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Exploring New Activating Groups for Reactive Cysteine NCAs
Abstract
Due to its ability to reversibly crosslink proteins, cysteine has a unique role as an amino acid in nature. For controlled, asymmetric formation of disulfides from two thiols, one thiol needs to be activated. While few activating groups for cysteine have been proposed, they are usually not stable against amines making them unsuitable for solid phase peptide synthesis or amine initiated polymerization of α-amino acid-N-carboxy-anhydrides (NCAs). In this report we describe a series of new thiol activated cysteines, as well as their NCAs and explore the link between electron deficiency of the leaving group and control over NCA polymerization.
... In the light of these considerations, activated cysteines were employedi nNCA synthesis and polymerization, first equipped with protecting groups of varying potency as electron-withdrawing groups (Scheme 4B). [163] The findings were, however,h umbling, because the protecting groups in question are highly reactive like most activatedt hiols or disulfides and were pronet oh ydrolysis and aminolysis during NCA synthesis as well as during polymerization. At this point, a related class of sulfur-sulfur-containingb onds known for its potencyi nt hiol activation was explored:t he thiosulfonyl group. ...
... At this point, a related class of sulfur-sulfur-containingb onds known for its potencyi nt hiol activation was explored:t he thiosulfonyl group. [39,172,173] Starting with S-pheylsulfonyl-l-cysteine [163] and followed by S-alkylsulfonyl-l-cysteines [42] (Scheme 4C), thiosulfonyl protecting groups were first explored in NCA polymerization to successfully bridge the gap between stabilitya nd reactivity. ...
... Reproduced with permissionfrom refs. [49,163].Copyright2 016 and 2017 Elsevier. ated hydrolysis needs to be taken into account due to the oxophilic character of sulfur,a sr eflected in increasing rate constants upon higherp Hl evels.A saresult,m ildly acidic conditions are preferred when reactions are performed in aqueous media. ...
In poly(S‐alkylsulfonyl)‐l‐cysteines, an exciting combination of properties is merged to enable asymmetric disulfide formation in a chemoselective manner, while promoting directed self‐assembly in solution by β‐sheet formation. These properties allow the straightforward synthesis of core–shell nanoparticles with decoupled control over morphology and functionality. For more information on the latest results in this field, see the Concept article by O. Schäfer and M. Barz on page 12131 ff.
... Reproduced with permission from refs. [49,163]. ...
... Thus, activated cysteines were employed in NCA synthesis and polymerization, first equipped with protecting groups of varying potency as electron-withdrawing groups (Scheme 4 B). [163] The findings were, however, humbling because the protecting groups in question are highly reactive like most activated thiols and disulfides and were prone to hydrolysis and aminolysis during NCA synthesis as well as during polymerization. At this point, a related class of sulfur-sulfur-containing bonds known for its potency in thiol activation was explored: the thiosulfonyl group. ...
... At this point, a related class of sulfur-sulfur-containing bonds known for its potency in thiol activation was explored: the thiosulfonyl group. [39,172,173] Starting with S-pheylsulfonyl-l-cysteine [163] and followed by Salkylsulfonyl-l-cysteines [42] (Scheme 4 C), thiosulfonyl protecting groups were first explored in NCA polymerization to successfully bridge the gap between stability and reactivity. ...
With regard to protein‐ or peptide chemistry, thiols are frequently chosen as a chemical entity for chemo‐selective modification reactions. And while it is a well established methodology to address cysteines and homocysteines in aqueous media to form S‐C bonds, possibilities for the chemoselective formation of asymmetric disulfide bonds are much less approached. Focusing on bioreversibility in conjugation chemistry, the formation of disulfide bonds is highly desirable for the attachment of thiol‐bearing bioactive agents to proteins or in cross‐linking reactions, since disulfide bonds can combine stability in blood with degradability inside cells. In this concept article recent approaches in the field of activating groups for thiol moieties incorporated in polymeric and polypeptide materials are highlighted. Advantageous combinations of stability during synthesis of the material with high reactivity towards thiols are explored focusing on simplification and prevention of side reactions as well as additional deprotection and activation steps prior to disulfide formation. Moreover, applications of this chemistry are highlighted and future perspectives are envisioned.
... Cytotoxicity assessment 3. NMR spectra for new compounds a. Figure S3. 1 H-NMR of FTS-PSarc b. Figure S4. 1 H-NMR of PSarc-b-PPS 4. Polymer formulation optimizations a. Table S1. Linker selection b. ...
... Cytotoxicity assessment 3. NMR spectra for new compounds a. Figure S3. 1 H-NMR of FTS-PSarc b. Figure S4. 1 H-NMR of PSarc-b-PPS 4. Polymer formulation optimizations a. Table S1. Linker selection b. ...
A combinatorial copolymer library was created to rapidly screen the landscape of self-assembed nanostructure morphologies formed by block copolymers composed of hydrophilic peptoid polysarcosine (PSarc) and hydrophobic poly(propylene sulfide) (PPS)...
... [30,[33][34][35] The polypeptidelp-CysSO 2 Etenables secondary structure directed self-assembly and rapid (reaction rates: k SH >> 1 s −1 ) chemo-selective core cross-linking by formation of asymmetric disulfides with dior oligo-thiols providing control over particle morphology and functionality. [36][37][38] While microfluidics have evolved to the state-of-the-art technique for the production of lipid nanoparticles (LNPs) and colloidal nanoparticles, polymeric micelles and in particular CCPMs are still synthesized in batch-mode using either film rehydration, solvent exchange, temperature-induced aggregation, or precipitation techniques. [39][40][41][42][43][44][45] In contrast to such methods, micromixers enable continuous-flow processes and offer automated manufacturing increasing production rates and reproducibility counteracting an advancing complexity, whereby the closed setup facilitates sterile particle preparation. ...
