Exploiting the Potential of Meroterpenoid Cyclases to Expand the Chemical Space of Fungal Meroterpenoids

Fungal meroterpenoids are a diverse group of hybrid natural products with impressive structural complexity and high potential as drug candidates. In this work, we evaluate the promiscuity of the early structure diversity-generating step in fungal meroterpenoid biosynthetic pathways: the multibond-forming polyene cyclizations catalyzed by the yet poorly understood family of fungal meroterpenoid cyclases. In total, 12 unnatural meroterpenoids were accessed chemoenzymatically using synthetic substrates. Their complex structures were determined by 2D NMR studies as well as crystalline-sponge-based X-ray diffraction analyses. The results obtained revealed a high degree of enzyme promiscuity and experimental results which together with quantum chemical calculations provided a deeper insight into the catalytic activity of this new family of non-canonical, terpene cyclases. The knowledge obtained paves the way to design and engineer artificial pathways towards second generation meroterpenoids with valuable bioactivities based on combinatorial biosynthetic strategies.     

Direct Synthesis of Pyridyl Disulfide‐Terminated Polymers by RAFT Polymerization

A trithiocarbonate RAFT agent was modified with a pyridyl disulfide group and used in the direct synthesis of endgroup pyridyl disulfide‐functionalized homo‐ and amphiphilic block copolymers of oligo(ethyleneglycol) acrylate (PEG‐A) and butyl acrylate (BA). Both the homo‐ and copolymerizations were found to be well controlled via the RAFT mechanism. The NMR analysis indicated that both the homopolymers of PEG‐A and the amphiphilic diblock copolymers of PEG‐A and BA possessed pyridyl disulfide terminal groups. A UV‐Vis absorption test revealed that the pyridyl disulfide endgroup of the polymer could be efficiently used to couple thiol‐bearing molecules to the polymer without the need for any post‐polymerization modification. This communication presents the first efficient direct synthesis of thiol‐reactive endgroup‐functionalized well‐defined polymers via the RAFT technique.

     

Competition for cores in remanufacturing

We study competition between an original equipment manufacturer (OEM) and an independently operating remanufacturer (IO). Different from the existing literature, the OEM and IO compete not only for selling their products but also for collecting returned products (cores) through their acquisition prices. We consider a two-period model with manufacturing by the OEM in the first period, and manufacturing as well as remanufacturing in the second period. We find the optimal policies for both players by establishing a Nash equilibrium in the second period, and then determine the optimal manufacturing decision for the OEM in the first period. This leads to a number of managerial insights. One interesting result is that the acquisition price of the OEM only depends on its own cost structure, and not on the acquisition price of the IO. Further insights are obtained from a numerical investigation. We find that when the cost benefits of remanufacturing diminishes and the IO has more chance to collect the available cores, the OEM manufactures less in the first period as the market in the second period gets larger to protect its market share. Finally, we consider the case where consumers have lower willingness to pay for the remanufactured products and find that in that case remanufacturing becomes less profitable overall.     

pH-labile sheddable block copolymers by RAFT polymerization: Synthesis and potential use as siRNA conjugates

Well-defined amphiphilic block copolymers composed of hydrophilic and hydrophobic blocks linked through an acid-labile acetal bond were synthesized directly by RAFT polymerization using a new poly(ethylene glycol) (PEG) macroRAFT agent modified with an acid-labile group at its R-terminal. The new macroRAFT agent was used for polymerization of poly(t-butyl methacrylate) (PtBMA) or poly(cholesterol-methacrylate) (PCMA) to synthesize well-defined block copolymers with a PEG block sheddable under acidic conditions. The chain extension polymerization kinetics showed known traits of RAFT polymerization. The molecular weight distributions of the copolymers prepared using the new macroRAFT agent remained below 1.2 during the polymerizations and the molecular weight of the copolymers was linearly proportional to monomer conversions. The acid-catalyzed hydrolysis behavior of the PEG-macroRAFT agent and the PEG-b-PtBMA (Mn = 13,600 by GPC, PDI = 1.10) was studied by GPC, ¹H NMR and UV–vis spectroscopy. The half-life of acid-hydrolysis was 70 min at pH 2.2 and 92 h at pH 4.0. The potential use of the pH-labile shedding behavior of the copolymers was demonstrated by conjugating a thiol-modified siRNA to ω-pyridyldisulfide modified PEG-b-PCMA. The resultant PEG-b-PCMA-b-siRNA triblock modular polymer released PCMA-b-siRNA segment in acidic and siRNA segment in reductive conditions, as confirmed by polyacrylamide gel electrophoresis.     

