Respective Monomers Research Articles

Novel Bispecific PSMA/GRPr Targeting Radioligands with Optimized Pharmacokinetics for Improved PET Imaging of Prostate Cancer

A new series of bispecific radioligands (BRLs) targeting prostate-specific membrane antigen (PSMA) and gastrin releasing peptide receptor (GRPr), both expressed on prostate cancer cells, was developed. Their design was based on the bombesin (BN) analogue, H2N-PEG2-[D-Tyr(6),β-Ala(11),Thi(13),Nle(14)]BN(6-14), which binds to GRPr with high affinity and specificity, and the peptidomimetic urea-based pseudoirreversible inhibitor of PSMA, Glu-ureido-Lys. The two pharmacophores were coupled through copper(I)-catalyzed azide-alkyne cycloaddition to the bis(tetrafluorophenyl) ester of the chelating agent HBED-CC via amino acid linkers made of positively charged His (H) and negatively charged Glu (E): -(HE)n- (n = 0-3). The BRLs were labeled with (68)Ga, and their preliminary pharmacological properties were evaluated in vitro (competitive and time kinetic binding assays) on prostate cancer (PC-3, LNCaP) and rat pancreatic (AR42J) cell lines and in vivo by biodistribution and small animal PET imaging studies in both normal and tumor-bearing mice. The IC50/Ki values determined for all BRLs essentially matched those of the respective monomers. The maximal cellular uptake of the BLRs was observed between 20 and 30 min. The BRLs showed a synergistic ability in vivo by targeting both PSMA (LNCaP) and GRPr (PC-3) positive tumors, whereas the charged -(HE)n- (n = 1-3) linkers significantly reduced the kidney and spleen uptake. The bispecific (PSMA and GRPr) targeting ability and optimized pharmacokinetics of the compounds developed in this study could lead to their future application in clinical practice as more sensitive radiotracers for noninvasive imaging of prostate cancer (PCa) by PET/CT and PET/MRI.

Recent advances in metathesis-derived polymers containing transition metals in the side chain.

This account critically surveys the field of side-chain transition metal-containing polymers as prepared by controlled living ring-opening metathesis polymerization (ROMP) of the respective metal-incorporating monomers. Ferrocene- and other metallocene-modified polymers, macromolecules including metal-carbonyl complexes, polymers tethering early or late transition metal complexes, etc. are herein discussed. Recent advances in the design and syntheses reported mainly during the last three years are highlighted, with special emphasis on new trends for superior applications of these hybrid materials.

Synthesis and characterization of well‐defined random and block copolymers of ε‐caprolactone with l‐lactide as an additive for toughening polylactide: Influence of the molecular architecture

ABSTRACTWell‐defined multiarmed star random and block copolymers of ε‐caprolactone with l‐lactide with controlled molecular weights, low polydispersities, and precise numbers of arms were synthesized by the ring‐opening polymerization of respective cyclic ester monomers. The polymers were characterized by 1H‐NMR and 13C‐NMR to determine their chemical composition, molecular structure, degree of randomness, and proof of block copolymer formation. Gel permeation chromatography was used to establish the degree of branching. Star‐branched random copolymers exhibited lower glass‐transition temperatures (Tg's) compared to a linear random copolymer. When the star random copolymers were melt‐blended with poly(l‐lactic acid) (PLA), we observed that the elongation of the blend increased with the number of arms of the copolymer. Six‐armed block copolymers, which exhibited higher Tg's, caused the maximum improvement in elongation. In all cases, improvements in the elongation were achieved with no loss of stiffness in the PLA blends. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43267.

Combination of six enzymes of a marine Novosphingobium converts the stereoisomers of β-O-4 lignin model dimers into the respective monomers.