Translating innovative nanomaterials to medical products requires efficient manufacturing techniques that enable large-scale high-throughput synthesis and high reproducibility. Drug carriers in medicine embrace a complex subset of tasks calling for multi-functionality. Here, we report the synthesis of pro-drug-loaded core cross-linked polymeric micelles (CCPMs) in a continuous flow process, which combines the commonly separated steps of micelle formation, core cross-linking, functionalization, and purification into a single process. Redox-responsive CCPMs are formed from thiol-reactive polypept(o)ides of polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) and functional cross-linkers based on dihydrolipoic acid hydrazide for pH-dependent release of paclitaxel. The precisely controlled microfluidic self-assembly and purification process allows the production of spherical micelles (Dh = 35 nm) with low polydispersity values (PDI<0.1) while avoiding toxic organic solvents and additives with unfavorable safety profiles. Self-assembly and cross-linking via slit interdigital micromixers produce 350-700 mg of CCPMs/h per single system, while purification by online tangential flow filtration successfully removes impurities (unimer ≤ 0.5%). The formed paclitaxel-loaded CCPMs possess the desired pH-responsive release profile, display stable drug encapsulation, an improved toxicity profile compared to Abraxane, and therapeutic efficiency in the B16F1-xenotransplanted zebrafish model. The combination of reactive polymers, functional cross-linkers, and microfluidics enables the continuous-flow synthesis of therapeutically active CCPMs in a single process. This article is protected by copyright. All rights reserved.
... S-Sulfonyl-cysteines 5 and 6 were prepared on multigram scale by slight modification of reported procedures ( Figure 2). 23,24 Our initial investigations into the diazotization of 5 with 2 equiv of nitrite, 4 equiv of sulfuric acid, and 24 h reaction time produced the targeted α-hydroxy acid 7 in 33% yield, according to 1 H NMR ( Figure 2). This promising result demonstrated that the thiosulfonate was more stable to the acidic and oxidizing reaction conditions than the other thiol protecting groups tested. ...
We report the preparation of enantiomerically enriched β-thio-α-hydroxy and α-chloro carboxylic acid and ester building blocks by diazotization of S-sulfonyl-cysteines. The thiosulfonate protecting group demonstrated resistance to oxidation and attenuation of sulfur’s nucleophilicity by the anomeric effect. The key transformation was optimized by a 2² factorial design of experiment, highlighting the unique reactivity of cysteine derivatives in comparison with aliphatic amino acids.
Core cross-linked polymeric micelles (CCPMs) are designed to improve the therapeutic profile of hydrophobic drugs, reduce or completely avoid protein corona formation, and offer prolonged circulation times, a prerequisite for passive or active targeting. In this study, we tuned the CCPM stability by using bifunctional or trifunctional cross-linkers and varying the cross-linkable polymer block length. For CCPMs, amphiphilic thiol-reactive polypept(o)ides of polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) [pSar-b-pCys(SO2Et)] were employed. While the pCys(SO2Et) chain lengths varied from Xn = 17 to 30, bivalent (derivatives of dihydrolipoic acid) and trivalent (sarcosine/cysteine pentapeptide) cross-linkers have been applied. Asymmetrical flow field-flow fraction (AF4) displayed the absence of aggregates in human plasma, yet for non-cross-linked PM and CCPMs cross-linked with dihydrolipoic acid at [pCys(SO2Et)]17, increasing the cross-linking density or the pCys(SO2Et) chain lengths led to stable CCPMs. Interestingly, circulation time and biodistribution in mice of non-cross-linked and bivalently cross-linked CCPMs are comparable, while the trivalent peptide cross-linkers enhance the circulation half-life from 11 to 19 h.
Synthetic polypeptides from ring‐opening polymerization of α‐amino acid N‐carboxyanhydrides (NCA) are versatile materials for various biomedical applications due to their biocompatibility, biodegradability, and bioactivity. This article overviews recent progress in NCA polymerizations and side‐chain functionalization of polypeptides. Initiating systems, mechanism studies, and optimized polymerization conditions for the NCA polymerization are comprehensively reviewed and evaluated here. The substituents of NCA monomers are also described. Finally, the design and synthesis of functional polypeptides is summarized and discussed.
Homocysteine and cysteine are the only natural occurring amino acids that are capable of disulfide bond formations in peptides and proteins. The chemoselective formation of asymmetric disulfide bonds, however, is chemically challenging and requires an activating group combining stability against hard nucleophiles, e.g., amines, with reactivity toward thiols and soft nucleophiles. In light of these considerations, we introduced the S-alkylsulfonyl cysteines in our previous work. Here, we present the synthesis and ring-opening polymerization of S-ethylsulfonyl-l-homocysteine N-carboxyanhydrides. We demonstrate that the polymerization leads to narrowly distributed polypeptides (Đ = 1.1–1.3) with no detectable side reactions in a chain length regime from 11 to 165. In contrast to the already reported cysteine derivatives, poly(S-ethylsulfonyl-l-homocysteine)s do not form β-sheets, which reduce solubility and limit the degree of polymerization of poly(S-ethylsulfonyl-l-cysteine)s to 50. Instead, these polymers form α-helices as confirmed by circular dicroism (CD) experiments and infrared spectroscopy (FT-IR). In comparison to the cysteine derivatives, the α-helix formation leads to slightly faster polymerization kinetics (rate constants from 1.44 × 10–5 to 5.29 × 10–5 s–1). In addition, the ability for the chemoselective formation of asymmetric disulfides is preserved as monitored via ¹H NMR experiments. Consequently, this new polypeptide overcomes the chain length limitations of poly(S-ethylsulfonyl-l-cysteine)s and thus provides convenient access to reactive poly(S-ethylsulfonyl-l-homocysteine)s for chemoselective disulfide formation.