Insight into serum protein interactions with functionalized magnetic nanoparticles in biological media

Surface modification with linear polymethacrylic acid (20 kDa), linear and branched polyethylenimine (25 kDa), and branched oligoethylenimine (800 Da) is commonly used to improve the function of magnetite nanoparticles (MNPs) in many biomedical applications. These polymers were shown herein to have different adsorption capacity and anticipated conformations on the surface of MNPs due to differences in their functional groups, architectures, and molecular weight. This in turn affects the interaction of MNPs surfaces with biological serum proteins (fetal bovine serum). MNPs coated with 25 kDa branched polyethylenimine were found to attract the highest amount of serum protein while MNPs coated with 20 kDa linear polymethacrylic acid adsorbed the least. The type and amount of protein adsorbed, and the surface conformation of the polymer was shown to affect the size stability of the MNPs in a model biological media (RPMI-1640). A moderate reduction in r(2) relaxivity was also observed for MNPs suspended in RPMI-1640 containing serum protein compared to the same particles suspended in water. However, the relaxivities following protein adsorption are still relatively high making the use of these polymer-coated MNPs as Magnetic Resonance Imaging (MRI) contrast agents feasible. This work shows that through judicious selection of functionalization polymers and elucidation of the factors governing the stabilization mechanism, the design of nanoparticles for applications in biologically relevant conditions can be improved.     

Modification of RAFT‐polymers via thiol‐ene reactions: A general route to functional polymers and new architectures

An investigation into the aminolysis of ω‐end groups of RAFT‐polymers and simultaneous thiol‐ene reactions with ene‐bearing compounds is described. Three different polymers, P(MMA), P(HPMA), and P(NIPAAm), with low PDIs were synthesized using dithiobenzoate and trithiocarbonate RAFT agents. P(NIPAAm) synthesized with trithiocarbonate RAFT agent and P(HPMA) synthesized with dithiobenzoate RAFT agent were both functionalized with a methacrylate‐modified mannose and a maleimide‐modified biotin via one‐pot simultaneous aminolysis and thiol‐ene reactions with product yields above 85%. The presence of ene‐compounds during aminolysis was shown to prevent the formation of disulfide interchain crosslinking. Using the same approach, P(MMA), P(HPMA), and P(NIPAAm) were converted to (meth)acrylate macromonomers with high yields (>80%). In the case of P(MMA), the simultaneous aminolysis and thiol‐ene addition prevented any intrachain side reactions, i.e., thiolactone formation. New architectures such as graft and block copolymers were successfully generated from the macromonomers. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3773–3794, 2009     

A simple methodology for the synthesis of heterotelechelic protein–polymer–biomolecule conjugates

A synthetic protocol for the preparation of hetero‐biofunctional protein–polymer conjugates is described. A chain transfer agent, S,S‐bis (α,α′‐dimethyl‐α″‐acetic acid) trithiocarbonate was functionalized with α,ω‐pyridyl disulfide (PDS) groups, Subsequently, one of the PDS groups was covalently attached to bovine serum albumin (BSA) at the specific free thiol group on the cysteine residue through a disulfide linkage. The second PDS group remained intact, as it was found to be inaccessible to further BSA functionalization. The BSA‐macro‐reversible addition‐fragmentation chain transfer (RAFT) agent was then used to prepare BSA‐polymer conjugates via in situ polymerization of oligo (ethyleneglycol) acrylate and N‐(2‐hydroxypropyl) methacrylamide using an ambient temperature initiator, 4,4′‐azobis [2,9‐imidazolin‐2‐ethyl)propane] dihydrochloride in an aqueous medium. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) confirmed that the in situ polymerization occurred at the protein surface where the RAFT agent was attached and the molecular weights of the BSA–polymer conjugates were found to increase concomitantly with monomer conversion and polymerization time. After polymerization the remaining terminal PDS groups were then utilized to attach thiocholesterol and a flurophore, rhodamine B to the protein–polymer conjugates via disulfide coupling. UV–Vis and fluorescence analyses revealed that ～80% of the protein conjugates were found to retain integral PDS end groups for further attachment to free thiol‐tethered precursors. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1399–1405, 2010     