Lignin, an aromatic polymer of phenylpropane units joined predominantly by β-O-4 linkages, is the second most abundant biomass component on Earth. Despite the continuous discharge of terrestrially produced lignin into marine environments, few studies have examined lignin degradation by marine microorganisms. Here, we screened marine isolates for β-O-4 cleavage activity and determined the genes responsible for this enzymatic activity in one positive isolate. Novosphingobium sp. strain MBES04 converted all four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGGE), a structural mimic of lignin, to guaiacylhydroxypropanone as an end metabolite in three steps involving six enzymes, including a newly identified Nu-class glutathione-S-transferase (GST). In silico searches of the strain MBES04 genome revealed that four GGGE-metabolizing GST genes were arranged in a cluster. Transcriptome analysis demonstrated that the lignin model compounds GGGE and (2-methoxyphenoxy)hydroxypropiovanillone (MPHPV) enhanced the expression of genes in involved in energy metabolism, including aromatic-monomer assimilation, and evoked defense responses typically expressed upon exposure to toxic compounds. The findings from this study provide insight into previously unidentified bacterial enzymatic systems and the physiological acclimation of microbes associated with the biological transformation of lignin-containing materials in marine environments.

Atomistic details of the molecular recognition of DNA-RNA hybrid duplex by ribonuclease H enzyme

Bacillus halodurans (Bh) ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and is a prototypical member of a large family of enzymes that use two-metal ion (Mg2+ or Mn2+) catalysis to cleave nucleic acids. Long timescale molecular dynamics simulations have been performed on the BhRNase H-DNA-RNA hybrid complex and the respective monomers to understand the recognition mechanism, conformational preorganization, active site dynamics and energetics involved in the complex formation. Several structural and energetic analyses were performed and significant structural changes are observed in enzyme and hybrid duplex during complex formation. Hybrid molecule binding to RNase H enzyme leads to conformational changes in the DNA strand. The ability of the DNA strand in the hybrid duplex to sample conformations corresponding to typical A- and B-type nucleic acids and the characteristic minor groove widthseem to be crucial for efficient binding. Sugar moieties in certain positions interacting with the protein structure undergo notable conformational transitions. The water coordination and arrangement around the metal ions in active site region are quite stable, suggesting their important role in enzymatic catalysis. Details of key interactions found at the interface of enzyme-nucleic acid complex that are responsible for its stability are discussed.

Synthesis of Soluble Ferrocene-Based Polythiophenes and Their Properties

Ferrocene-based polythiophenes are the promising class of the functional materials, but mostly the polythiophenes are insoluble in common solvents except some derivatives of polythiophenes like poly(3-hexylthiophene) (P3HT). Herein, two new ferrocene-based thiophene monomers, 2-(thiophen-3-yl) ethyl ferrocenoate and 2-(thiophen-3-yl) methyl ferrocenoate were synthesized and copolymerized with 3-hexylthiophene (3HT) respectively. The resulting polymers were soluble (in common organic solvents) and processable. The synthesis and the molecular weights were confirmed by the 1H-NMR and GPC studies respectively. Poly (TEF-co-3HT) was subjected to study the electrochemical properties in comparison of respective monomers and ferrocene. It was found that the resulting copolymer had the combined redox properties of its monomers. Also, very interestingly, the copolymer showed improved reversible redox properties which were not only better than its parent monomers but even better than the pure ferrocene. Photoemission spectra of P3HT and Poly (TEF-co-3HT) were also rerecorded and compared with each other.

Next Step toward Optimization of GRP Receptor Avidities: Determination of the Minimal Distance between BBN(7-14) Units in Peptide Homodimers.

As the gastrin releasing peptide receptor (GRPR) is overexpressed on several tumor types, it represents a promising target for the specific in vivo imaging of these tumors using positron emission tomography (PET). We were able to show that PESIN-based peptide multimers can result in substantially higher GRPR avidities, highly advantageous in vivo pharmacokinetics and tumor imaging properties compared to the respective monomers. However, the minimal distance between the peptidic binders, resulting in the lowest possible system entropy while enabling a concomitant GRPR binding and thus optimized receptor avidities, has not been determined so far. Thus, we aimed here to identify the minimal distance between two GRPR-binding peptides in order to provide the basis for the development of highly avid GRPR-specific PET imaging agents. We therefore synthesized dimers of the GRPR-binding bombesin analogue BBN(7-14) on a dendritic scaffold, exhibiting different distances between both peptide binders. The homodimers were further modified with the chelator NODAGA, radiolabeled with (68)Ga, and evaluated in vitro regarding their GRPR avidity. We found that the most potent of the newly developed radioligands exhibits GRPR avidity twice as high as the most potent reference compound known so far, and that a minimal distance of 62 bond lengths between both peptidic binders within the homodimer can result in concomitant peptide binding and optimal GRPR avidities. These findings answer the question as to what molecular design should be chosen when aiming at the development of highly avid homobivalent peptidic ligands addressing the GRPR.