We report on the synthesis of polysarcosine-block-poly(S-alkylsulfonyl)-l-cysteine block copolymers, which combine three orthogonal addressable groups enabling site-specific conversion of all reactive entities in a single step. The polymers are readily obtained by ring-opening polymerization (ROP) of corresponding α-amino acid N-carboxyanhydrides (NCAs) combining azide and amine chain ends, with a thiol-reactive S-alkylsulfonyl cysteine. Functional group interconversion of chain ends using strain-promoted azide–alkyne cycloaddition (SPAAC) and activated ester chemistry with NHS- and DBCO-containing fluorescent dyes could be readily performed without affecting the cross-linking reaction between thiols and S-alkylsulfonyl protective groups. Eventually, all three functionalities can be combined in the formation of multifunctional disulfide core cross-linked nanoparticles bearing spatially separated functionalities. The simultaneous attachment of dyes in core and corona during the formation of core-cross-linked nanostructures with controlled morphology is confirmed by fluorescence cross-correlation spectroscopy (FCCS).
Functional amino acids whose reactivity is compatible with the polymerization of α-amino acid-N-carboxyanhydrides (NCAs) have received a lot of attention in recent years. The appeal of these reactive monomers lies in the fact that the resulting polymers can be easily modified in one controlled post-polymerization step, leading to a variety of polypeptidic materials like helical non-natural polycations or glycopeptides. This review highlights recent developments in the field and focuses on the different reactive groups like alkynes, alkenes, azides, chlorides and S-alkylsulfonyls. Furthermore, the modifications after polymerization are discussed, pointing out advantages and challenges. Besides orthogonal functionalities in the side chain, different approaches are summarized to modify α- and ω-chain ends with orthogonal functionalities for grafting to and grafting from applications. Thus, new materials can be produced through mild modifications as presented. Finally, we also highlight the development of orthogonally reactive NCAs for the synthesis of polypeptoids, a field that is relatively unexplored, but offers great possibilities for example for polypept(o)idic hybrid materials.
The amino acid cysteine possesses a unique role in nature due to its ability to reversibly cross-link proteins. To transfer this feature to polypeptides and control the process of disulfide formation, a protective group needs to provide stability against amines during synthesis, combined with chemoselective reactivity toward thiols. A protective group providing these unique balance of stability and reactivity toward different nucleophiles is the S-alkylsulfonyl group. In this work we report the polymerization of S-ethylsulfonyl-l-cysteine N-carboxyanhydride and kinetic evaluations with respect to temperature (10, 0, and +10 °C) and monomer concentration. The polymerization degree of poly(S-ethylsulfonyl-l-cysteines) can be controlled within a range of 10-30, yielding well-defined polymers with molecular weights of 6900-12 300 g/mol with dispersity indices of 1.12-1.19 as determined by GPC and MALDI-TOF analysis. The limitation of chain length is, however, not related to side reactions during ring-opening polymerization, but to physical termination during β-sheet assembly. In the case of poly(S-ethylsulfonyl-l-cysteines), circular dichroism as well as FT-IR experiments confirm an antiparallel β-sheet conformation. The reaction of poly(S-ethylsulfonyl-l-cysteines) with thiols is completed in less than a minute, leading quantitatively to asymmetric disulfide bond formation in the absence of side reactions. Therefore, poly(S-ethylsulfonyl cysteines) are currently the only reactive cysteine derivative applicable to NCA synthesis and polymerization, which allows efficient and chemoselective disulfide formation in synthetic polypeptides, bypassing additional protective group cleavage steps.
The ability to reversibly cross-link proteins and peptides grants the amino acid cysteine its unique role in nature as well as in peptide chemistry. We report a novel class of S-alkylsulfonyl-l-cysteines and N-carboxy anhydrides (NCA) thereof for peptide synthesis. The S-alkylsulfonyl group is stable against amines and thus enables its use under Fmoc chemistry conditions and the controlled polymerization of the corresponding NCAs yielding well-defined homo- as well as block co-polymers. Yet, thiols react immediately with the S-alkylsulfonyl group forming asymmetric disulfides. Therefore, we introduce the first reactive cysteine derivative for efficient and chemoselective disulfide formation in synthetic polypeptides, thus bypassing additional protective group cleavage steps.
A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking
For small interfering RNA (siRNA)-based cancer therapies, we report an actively-targeted and stabilized polyion complex micelle designed to improve tumor accumulation and cancer cell uptake of siRNA following systemic administration. Improvement in micelle stability was achieved using two stabilization mechanisms; covalent disulfide cross-linking and non-covalent hydrophobic interactions. The polymer component was designed to provide disulfide cross-linking and cancer cell-targeting cyclic RGD peptide ligands, while cholesterol-modified siRNA (Chol-siRNA) provided additional hydrophobic stabilization to the micelle structure. Dynamic light scattering confirmed formation of nano-sized disulfide cross-linked micelles (<50 nm in diameter) with a narrow size distribution. Improved stability of Chol-siRNA-loaded micelles (Chol-siRNA micelles) was demonstrated by resistance to both the dilution in serum-containing medium and counter polyion exchange with dextran sulfate, compared to control micelles prepared with Chol-free siRNA (Chol-free micelles). Improved stability resulted in prolonged blood circulation time of Chol-siRNA micelles compared to Chol-free micelles. Furthermore, introduction of cRGD ligands onto Chol-siRNA micelles significantly facilitated accumulation of siRNA in a subcutaneous cervical cancer model following systemic administration. Ultimately, systemically administered cRGD/Chol-siRNA micelles exhibited significant gene silencing activity in the tumor, presumably due to their active targeting ability combined with the enhanced stability through both hydrophobic interactions of cholesterol and disulfide cross-linking.
Polypeptides are envisaged to achieve a major impact on a number of different relevant areas such as biomedicine and biotechnology. Acquired knowledge and the increasing interest on amino acids, peptides and proteins is establishing a large panel of these biopolymers whose physical, chemical and biological properties are ruled by their controlled sequences and composition. Polymer therapeutics has helped to establish these polypeptide-based constructs as polymeric nanomedicines for different applications, such as disease treatment and diagnostics. Herein, we provide an overview of the advantages of these systems and the main methodologies for their synthesis, highlighting the different polypeptide architectures and the current research towards clinical applications.