Synthesis of dendritic carbohydrate end‐functional polymers via RAFT: Versatile multi‐functional precursors for bioconjugations

Well‐defined pyridyl disulfide (PDS) end‐functionalized polymer‐dendritic carbohydrate scaffolds are reported as novel precursors for the attachment of biomolecules. This synthetic approach combines reversible addition fragmentation chain transfer (RAFT) polymerization and “click” reactions. Poly(N‐(2‐hydroxypropyl) methacrylamide) (PHPMA) with 2‐mercaptothiozalidine end‐groups was prepared by RAFT polymerization yielding molecular weights of M_n = 4300 and 9900, both with a polydispersity of less than 1.2. These polymers were then attached to dendritic mannose scaffolds preconstructed via consecutive “click” reactions. Finally, the ω‐dithiobenzoate RAFT end‐group of PHPMA was modified to yield PDS functionality, by aminolysis in the presence of 2,2′‐dithiodipyridine. This PDS end‐functionalized PHPMA‐dendritic carbohydrate scaffold is a versatile precursor for bioconjugations, as the synthetic procedure can easily accommodate a range of sugar functionalities. In addition, the PDS groups can be used to react with any thiol present in a biomolecule (e.g., cysteine residue in proteins, or ? SH terminal nucleotides). To demonstrate the utility of these scaffolds we describe their bioconjugation to short interfering RNA. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 4302–4313, 2009     

Well-defined protein-polymer conjugates via in situ RAFT polymerization

Biotechnology, biomedicine, and nanotechnology applications would benefit from methods generating well-defined, monodisperse protein-polymer conjugates, avoiding time-consuming and difficult purification steps. Herein, we report the in situ synthesis of protein-polymer conjugates via reversible addition-fragmentation chain transfer polymerization (RAFT) as an efficient method to generate well-defined, homogeneous protein-polymer conjugates in one step, eliminating major postpolymerization purification steps. A water soluble RAFT agent was conjugated to a model protein, bovine serum albumin (BSA), via its free thiol group at Cys-34 residue. The conjugation of the RAFT agent to BSA was confirmed by UV-visible spectroscopy, matrix-assisted laser desorption ionization--time of flight (MALDI-TOF), and 1H NMR. BSA-macroRAFT agent was then used to control the polymerization of two different water soluble monomers, N-isopropylacrylamide (NIPAAm) and hydroxyethyl acrylate (HEA), in aqueous medium at 25 degrees C. The growth of the polymer chains from BSA-macroRAFT agent was characterized by size exclusion chromatography (SEC), 1H NMR, MALDI-TOF, and polyacrylamide gel electrophoresis (PAGE) analyses. The controlled character of the RAFT polymerizations was confirmed by the linear evolution of molecular weight with monomer conversion. The SEC analyses showed no detectable free, nonconjugated polymer formation during the in situ polymerization. The efficiency of BSA-macroRAFT agent to generate BSA-polymer conjugates was found to be ca. 1 by deconvolution of the SEC traces of the polymerization mixtures. The structural integrity and the conformation-related esterase activity of BSA were found to be unaffected by the polymerization conditions and the conjugation of the polymer chain. BSA-poly(NIPAAm) conjugates showed hybrid temperature-dependent phase separation and aggregation behavior. The lower critical solution temperature values of the conjugates were found to increase with the decrease in molecular weight of poly(NIPAAm) block conjugated to BSA.