Artificial human Met agonists based on macrocycle scaffolds.

Hepatocyte growth factor (HGF) receptor, also known as Met, is a member of the receptor tyrosine kinase family. The Met–HGF interaction regulates various signalling pathways involving downstream kinases, such as Akt and Erk. Met activation is implicated in wound healing of tissues via multiple biological responses triggered by the above-mentioned signalling cascade. Here we report the development of artificial Met-activating dimeric macrocycles. We identify Met-binding monomeric macrocyclic peptides by means of the RaPID (random non-standard peptide integrated discovery) system, and dimerize the respective monomers through rational design. These dimeric macrocycles specifically and strongly activate Met signalling pathways through receptor dimerization and induce various HGF-like cellular responses, such as branching morphogenesis, in human cells. This work suggests our approach for generating dimeric macrocycles as non-protein ligands for cell surface receptors can be useful for developing potential therapeutics with a broad range of potential applications.

Tuning electron donor-acceptor hybrids by alkali metal complexation.

A zinc phthalocyanine endowed with four [18]-crown-6 moieties, ZnPcTeCr, has been prepared and self-assembled with either pyridyl-functionalized perylenebisimides (PDI-Py) or fullerenes (C60-Py) to afford a set of novel electron donor-acceptor hybrids. In the case of ZnPcTeCr, aggregation has been circumvented by the addition of potassium or rubidium ions to lead to the formation of monomers and cofacial dimers, respectively. From fluorescence titration experiments, which gave rise to mutual interactions between the electron donors and the acceptors in the excited state, the association constants of the respective ZnPcTeCr monomers and/or dimers with the corresponding electron acceptors were derived. Complementary transient-absorption experiments not only corroborated photoinduced electron transfer from ZnPcTeCr to either PDI-Py or C60-Py within the electron donor-acceptor hybrids, but also the unexpected photoinduced electron transfer within ZnPcTeCr dimers. In the electron donor-acceptor hybrids, the charge-separated-state lifetimes were elucidated to be close to 337 ps and 3.4 ns for the two PDI-Pys, whereas the longest lifetime for the photoactive system that contains C60-Py was calculated to be approximately 5.1 ns.

Tetrahydrothiophene and Tetrahydrofuran, Computational and X-ray Studies in the Crystalline Phase

Calculations at various levels of theory with different methods and respective evaluations confirm that the twist conformation (C2) is preferred for tetrahydrothiophene (THT) in the gas phase. In the crystalline phase, achieved by a laser assisted crystallization device, THT has C1 symmetry (slightly distorted C2 symmetry) in the chiral space group P212121. This is obviously a packing effect caused by the nonsymmetrical arrangement of neighboring molecules. The distortion from C2 symmetry costs very little energy as confirmed by computational methods in the gas phase. Only one enantiomer of the chiral THT is found in the cell which requires spontaneous crystallization, which results in a racemic mixture of crystals, or a racemization occurs prior to/during nucleation or in the “embryonic” state. The racemization happens by a mechanism that can be described as a partial pseudo rotation within a five-membered mono-heterocycle with a C2–CS–C2′ transition (C2 and C2′ are enantiomers) maintaining the heteroatom residing within the symmetry elements. While THT has the molecular symmetry of the gas phase almost also in the crystalline phase, THF has an envelope conformation (CS). This was also established by calculations at various levels of theory which agrees well with the previously experimentally found conformation by electron diffraction. However, in the X-ray crystal structure, previously determined by Luger & Buschmann, THF has C2 symmetry in the centrosymmetric space group C2/c with the oxygen atom situated on the crystallographic C2 polar axis, requesting a racemic crystal for the twisted conformers of the enantiomers. No solid-state phase transitions were detected within the experimental ranges for THT and THF. Following the stabilization by molecular clustering, and ending at the crystal lattice, we stepwise increased the number of molecules by calculation of the respective monomers, dimers, trimers, and tetramers for THF and THT. The starting point was taken from the arrangements as found in the respective crystal structures. Both conformational enantiomers are equal in energy. In such cases, a crystal may contain either a racemate of conformers or one of the conformational enantiomers only. The first case is observed in THF, the latter one in THT. It is quite likely that the selection of one enantiomeric conformer of THT from an equilibrium of conformers at the early stage of nucleation (“embryonic” stage) is responsible for the spontaneous crystallization. In order to check if THF could form a polymorph with the molecular packing of THT and vice versa, we first calculated THF and THT in their respective crystal lattices as determined by X-ray diffraction. Exchanging the compounds in the THT and THF crystal lattices (i.e., replacing O against S and vice versa) results in significantly worse lattice energies indicating that such a polymorph is not a probable option.