Keywords: polymer therapeutics; polypeptides; drug delivery; nanomedicine
The preparation of the thiolsulphonate derivatives 3 and 7 of L-cysteine and glutathione, respectively, their corresponding S-nitroso derivatives 2 and 6, is described.
Small interfering RNA (siRNA) has great therapeutic potential for the suppression of proteins associated with disease, but delivery methods are needed for improved efficacy. Here, we investigated the properties of micellar siRNA delivery vehicles prepared with poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLL) comprising lysine amines modified to contain amidine and thiol functionality. Lysine modification was achieved using 2-iminothiolane (2-IT) [yielding PEG-b-PLL(N2IM-IM)] or dimethyl 3,3'-dithiobispropionimidate (DTBP) [yielding PEG-b-PLL(MPA)], with modifications aimed to impart disulfide cross-linking ability without compromising cationic charge. These two lysine modification reagents resulted in vastly different chemistry contained in the reacted block copolymer, which affected micelle formation behavior and stability along with in vitro and in vivo performance. Amidines formed with 2-IT were unstable and rearranged into a noncharged ring structure lacking free thiol functionality, whereas amidines generated with DTBP were stable. Micelles formed with siRNA and PEG-b-PLL(N2IM-IM) at higher molar ratios of polymer/siRNA, while PEG-b-PLL(MPA) produced micelles only near stoichiometric molar ratios. In vitro gene silencing was highest for PEG-b-PLL(MPA)/siRNA micelles, which were also more sensitive to disruption under disulfide-reducing conditions. Blood circulation was most improved for PEG-b-PLL(N2IM-IM)/siRNA micelles, with a circulation half-life 3× longer than naked siRNA. Both micelle formulations are promising for siRNA delivery applications in vitro and in vivo.
Tables of ^1H and ^(13)C NMR chemical shifts have been compiled for common organic compounds often used as reagents or found as products or contaminants in deuterated organic solvents. Building upon the work of Gottlieb, Kotlyar, and Nudelman in the Journal of Organic Chemistry, signals for common impurities are now reported in additional NMR solvents (tetrahydrofuran-d_8, toluene-d_8, dichloromethane-d_2, chlorobenzene-d_5, and 2,2,2-trifluoroethanol-d_3) which are frequently used in organometallic laboratories. Chemical shifts for other organics which are often used as reagents or internal standards or are found as products in organometallic chemistry are also reported for all the listed solvents.
The synthesis of well-defined polypeptide-based materials through the ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCA) was reviewed. Normal Amine Mechanism (NAM) is generally applied for the polymerization of NCAs initiated by nonionic initiators having at least one mobile hydrogen atom (base-H), such as primary and secondary amines, alcohols, and water. Water is a common impurity, which can influence the ROP of NCAs. The temperature of the system is also critical for the hydrolysis of NCAs, since at low temperatures it proceeds slower. Activated monomer mechanism (AMM) was proposed to explain the ROP of DL-phenylalanine NCA, initiated by a tertiary amine. Bamford's group found that Sar-NCA was almost unreactive when diisopropylamine was used as initiator, while the polymerization of γethyl-L-glutamate NCA is even faster under these conditions. The new developments in the ROP of NCAs hold tremendous promise that well-defined polypeptides with controllable molecular weight, sequence, composition, and molecular weight distribution can be synthesized.
The protection of the thiol function of cysteine with the 3-nitro-2-pyridylsulfenyl (Npys) group has been successfully applied in the solid phase synthesis of nine peptides. A reexamination of the chemical stability of the protecting group has shown that, while Npys is essentially suitable for standard Boc/benzyl synthesis conditions, it is inadequate for the Fmoc strategy. Its proven stability to "high" HF acidolysis can not be extended to "low-high" conditions without significant thiol deprotection. On the other hand, the Npys group is quite compatible with standard photolytical cleavage conditions. The stability of Npys to HF and its thiol-activating character allow its application in peptide-carrier protein conjugation reactions by specific coupling through cysteine residues in the peptide.
Syntheses and polymerizations of alpha-amino acid N-carboxyanhydrides (NCAs) were reported for the first time by Hermann Leuchs in 1906. Since that time, these cyclic and highly reactive amino acid derivatives were used for stepwise peptide syntheses but mainly for the formation of polypeptides by ring-opening polymerizations. This review summarizes the literature after 1985 and reports on new aspects of the polymerization processes, such as the formation of cyclic polypeptides or novel organometal catalysts. Polypeptides with various architectures, such as diblock, triblock, and multiblock sequences, and star-shaped or dendritic structures are also mentioned. Furthermore, lyotropic and thermotropic liquid-crystalline polypeptides will be discussed and the role of polypeptides as drugs or drug carriers are reviewed. Finally, the hypothetical role of NCAs in molecular evolution on the prebiotic Earth is discussed.
An improved method for the synthesis of glycosylated N-carboxyanhydrides, which are monomers for glycopeptide synthesis, is presented.
This article reviews recent developments in the polymerization of ar-amino acid-N-carboxyanhydrides (NCAs) to form polypeptides. Traditional methods used to polymerize these monomers are described, and limitations in the utility of these systems for the preparation of polypeptides with controlled molecular weights and narrow molecular weight distributions are discussed. The development of transition-metal-based initiators, which activate the monomers to form covalent active species, permits the formation of polypeptides via the living polymerization of NCAs. In these systems, polymer molecular weights are controlled by monomer-to-initiator stoichiometry, polydispersities are low, and block copolypeptides can be prepared. The scope and limitations of these initiators and their key features and mode of operation are described in detail in this highlight. (C) 2000 John Wiley & Sons, Inc.
Disulfides and polysulfides have the structure R1SSnR2, in which chains of sulfur atoms are terminated by two groups that may be the same, different, or connected in a ring. Chapter 7 considers only substances where the S-R bond involves a carbon linkage; a review is available of structures such as ROSnOR and R2NSnNR2) Disulfides (n = 1) and their oxidation products are by far the most important subclass, but trisulfides and higher sulfides have received much attention. This chapter discusses these bivalent sulfur compounds, with some attention to their oxidation products.
