Catalytic Chain Research Articles

Biochemical Features of a Catalytic Antibody Light Chain, 22F6, Prepared from Human Lymphocytes

Human antibody light chains belonging to subgroup II of germ line genes were amplified by a seminested PCR technique using B-lymphocytes taken from a human adult infected with influenza virus. Each gene of the human light chains was transferred into the Escherichia coli system. The recovered light chain was highly purified using a two-step purification system. Light chain 22F6 showed interesting catalytic features. The light chain cleaved a peptide bond of synthetic peptidyl-4-methyl-coumaryl-7-amide (MCA) substrates, such as QAR-MCA and EAR-MCA, indicating amidase activity. It also hydrolyzed a phosphodiester bond of both DNA and RNA. From the analysis of amino acid sequences and molecular modeling, the 22F6 light chain possesses two kinds of active sites as amidase and nuclease in close distances. The 22F6 catalytic light chain could suppress the infection of influenza virus type A (H1N1) of Madin-Darby canine kidney cells in an in vitro assay. In addition, the catalytic light chain clearly inhibited the infection of the influenza virus of BALB/c mice via nasal administration in an in vivo assay. In the experiment, the titer in the serum of the mice coinfected with the 22F6 light chain and H1N1 virus became considerably lowered compared with that of 22F6-non-coinfected mice. Note that the catalytic light chain was prepared from human peripheral lymphocyte and plays an important role in preventing infection by influenza virus. Considering the fact that the human light chain did not show any acute toxicity for mice, our procedure developed in this study must be unique and noteworthy for developing new drugs.

Novel coating resins based on polycarbonates and poly(ester-co-carbonate)s made by catalytic chain growth polymerization of epoxides with CO2 and with anhydride/CO2

In this work we describe a possibly new generation of (powder) coating resins of the polycarbonate or poly(ester-co-carbonate) type, synthesized from epoxides like cyclohexene oxide (CHO), anhydrides like phthalic anhydride (PA) and carbon dioxide (CO2) by chain growth polymerization, catalyzed by a chromium-Salophen complex and using dimethylaminopyridine (DMAP) as a co-catalyst. The molecular structures of the polymers produced were characterized and especially MALDI-ToF-MS yielded important information on the end-groups and other functional groups which are crucial for the curing chemistry of these resins. One special type of copolycarbonate in this study carried pendent vinyl groups, introduced by copolymerization of CHO and CO2 with 4-vinylcyclohexene oxide (VCHO). This copolycarbonate was first casted from solution, after which the polymer film was successfully cured with a trithiol compound by UV- or thermally induced radical curing chemistry. These cured coatings showed a good acetone resistance (≥75 double rubs) and reversed impact toughness. A powder coating evaluation of this CHO/VCHO-based copolycarbonate showed excellent processability, high pencil hardness (6-8H), value zero in a Gitterschnitt test on aluminum, reasonable appearance in a ‘PCI-smoothness test’ (value 2–3) and good acetone resistance (≥75 double rubs). Most probably due to a too high Tg (85–104°C) of the cured coating the reverse impact resistance was poor. A similar powder coating evaluation of a poly(ester-co-carbonate) based on CHO, PA and CO2 showed less promising results due to poor flow properties and foaming above 140°C.

Theoretical Relationship between Quencher Diffusion Constant and Image Quality in Chemically Amplified Resists Used for Extreme Ultraviolet Lithography

Quenchers play an important role in the control of the acid catalytic chain reaction in chemically amplified resists, which is an indispensable technology in the high-volume production of semiconductor devices. In this study, the effects of quencher diffusion constants on the chemical gradient of latent images formed in chemically amplified resists were investigated assuming 16 nm line-and-space patterns. Although it has been reported that the chemical gradient of 22 nm line-and-space patterns was increased with quencher diffusion constant, it was found that the chemical gradient of 16 nm line-and-space patterns was increased with the decrease of quencher diffusion constant.

Acid Diffusion Length in Line-and-Space Resist Patterns Fabricated by Extreme Ultraviolet Lithography

Acids generated upon exposure to radiation induce the polarity change of the polymer through catalytic chain reactions in chemically amplified resists. With the reduction of feature size, the acid diffusion length increasingly becomes an important issue. In this study, we investigated the acid diffusion length in line-and-space patterns fabricated using a small field exposure tool for extreme ultraviolet (EUV) lithography and the EIDEC standard resist to clarify the acid diffusion length in a state-of-the-art resist. The acid diffusion length depended on the nominal line width and exposure dose. Upon exposure to EUV radiation with an exposure dose of 16 mJ cm-2, the acid diffusion length (three-dimensional) in a line-and-space pattern with 21 nm nominal line width was 9.5 nm.

Mechanistic Investigation into the Accelerated Synthesis of Methacrylate Oligomers via the Application of Catalytic Chain Transfer Polymerization and Selective Microwave Heating

The synthesis of methyl methacrylate (MMA) oligomers by catalytic chain transfer polymerization (CCTP) is demonstrated to be significantly accelerated by the use of microwave heating. The CCTP reactions, which use a cobalt-based catalyst to very efficiently control the molecular weight of the final polymer, were conducted in both a conventional oil bath and a CEM Discover microwave reactor with a target set point of 80 °C. The required reaction time was shown to be reduced from 300 to 3 min, while also retaining control over the polymerization. Additionally, for the first time the bulk temperature of these catalyzed polymerizations was monitored in both heating methods by the use of internal optical fiber sensors. It was demonstrated that, to monitor the temperature of the reaction correctly, it is essential to use an optical fiber sensor rather than the external IR sensor supplied with the reactor. The acceleration in the synthesis during microwave heating was attributed to selective heating of the radical and oligomeric species within the reaction, which lead to both rapid heating of the reaction bulk to reaction temperature and average reaction temperatures that were higher than the chosen set point. However, comparative reactions carried out under conventional heating (CH) conditions at the true reaction temperature of the microwave experiments (MWH) showed that MWH was able to produce significantly greater yields than the CH experiments after only 3 min, indicating the existence of a real selective heating effect during the reaction. Three methods have been investigated to optimize the acceleration achieved in the MWH experiments while retaining control and yield levels within the MWH experiments. These were varying the; solvent concentration, initiator concentration and chain transfer agent concentration. It was demonstrated that by understanding the influence of the microwave heating that it was possible to retain control over the molecular structure of the product polymer at the accelerated rate.