Ratiometric fluorescent nanosensors for copper(II) based on bis(rhodamine)-derived PMOs with J-type aggregates.

By using pentyl-linked bis(rhodamine)-derived tetra-siloxane (PRh-Si4 ) as the organosilica precursor, highly ordered PRh-bridged periodic mesoporous organosilicas (PRhPMOs) were prepared. When excited at λ=500 nm, the PRhPMO suspension that contained metal ions showed two separate emission peaks at λ=550 and 623 nm. The first peak, located at λ=550 nm, was due to ring-opening of the spiro structure in the rhodamine moiety and the second, located at λ=623 nm, originated from fluorescent aggregates of the PRh units embedded in the silica framework of the PRhPMO. By using the different intensity ratios of the two fluorescence signals (FI550/623), PRhPMOs could be used as turn-ON type fluorescent ratiometric chemosensors for Cu(2+). Furthermore, based on the single-exciton theory, it was deduced that the fluorescent aggregates formed were of the J-type and had a coplanar configuration. Consequently, PRhPMOs display a longer fluorescence lifetime and greater fluorescent quantum yield than the respective monomers dissolved in solution, which is consistent with the experimental results.

Corrole-ferrocene and corrole-anthraquinone dyads: synthesis, spectroscopy and photochemistry.

Two different donor-acceptor systems based on corrole-ferrocene and corrole-anthraquinone having the 'Olefin Bridge' at the β-pyrrole position have been designed and synthesized. Both the dyads corrole-ferrocene () and corrole-anthraquinone () are characterized by elemental analysis, ESI-MS, (1)H NMR, UV-Visible, fluorescence spectroscopies (steady-state, femtosecond time-resolved), femtosecond transient absorption spectroscopy (fs-TA) and electrochemical methods. (1)H-NMR shows that two doublets at 6.50 and 7.25(δ) ppm belong to vinylic protons, which are characteristic of the formation of dyads. UV-Visible absorption spectra showed that dyads are merely superpositions of their respective constituent monomers and dominated by corrole S1 ← S0 (Q-band) and S2 ← S0 (Soret band) transitions with a systematic red-shift of both Soret and Q-bands along with the broadening of the bands. A prominent splitting of the Soret band for both the dyads is observed due to bulky substitutions at the peripheral position, which deviate from the planarity of the corrole macrocycle. Both the dyads exhibit significant fluorescence emission quenching (95-97%) of corrole emission compared to the free-base corrole monomer. Emission quenching is attributed to the excited-state intramolecular photoinduced electron transfer (PET) from corrole to anthraquinone in the dyad, whereas in the dyad it is reversed. The electron-transfer rates (kET) for and were found to be 3.33 × 10(11) and 2.78 × 10(10) s(-1), respectively. Despite their very different driving forces, charge separation (CS) and charge recombination (CR) are found to be in identical timescales.