Polypept(o)ides combine the multifunctionality and intrinsic stimuli-responsiveness of synthetic polypeptides with the "stealth"-like properties of the polypeptoid polysarcosine (poly(N-methyl glycine)). This class of block copolymers can be synthesized by sequential ring opening polymerization of α-amino acid N-carboxy-anhydrides (NCAs) and correspondingly of the N-substituted glycine N-carboxyanhydride (NNCA). The resulting block copolymers are characterized by Poisson-like molecular weight distributions, full end group integrity, and dispersities below 1.2. While polysarcosine may be able to tackle the currently arising issues regarding the gold standard PEG, including storage diseases in vivo and immune responses, the polypeptidic block provides the functionalities for a specific task. Additionally, polypeptides are able to form secondary structure motives, e.g., α-helix or β-sheets, which can be used to direct self-assembly in solution. In this feature article, we review the relatively new field of polypept(o)ides with respect to synthesis, characterization, and first data on the application of block copolypept(o)ides in nanomedicine. The summarized data already indicates the great potential of polypept(o)ides.
Polymeric micelles (PM) are extensively used to improve the delivery of hydrophobic drugs. Many different PM have been designed and evaluated over the years, and some of them have steadily progressed through clinical trials. Increasing evidence suggests, however, that for prolonged circulation times and for efficient EPR-mediated drug targeting to tumors and to sites of inflammation, PM need to be stabilized, to prevent premature disintegration. Core-crosslinking is among the most popular methods to improve the in vivo stability of PM, and a number of core-crosslinked polymeric micelles (CCPM) have demonstrated promising efficacy in animal models. The latter is particularly true for CCPM in which (pro-) drugs are covalently entrapped. This ensures proper drug retention in the micelles during systemic circulation, efficient drug delivery to pathological sites via EPR, and tailorable drug release kinetics at the target site. We here summarize recent advances in the CCPM field, addressing the chemistry involved in preparing them, their in vitro and in vivo performance, potential biomedical applications, and guidelines for efficient clinical translation.
Core-shell structures based on polypept(o)ides combine stealth-like properties of the corona material polysarcosine with adjustable functionalities of the polypeptidic core. Mannose-bearing block copolypept(o)ides (PSar-block-PGlu(OBn)) have been synthesized using 11-amino-3,6,9-trioxa-undecyl-2,3,4,6-tetra-O-acetyl-O-α-D-mannopyranoside as initiator in the sequential ring-opening polymerization of α-amino acid N-carboxyanhydrides. These amphiphilic block copolypept(o)ides self-assemble into multivalent PeptoMicelles and bind to mannose-binding receptors as expressed by dendritic cells. Mannosylated micelles showed enhanced cell uptake in DC 2.4 cells and in bone marrow-derived dendritic cells (BMDCs) and therefore appear to be a suitable platform for immune modulation.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
The synthesis of triblock copolymers based on polysarcosine, poly-N-ε-t-butyloxycarbonyl-l-lysine, and poly-N-ε-t-trifluoroacetyl-l-lysine by ring-opening polymerization of the corresponding α-amino acid N-carboxyanhydrides (NCAs) is described. For the synthesis of N-ε-t-butyloxycarbonyl-l-lysine (lysine(Boc)) NCAs, an acid-free method using trimethylsilylchloride/triethylamine as hydrochloric acid (HCl) scavengers is presented. This approach enables the synthesis of lysine(Boc) NCA of high purity (melting point 138.3 °C) in high yields. For triblock copolypept(o)ides, the degree of polymerization (Xn ) of the polysarcosine block is varied between 200 and 600; poly-N-ε-t-butyloxycarbonyl-l-lysine and poly-N-ε-t-trifluoroacetyl-l-lysine blocks are designed to have a Xn in the range of 10-50. The polypeptide-polypeptoid hybrids (polypept(o)ides) can be synthesized with precise control of molecular weight, high end group integrity, and dispersities indices between 1.1 and 1.2. But more important, the use of tert-butyloxycarbonyl- and trifluoroacetyl-protecting groups allows the selective, orthogonal deprotection of both blocks, which enables further postpolymerization modification reactions in a block-selective manner. Therefore, the presented synthetic approach provides a versatile pathway to triblock copolypept(o)ides, in which functionalities can be separated in specific blocks.
Vinyl sulfone-substituted l-cysteine N-carboxyanhydride (VSCys-NCA) monomer was designed and developed to afford a novel and versatile family of vinyl sulfone (VS)-functionalized polypeptides, which further offer a facile access to functional polypeptide-based materials including glycopolypeptides, functional polypeptide coatings, and in situ forming polypeptide hydrogels through Michael-type addition chemistry under mild conditions. VSCys-NCA was obtained in two straightforward steps with a high overall yield of 76%. The copolymerization of γ-benzyl l-glutamate NCA (BLG-NCA), N-benzyloxycarbonyl-l-lysine NCA (ZLL-NCA), or l-leucine NCA (Leu-NCA) with VSCys-NCA using 1,1,1-trimethyl-N-2-propenylsilanamine (TMPS) as an initiator proceeded smoothly in DMF at 40 °C, yielding P(BLG-co-VSCys), P(ZLL-co-VSCys), or P(Leu-co-VSCys) with defined functionalities, controlled molecular weights, and moderate polydispersities (PDI = 1.15–1.50). The acidic deprotection of P(BLG-co-VSCys) and P(ZLL-co-VSCys) furnished water-soluble VS-functionalized poly(l-glutamic acid) (P(Glu-co-VSCys)) and VS-functionalized poly(l-lysine) (P(LL-co-VSCys)), respectively. These VS-functionalized polypeptides were amenable to direct, efficient, and selective postpolymerization modification with varying thiol-containing molecules such as 2-mercaptoethanol, 2-mercaptoethylamine hydrochloride, l-cysteine, and thiolated galactose providing functional polypeptides containing pendant hydroxyl, amine, amino acid, and saccharide, respectively. The contact angle and fluorescence measurements indicated that polymer coatings based on P(Leu-co-VSCys) allowed direct functionalization with thiol-containing molecules under aqueous conditions. Moreover, hydrogels formed in situ upon mixing aqueous solutions of P(Glu-co-VSCys) and thiolated glycol chitosan at 37 °C. These vinyl sulfone-functionalized polypeptides have opened a new avenue to a broad range of advanced polypeptide-based materials.
