PLL­(diC12P) complexes, respectively. The complexes contain PBLG α-helices of fixed diameter and PLL-surfactant complexes adopting either α-helices of tunable diameters or β-sheets. For PBLG-b-PLL­(diC4P), that is, using a surfactant with short n-butyl tails, both blocks were α-helical, of roughly equal diameter and thus with minor packing frustrations, leading to alternating PBLG and PLL­(diC4P) smectic layers of approximately perpendicular alignment of both types of α-helices. Surfactants with longer and branched alkyl tails lead to an increased diameter of the PLL-surfactant α-helices. Smectic alternating PBLG and PLL­(diC2/6P) layers involve larger packing frustration, which leads to poor overall order and suggests an arrangement of tilted PBLG α-helices. In PBLG-b-PLL­(diC8/12P), the PLL­(diC8/12P) α-helices are even larger and the overall structure is poor. Using a surfactant with two linear n-dodecyl tails leads to well-ordered β-sheet domains of PLL­(diC12P), consisting of alternating PLL and alkyl chain layers. This dominates the whole assembly, and at the block copolypeptide length scale, the PBLG α-helices do not show internal order and have poor organization. Packing frustration becomes an important aspect to design block copolypeptide assemblies, even if frustration could be relieved by conformational imperfections. The results suggest pathways to control hierarchical liquid-crystalline assemblies by competing interactions and by controlling molecular packing frustrations

Architecturally Induced Multiresponsive Vesicles from Well-Defined Polypeptides. Formation of Gene Vehicles

A series of novel, partially labeled amphiphilic triblock copolypeptides, PLL-b-PBLG-d7-b-PLL, has been synthesized, where PLL and PBLG-d7 are poly(L-lysine hydrochloride) and poly(γ-benzyl-d7-L-glutamate), respectively. The synthetic approach involved the sequential ring-opening polymerization (ROP) of γ-benzyl-L-glutamate and ε-Boc-L-lysine N-carboxy anhydrides by a diamino initiator using high-vacuum techniques, followed by the selective deprotection of the Boc groups. Combined characterization results showed that the copolypeptides exhibit high degrees of molecular and compositional homogeneity. The synthesized copolypeptides had similar molecular weights, while the composition of the middle block ranged between 19 and 74% with respect to the monomeric units. Due to the macromolecular architecture of the copolypeptide and the rigid nature of the middle block, the formation of monolayers was favored, and, surprisingly, vesicles were formed in water at neutral pH over the entire compositional range. The vesicular structures were extensively characterized by static and dynamic light scattering, small-angle neutron scattering, atomic force microscopy, cryo-transmission electron microscopy, scanning electron microscopy, UV and Fourier transform infrared spectroscopy, and circular dichroism. In contrast to other vesicular structures derived from conventional polymers, the formed polypeptidic vesicles possess the unique feature of being stimuli-responsive to pH and temperature. When the copolypeptides were mixed with plasmid DNA (pDNA), large vesicular structures were also formed. The molecular characterization of the vectors was performed with most of the methods mentioned above, and indicated that the pDNA is both partially condensed on the PLL phase and partially encapsulated inside the vesicle. Consequently, the synthesized vectors combine the advantages of the polylysine−DNA systems to condense large amounts of genes, as well as those of the liposome−DNA systems to better protect the encapsulated DNA. These vectors are expected to present better gene transfection efficiency to the cell nucleus

Tunable Hydrogels with Improved Viscoelastic Properties from Hybrid Polypeptides

Hydrogels that can respond to a number of external stimuli and at the same time show impressive rheological properties are promising materials for a wide range of bioapplications. Here, we present a series of well-defined linear amphiphilic pentablock hybrid polypeptides of the ABCBA type, where A is poly­(l-lysine), B is poly­(l-histidine)-co-poly­(γ-benzyl-l-glutamate), and C is poly­(ethylene oxide) (PEO). The polymers were synthesized by the sequential primary amine ring-opening polymerization of N-carboxy anhydrides using bis amine poly­(ethylene oxide) (PEO) as a bifunctional macroinitiator, and the length of all of the blocks was varied. The resulting materials formed novel extrudable in situ forming quickly self-healing hydrogels, responsive to the alteration of pH and increase of temperature. The connection between the alteration of the secondary structure of the polypeptides with the viscoelastic behavior was revealed by means of rheology and circular dichroism. Small-angle neutron scattering and scanning electron microscopy were employed to shed light on the structure of the polymers and how it affects their rheological properties. The obtained polymers were subjected to enzymatic degradation tests with trypsin and leucine aminopeptidase. The results suggest that these biomaterials have the potential to be used in a number of bioapplications like drug delivery, 3D printing, and tissue engineering

Self-Healing pH- and Enzyme Stimuli-Responsive Hydrogels for Targeted Delivery of Gemcitabine To Treat Pancreatic Cancer

A novel, multifunctional hydrogel that exhibits a unique set of properties for the effective treatment of pancreatic cancer (PC) is presented. The material is composed of a pentablock terpolypeptide of the type PLys-b-(PHIS-co-PBLG)-PLys-b-(PHIS-co-PBLG)-b-PLys, which is a noncytotoxic polypeptide. It can be implanted via the least invasive route and selectively delivers gemcitabine to efficiently treat PC. Simply mixing the novel terpolypeptide with an aqueous solution of gemcitabine within a syringe results in the facile formation of a hydrogel that has the ability to become liquid under the shear rate of the plunger. Upon injection in the vicinity of cancer tissue, it immediately reforms into a hydrogel due to the unique combination of its macromolecular architecture and secondary structure. Because of its pH responsiveness, the hydrogel only melts close to PC; thus, the drug can be delivered directionally toward the cancerous rather than healthy tissues in a targeted, controlled, and sustained manner. The efficacy of the hydrogel was tested in vivo on human to mouse xenografts using the drug gemcitabine. It was found that the efficacy of the hydrogel loaded with only 40% of the drug delivered in one dose was equal to or slightly better than the peritumoral injection of 100% of the free drug delivered in two doses, the typical chemotherapy used in clinics so far. This result suggests that the hydrogel can direct the delivery of the encapsulated drug effectively in the tumor tissue. Enzymes lead to its biodegradation, avoiding removal by resection of the polypeptidic carrier after cargo delivery. The unique properties of the hydrogel formed can be predetermined through its molecular characteristics, rendering it a promising modular material for many biological applications