Coaxial conducting polymer nanotubes: polypyrrole nanotubes coated with polyaniline or poly(p-phenylenediamine) and products of their carbonisation

AbstractPolypyrrole nanotubes were prepared by the oxidation of pyrrole with iron(III) chloride in a reaction mixture containing methyl orange. They were subsequently coated with polyaniline or poly(p-phenylenediamine) in situ during the oxidation of respective monomers in their presence. A part of the coaxial nanotubes was deprotonated using ammonia solution. The conductivity of polypyrrole nanotubes of 60 S cm

Modified Nonconventional Synthesis of New Terpoly(Aniline, o-Anthranilic Acid and o-Phenylenediamine)/Bentonite Composites

Terpolymers of aniline, anthranilic acid and o-phenylenediamine/Hydrophilic bentonite composites were synthesized using a 1:1:1 molar ratio of the respective monomers with different percentages of clay via modified in situ chemical terpolymerization. The spectral characteristics upon incorporation of anthranilic acid units into the copoly(aniline o-phenylenediamine) backbone in absence and in presence of bentonite nanoparticles were investigated. Moreover, terpolymer/bentonite composites were thermally more stable than pure terpolymer under nonoxidative condition. On the basis of surface morphological studies of terpolymer composites, a platy network texture can be explained, and it can be clearly seen in the SEM photomicrographs.

New strategy for high-level expression and purification of biologically active monomeric TGF-β1/C77S in Escherichia coli.

Mature transforming growth factor beta1 (TGF-β1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-β1/C77S (further termed TGF-β1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding enteropeptidase recognition site. High-level expression (~1.5 g l(-1)) of Trx/TGF-β1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-β1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-β1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-β1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-β1m from bacteria with a yield of about 2.8 mg from 100 ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-β1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.

Terpolymers as precursors for CuO nanoparticles synthesis

ABSTRACTThis work describes the synthesis of terpolymers of aniline, diphenylamine, and o‐anthranilic acid (PANIDPAA) by 1 : 1 : 1 molar ratio of the respective monomers doped by different concentration of copper ions via in situ chemical terpolymerization. The results are justified by measuring spectral characteristics namely, UV‐vis absorption spectra, FTIR, and TGA. Calcining these PANIDPAA terpolymers doped by copper at temperatures in the range of 700°C led to the formation of CuO nanoparticles in the nanoscale by thermal decomposition in air directly. The stages of decompositions and the calcination temperature of the precursors have been determined from thermal analysis data sheet. The obtained CuO nanoparticles have been characterized by X‐ray diffraction and transmission electron microscope (TEM). TEM showed a particle size less than 40 nm. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 41150.

New Crystalline Aniline and o-Anthranilic Acid Copolymer/Kaolinite Composites

Copolymers of aniline and o-anthranilic acid (PANAA)/kaolinite clay composites were synthesized by 5:1 molar ratios of the respective monomers with different percentages of kaolinite clay particles via in situ chemical co-polymerization. The spectral characteristics of the resulting copolymers were investigated. The results were justified by measuring the UV-Vis absorption spectra, FT-IR for PANAA copolymer, and the in situ PANAA/kaolinite clay composite. The thermal gravimetric analysis for the isolated copolymer powder in absence and in presence of kaolinite clay was carried out. Moreover, the morphology of the polymer clay composites system was studied by the scanning electron microscope micrographs at different magnifications. X-ray diffraction was used to measure the nature of the copolymer and extent of crystallinity present in the neat copolymer and copolymer/kaolinite composite materials.