Two series (degree of polymerization: 20−200)
of polylysines with Z and TFA protecting groups were
synthesized, and their behavior in a range of analytical
methods was investigated. Gel permeation chromatography
of the smaller polypeptides reveals a bimodal distribution,
which is lost in larger polymers. With the help of GPC, NMR,
circular dichroism (CD), and MALDI-TOF, it was demon-
strated that the bimodal distribution is not due to terminated
chains or other side reactions. Our results indicate that the
bimodality is caused by a change in secondary structure of the
growing peptide chain that occurs around a degree of
polymerization of about 15. This change in secondary
structure interferes strongly with the most used analysis method for polymersGPCby producing a bimodal distribution as an artifact. After deprotection, the polypeptides were found to exhibit exclusively random coil conformation, and thus a monomodal GPC elugram was obtained. The effect can be explained by a 1.6-fold increase in the hydrodynamic volume at the coil−helix transition. This work demostrates that secondary structures need to be carefully considered when performing standard analysis on polypeptidic systems.
We report the synthesis of polysarcosine-block-polyglutamic acid benzylester (PSar-block-PGlu(OBn)) and polysarcosine-block-polylysine-ε-N-benzyloxycarbonyl (PSar-block-PLys(Z)) copolymers. The novel polypeptoid-block-polypeptide copolymers have been synthesized by ring opening polymerization (ROP) of N-carboxyanhydrides (NCAs). Polymerization conditions were optimized regarding protecting groups, block sequence and length. While the degree of polymerization of the PSar block length was set to be around 200 or 400, PGlu(OBn) and PLys(Z) block lengths were varied between 20 to 75. The obtained block copolypeptides had a total degree of polymerization of 220-475 and dispersity indices between 1.1 and 1.2. Having ensured a non-toxic behavior up to a concentration of 3 mg/mL in HEK293 cells the novel block copolymers have been applied to the synthesis of organic colloids (by miniemulsion polymerization and miniemulsion solvent evaporation process). Colloids of around 100 nm (miniemulsion polymerization) to 200 nm (miniemulsion process) have been prepared. Additionally, PSar-block-PGlu(OBn) copolymers have been used in a drug formulation of an adenylate cyclase inhibitor increasing solubility of the drug, which enhances its bioavailability reducing of intracellular cAMP levels.
Polystyrene sodium sulfinate reacted with thionitrites gave polystyrene thiolsulfonates. These immobilised thioalkylation reagents were used for an effective synthesis of mixed disulfides.
Five rare earth complexes are first introduced to catalyze ring opening polymerizations (ROPs) of ?-benzyl-L-glutamate N-carboxyanhydride (BLG NCA) and L-alanine NCA (ALA NCA) including rare earth isopropoxide (RE(OiPr)3), rare earth tris(2,6-di-tert-butyl-4-methylphenolate) (RE(OAr)3), rare earth tris(borohydride) (RE(BH4)3(THF)3), rare earth tris[bis(trimethylsilyl)amide] (RE(NTMS)3), and rare earth trifluoromethanesulfonate. The first four catalysts exhibit high activities in ROPs producing polypeptides with quantitative yields (>90%) and moderate molecular weight (MW) distributions ranging from 1.2 to 1.6. In RE(BH4)3(THF)3 and RE(NTMS)3 catalytic systems, MWs of the produced polypeptides can be controlled by feeding ratios of monomer to catalyst, which is in contrast to the systems of RE(OiPr)3 and RE(OAr)3 with little controllability over the MWs. End groups of the polypeptides are analyzed by MALDI-TOF MS and polymerization mechanisms are proposed accordingly. With ligands of significant steric hindrance in RE(OiPr)3 and RE(OAr)3, deprotonation of 3-NH of NCA is the only initiation mode producing a N-rare earth metallated NCA (i) responsible for further chain growth, resulting in a-carboxylic-?-aminotelechelic polypeptides after termination. In the case of RE(BH4)3(THF)3 with small ligands, another initiation mode at 5-CO position of NCA takes place simultaneously, resulting in a-hydroxyl-?-aminotelechelic polypeptides. In RE(NTMS)3 system, the protonated ligand hexamethyldisilazane (HMDS) initiates the polymerization and produces a-amide-?-aminotelechelic polypeptides.
The synthesis and chemical properties of a series of amino acid derivatives containing the thiolsulphonate functionality are described.
The kinetics of the reaction of mercaptans with both phenyl benzenethiolsulfinate (1) and phenyl benzenethiolsulfonate (2) have been studied in a series of carboxylate buffers in 60% aqueous dioxane as solvent. The dependence of reaction rate on pH shows that in each instance the mercaptide ion RS - is at least 10 7 more reactive toward 1 or 2 than is the corresponding RSH, a much larger difference in reactivity than might have been expected based on their relative reactivity in other substitutions. The thiolsulfonate 2 reacts considerably faster with mercaptide ion than does the thiolsulfinate 1, in marked contrast to the thermal stability of the two compounds, where 1 undergoes homolytic dissociation of the S-S bond much more readily than 2. As might be expected from the fact that attack of -S - anions on dicoordinate sulfur is generally very facile, the rate constants for reaction of mercaptide ions with 1 or 2 are all very large (10 5 to 2 × 10 7 M -1 sec -1).
Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is an important bioactive target for the design of environmentally benign herbicides. On the basis of previous virtual screening, 50 asymmetric aryl disulfides containing [1,2,4]triazole groups were synthesized and characterized by (1)H NMR, HRMS, and crystal structure. Compounds I-a, I-b, and I-p show K(i) values of 1.70, 4.69, and 5.57 μM, respectively, for wild type Arabidopsis thaliana AHAS (AtAHAS) and low resistance against mutant type AtAHAS W574L. At 100 mg L(-1) concentration, compounds I-a, II-a, and II-b exhibit 86.6, 81.7, and 87.5% in vivo rape root growth inhibition. CoMFA steric and electrostatic contour maps were established, and a possible binding mode was suggested from molecular docking, which provide valuable information to understand the key structural features of these disulfide compounds. To the authors' knowledge, this is the first comprehensive case suggesting that asymmetric aryl disulfides are novel AHAS inhibitors.
This article reviews recent developments in the polymerization of α-amino acid-N-carboxyanhydrides (NCAs) to form polypeptides. Traditional methods used to polymerize these monomers are described, and limitations in the utility of these systems for the preparation of polypeptides with controlled molecular weights and narrow molecular weight distributions are discussed. The development of transition-metal-based initiators, which activate the monomers to form covalent active species, permits the formation of polypeptides via the living polymerization of NCAs. In these systems, polymer molecular weights are controlled by monomer-to-initiator stoichiometry, polydispersities are low, and block copolypeptides can be prepared. The scope and limitations of these initiators and their key features and mode of operation are described in detail in this highlight.
A photoresponsive S-(o-nitrobenzyl)-l-cysteine N-carboxyanhydride (NBC-NCA) monomer was for the first time designed, and the related poly(S-(o-nitrobenzyl)-l-cysteine)-b-poly(ethylene glycol) (PNBC-b-PEO) block copolymers were synthesized from the ring-opening polymerization (ROP) of NBC-NCA in DMF solution at 25 °C. Their molecular structures, physical properties, photoresponsive self-assembly, and drug release of PNBC-b-PEO were thoroughly investigated. The β-sheet conformational PNBC block within copolymers presented a thermotropic liquid crystal phase behavior, and the crystallinity of PEO block was progressively suppressed over the PNBC composition. The characteristic absorption peaks of these copolymers at about 310 and 350 nm increased over UV irradiation time and then leveled off, indicating that the o-nitrobenzyl groups were gradually photocleaved from copolymers until the completion of photocleavage. The PNBC-b-PEO copolymers self-assembled into spherical nanoparticles in aqueous solution, presenting a photoresponsive self-assembly behavior, together with a size reduction of nanoparticles after irradiation. The anticancer drug doxorubicin can be released in a controlled manner by changing the light irradiation time, which was induced by gradually photocleaving the PNBC core of nanoparticles. This work provides a facile strategy not only for the synthesis of photoresponsive polypeptide-based block copolymers but also for the fabrication of photoresponsive nanomedicine potential for anticancer therapy.
Conformation-switchable glycopolypeptides were prepared by the living polymerization of glycosylated L-cysteine-N-carboxyanhydride (glyco-C NCA) monomers. These new monomers were prepared in high yield by coupling of alkene-terminated C-linked glycosides of D-galactose or D-glucose to L-cysteine using thiol-ene "click" chemistry, followed by their conversion to the corresponding glyco-C NCAs. The resulting glycopolypeptides were found to be water-soluble and α-helical in solution. Aqueous oxidation of the side-chain thioether linkages in these polymers to sulfone groups resulted in disruption of the α-helical conformations without loss of water solubility. The ability to switch chain conformation and remain water-soluble is unprecedented in synthetic polymers, and allows new capabilities to control presentation of sugar functionality in subtly different contexts.
We describe the synthesis of homo-, block, and clickable copolypeptide brushes from low surface area substrates using nickel-mediated surface-initiated polymerization of α-amino N-carboxyanhydrides.
A novel disulfide core cross-linked PEGylated polypeptide nanogel has been synthesized by a one-step ring opening copolymerization of γ-benzyl L-glutamate N-carboxyanhydride and L-cystine N-carboxyanhydride using an amino group-terminated poly(ethylene glycol) methyl ether as initiator. Characterization of products confirms the formation of a core cross-linked PEGylated nanogel with disulfide linkages with a size of about 250 nm, and these studies also confirm that this nanogel can easily be broken by glutathione. Cell viability experiments show the good biocompatibility of the as-prepared polymer. Studies relating to loading and controlled release of indomethacin under reduction-sensitive conditions reveal that the nanogel is a good candidate for drug delivery systems.
Well-defined cysteine-containing synthetic polypeptides were synthesised and the versatility of various chemical reactions on these thiol groups was investigated.
We report a new strategy that uses N-trimethylsilyl (N-TMS) amine to mediate controlled ring-opening polymerization of amino acid N-carboxyanhydrides (NCAs). This polymerization proceeds via a unique, trimethylsilyl carbamate (TMS-CBM) propagating group that results from the cleavage of the Si-N bond of N-TMS amine followed by NCA ring opening. Propagation of the polypeptide chains proceeds through the transfer of the TMS group from the terminal TMS-CBM to the incoming monomer followed by the formation of a new TMS-CBM propagating group. Formation of the TMS-CBM was confirmed by MS and NMR. Polypeptides formed by the N-TMS amine-mediated polymerization have the expected molecular weights, narrow molecular weight distributions, and controlled functional groups at the C-termini of the polypeptides.
A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).
The reaction of NCA's with some amino acids having a nucleophilic functional group on the side chain was studied in a heterogeneous reaction medium (acetonitrile-water). Glutamic acid and aspartic acid, having a free carboxyl group on the side chain, were successfully used to synthesize oligopeptides without interactions of the γ- and β-carboxyl group with NCA's. Two products were obtained by the reaction of NCA with L-lysine, which contains a free amino group on the side chain. ε-Protected lysine was used to prepare α-peptides as a nucleophile in the reaction. No racemization was observed in the synthesis of peptides by the NCA method in the heterogeneous solvent system. Oligopeptides with some polar side chains were synthesized by the NCA method.