PESIN Multimerization Improves Receptor Avidities and in Vivo Tumor Targeting Properties to GRPR-Overexpressing Tumors

The gastrin releasing peptide receptor (GRPR), being overexpressed on several tumor types, represents a promising target for specific noninvasive in vivo tumor imaging using positron emission tomography. Many of the radiolabeled bombesin analogs being applied in tumor imaging, however, suffer from shortcomings such as limited in vivo stability and poor tumor to background ratios. These obstacles can be overcome by peptide multimerization, as this approach results in constructs comprising several copies of the same peptide, thus retaining the ability to specifically bind to the target structure even if one peptide is cleaved. Furthermore, peptide multimers can result in increased binding avidities to the target, which can entail higher absolute tumor uptakes and also tumor to background levels. We therefore synthesized monomers and multimers of the peptide PESIN on dendrimer scaffolds comprising linkers of different lengths. The monomers/multimers were functionalized with the chelator NODAGA, efficiently radiolabeled with (68)Ga and evaluated in vitro regarding their GRPR binding affinity. The results show that shorter distances between the peptide moieties result in substantially higher binding affinities/avidities of the monovalent/multivalent PESIN ligands to the GRPR. Furthermore, the bivalent ligands gave the best results in terms of binding avidity, achieving a 2.5-fold increase in avidity compared to the respective monomer. Moreover, the most potent bivalent ligand showed an about 2-fold higher absolute tumor uptake and twice as high tumor-to-background ratios than the monomeric reference DOTA-PESIN in an initial animal PET study in tumor-bearing mice. Thus, besides high avidities, multivalency also positively influences the in vivo pharmacokinetics of peptide multimers.

Temperature‐Induced Phase Separation and Hydration in Aqueous Polymer Solutions Studied by NMR and IR Spectroscopy: Comparison of Poly(N‐vinylcaprolactam) and Acrylamide‐Based Polymers

SummaryUsing NMR and IR spectroscopy we compared the LCST behaviour of two types of thermoresponsive polymer systems, poly(N‐vinylcaprolactam) (PVCL) and acrylamide‐based polymers in aqueous solutions. We confirmed that during phase separation both PVCL and poly(N‐isopropylmethacrylamide) (PNIPMAm) chains undergo coil‐globule transition. At the same time some important differences were found for PVCL in comparison with PNIPMAm and poly(N‐isopropylacrylamide) (PNIPAm), especially a much lower level of dehydration of PVCL segments at temperatures above the phase transition. We assume that the origin of these differences can be in different structure of respective monomer units. From time dependences of ATR FTIR spectra it follows that the low degree of dehydration of PVCL segments is not changed with time. Small increase in the wavenumbers of the Amide I components of the hydrated PVCL carbonyl groups with time indicates that water released from PVCL mesoglobules with time, as revealed from NMR T2 measurements, might be mainly indirectly bound water.

Preclinical evaluation of a bispecific low‐molecular heterodimer targeting both PSMA and GRPR for improved PET imaging and therapy of prostate cancer

It has recently been reported that metastases of prostate cancer usually show highly heterogeneous or partly lost prostate-specific membrane antigen (PSMA) expression. In order to image and treat both PSMA positive and negative tissues PSMA targeting probes need to be extended by a further specificity. Since prostate cancer cells usually express both PSMA and gastrin-releasing peptide receptor (GRPR) a bispecific low-molecular heterodimeric molecule, addressing both targets at the same time, may significantly improve prostate cancer imaging and therapy. The nonapeptide BZH3 representing the GRPR binding part was combined with the urea-based PSMA inhibitor Glu-urea-Lys(Ahx)-HBED-CC. The syntheses of the compounds were performed according to standard Fmoc-solid phase peptide synthesis. The binding properties were analyzed by competitive cell binding and internalization experiments. The in vivo targeting properties were investigated by means of biodistribution studies. Cell binding experiments revealed high binding affinities to both GRPR and PSMA expressing cell lines. The heterodimer bound with IC50 -values essentially matching the IC50 values of the respective monomers (25.0 ± 5.4 nM for PSMA and 9.0 ± 1.8 nM for GRPR, respectively). In vivo, the heterodimer showed dual targeting of PSMA (5.4%ID/g for PSMA-positive tumors) and GRPR receptors (3.3% ID/g for GRPR-positive tumors) while exhibiting fast pharmacokinetic properties. The clearance from background was comparable to the monomeric PSMA-targeting reference. The heterodimeric molecule is a promising agent for PET imaging of primary and recurrent prostate cancer covering two receptor entities which might lead to an improved diagnostic sensitivity and therapeutic efficiency.