Oligopeptides were synthesized in high yields by the controlled coupling reaction of N-carboxy α-amino acid anhydrides (NCA's) with amino acid and peptide sodium salts in the heterogeneous reaction medium of acetonitrile–water which contained sodium carbonate. In this solvent, it could be considered that the reaction could occur at the interface of acetonitrile and aqueous layers, and that NCA's could be protected by the organic layer from side reactions such as hydrolysis and polymerization. The careful control of reaction conditions such as pH of the solution was not necessary when the heterogeneous mixed solvent was used. Sodium carbonate which had not been used for a reaction of this kind could be satisfactorily used for stabilizing the carbamate intermediates in the aqueous layer of the heterogeneous reaction medium.
When the N-o-nitrophenylsulphenyl group is removed from cysteine peptides by means of hydrogen chloride in methanol or non-polar solvents, or by means of acids in aqueous methanol or acetone, an N → S transfer of the o-nitrophenylsulphenyl-group takes place, to give the corresponding S-o-nitrophenylsulphenyl derivative. Even in alkaline solution transfer of the o-nitrophenylsulphenyl group from the α-amino-group to the thiol can occur to some extent.
The novel 3-nitro-2-pyridinesulfenyl (Npys) group, which is useful for the protection and the activation of amino and hydroxyl groups for peptide synthesis, is reported. The Npys group is readily introduced by treatment of amino acids with 3-nitro-2-pyridinesulfenyl chloride. The Npys group is easily removed by treatment with very dilute HCl, e.g. 0.1-0.2 N HCl in dioxane, but is is resistant to trifluoroacetic acid and 88% formic acid. Npys is also selectively removed under neutral conditions using triphenylphosphine or 2-pyridinethiol 1-oxide without affecting benzyloxycarbonyl (Z), tert-butyloxycarbonyl (Boc), 2-(4-biphenylyl)propyl(2)oxycarbonyl (Bpoc), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyl (Bzl) or tert-butyl (tBu) protecting groups. The N-Npys and O-Npys groups when activated in the presence of RCOOH by the addition of tertiary phosphine form peptide or ester bonds via oxidation-reduction condensation. The important features of the Npys group are demonstrated through the synthesis of peptides in solution and by solid phase methodology without a formal deprotection procedure. In solid phase synthesis, 4-(Npys-oxymethyl) phenylacetic acid is used as the key intermediate for the introduction of the trifluoroacetic acid resistant 4-(oxymethyl) phenylacetamido linking group to the resin.
Many natural polymeric materials (particularly structural proteins) display a hierarchy of structure over several length scales. Block copolymers are able to self-assemble into ordered nanostructures, but the random-coiled nature of their polymer chains usually suppresses any further levels of organization. The use of components with regular structures, such as rigid-rod polymers, can increase the extent of spatial organization in self-assembling materials. But the synthesis of such polymeric components typically involves complicated reaction steps that are not suitable for large-scale production. Proteins form hierarchically organized structures in which the fundamental motifs are generally alpha-helical coils and beta-sheets. Attempts to synthesize polypeptides with well-defined amino-acid sequences, which might adopt similar organized structures, have been plagued by unwanted side reactions that give rise to products with a wide range of molecular weights, hampering the formation of well-defined peptide block copolymers. Here I describe a polymerization strategy that overcomes these difficulties by using organonickel initiators which suppress chain-transfer and termination side reactions. This approach allows the facile synthesis of block copolypeptides with well-defined sequences, which might provide new peptide-based biomaterials with potential applications in tissue engineering, drug delivery and biomimetic composite formation.
A block catiomer polyplex, showing a high stability in the extracellular medium and an efficient release of plasmid DNA (pDNA) in the intracellular compartment, was developed by controlling both the cationic charge and disulfide cross-linking densities of the backbone polycations. Poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) was thiolated using either of two thiolation reagents, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or 2-iminothiolane (Traut's reagent), to investigate the effects of both the charge and disulfide cross-linking densities on the properties of the polyplexes. The introduction of thiol groups by SPDP proceeded through the formation of amide linkages to concomitantly decrease the cationic charge density of PLL segment, whereas Traut's reagent promoted the thiolation with the introduction of cationic imino groups to keep the charge density constant. These thiolated PEG-PLLs were complexed with pDNA to form the disulfide cross-linked block catiomer polyplexes, which had the size of approximately 100 nm. Both thiolation methods were similarly effective in introducing disulfide cross-links to prevent the polyplex from the dissociation through a counter polyanion exchange in the extracellular oxidative condition. On the other hand, the efficient release of pDNA responding to the reductive condition mimicking the intracellular environment was only achieved for the polyplex thiolated with SPDP, a system compensating for the decrease in the charge density with the disulfide cross-linking. This distinctive sensitivity toward oxidative and reductive environments was nicely correlated with the remarkable difference in the transfection efficiency between these two types of thiolated polyplexes (SPDP and Traut's reagent types): the former revealed approximately 50 times higher transfection efficiency toward 293T cells than the latter. Obviously, the balance between the densities of the cationic charge and disulfide cross-linking in the thiolated polyplex played a crucial role in the delivery and controlled release of entrapped pDNA into the microenvironment of intracellular compartment to achieve the high transfection efficiency.
Ring-opening polymerizations of alpha-amino acid N -carboxyanhydrides (NCAs) initiated with amines typically form polypeptides with uncontrolled molecular weights and broad molecular weight distributions. However, we found that hexamethyldisilazane (HMDS)mediated controlled NCA polymerizations gave polypeptides with predictable molecular weights and narrow molecular weight distributions. Using MS, NMR, and FT-IR, we demonstrated that the initiation step involved the cleavage of the N-Si bond of HMDS and the formation of a trimethylsilyl carbamate (TMS-CBM) terminal group. Polypeptide chains were propagated through the migration of TMS of the TMS-CBM end group to the incoming monomer and formed a new TMS-CBM terminal group. This organosilicon reagent mediated NCA polymerization offers a metal-free strategy for the convenient synthesis of homo- or block polypeptides with predictable molecular weights and narrow molecular weight distributions.
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