First Structure of Protein Kinase CK2 Catalytic Subunit with an Effective CK2β-Competitive Ligand

The constitutively active Ser/Thr kinase CK2 (casein kinase 2) is used by tumor cells to acquire apoptosis resistance. CK2 exists as a heterotetrameric holoenzyme with two catalytic chains (CK2α) attached to a dimer of noncatalytic subunits (CK2β). A druggable cavity at the CK2β interface of CK2α allows the design of small molecules disturbing the CK2α/CK2β interaction and thus affecting activity, stability, and substrate specificity. We describe here the first structure of CK2α with an effective CK2β-competitive compound, namely, a 13-meric cyclic peptide derived from the C-terminal CK2β segment. Some well-ordered water molecules not visible in CK2 holoenzyme structures were detected at the interface. Driven mainly by enthalpy, the peptide binds with submicromolar affinity to CK2α, stimulates its catalytic activity, and reduces effectively the CK2α/CK2β affinity. The results provide a thermodynamic and structural rationalization of the peptide's CK2β-competitive functionality and pave thus the way to a peptidomimetic drug addressing the CK2α/CK2β interaction.

Conserved Structural Chemistry for Incision Activity in Structurally Non-homologous Apurinic/Apyrimidinic Endonuclease APE1 and Endonuclease IV DNA Repair Enzymes

Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 Å resolution APE1-DNA product complex with Mg(2+) and a 0.92 Å Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.

Polymerization of long chain [meth]acrylates by Cu(0)-mediated and catalytic chain transfer polymerisation (CCTP): high fidelity end group incorporation and modification

The Cu(0)-mediated polymerization of lauryl (C12) and stearyl (C18) acrylate is reported under a range of reaction conditions. Good first order kinetics, a linear evolution of number average molecular weight (Mn) with conversion and dispersity (∼1.10) are observed. The polymerization of lauryl acrylate proceeds either homogenously or in a self-generated biphasic system, depending on the solvent employed, with little deviation in overall polymerization control. The near quantitative retention of ω-bromo end groups is exploited via nucleophilic thio-bromine substitution with thioglycerol to yield highly hydrophobic polymers with polar head groups. Modification is spectroscopically confirmed by both NMR and MALDI-TOF-MS. Highly ω-chain end functional methacrylic analogues have also been prepared by Co(II) mediated catalytic chain transfer polymerisation (CCTP). Reactive vinyl groups at the ω-chain end of poly(lauryl methacrylate) have been targeted with thioglycerol for nucleophilic thio-Michael addition with quantitative modification possible.

Direct ‘in situ’, low VOC, high yielding, CO2expanded phase catalytic chain transfer polymerisation: towards scale-up

The successful application of catalytic chain transfer polymerisation (CCTP) by adopting an 'in situ' catalyst preparation methodology in several polymerisation media is described. More specifically, this study is focused on reporting the development of 'in situ' CCTP within a CO(2) expanded phase polymerisation process, which achieved high yields of polymer whilst minimising both VOC footprint and CO(2) compression costs. The 'in situ' method is shown to be effective in controlling polymerisations conducted in both conventional solvents and bulk under inert atmosphere, delivering molecular weight reductions and a Cs value of appropriate similar magnitude to those achieved by the benchmark, commercially sourced CoPhBF catalyst. The 'in situ' effect has been achieved with equal efficiency when both using catalysts with different axial ligands and where the complex is required to undergo a facile ligand dissociation in order to create the required catalyst necessary to achieve CCTP control. Furthermore, both catalysts are shown to effectively control polymerisations in a CO(2) expanded phase process, in which a small amount of compressed CO(2) is introduced to reduce the viscosity of the reaction mixture, allowing for easy heat transfer and good catalyst diffusion during reaction. In this way, yield limitations imposed to avoid the Trommsdorff effect required in bulk processing and the need for post precipitation have been successfully overcome. Both of these factors further improve the sustainability of such a polymerisation process. However, the 'in situ', high pressure expanded phase environment was observed to retard the ligand dissociation required for catalyst activation.

Optical Studies on Methyl Methacrylate Polymerized via Metal Carbazone Complexes

Optical studies are done on Poly methyl-methacrylate (PMMA) sheets. Sampleswere previously prepared by catalytic chain transfer polymerization via metal carbazone of Cd(II) and Hg(II). Transmission and absorbance are recorded (between 190 nm to 1100 nm) as a function of incident energy using UV-VIS spectrophotometer. Second derivative of absorbance spectra is calculated to enhance differences among spectra. The optical band gap, width of band tail and dispersion of refractive index of the sheets are determined.

Photopatterning Based on a Chemically Amplified Mechanism Nano-Sheet Films

In the paper, an approach for introducing a photoacid generator (PAG) into Langmuir- Blodgett (LB) films to draw photopatterns is described. The chemically amplified positive-tone resist system used here consisted of two components: a copolymer, poly(iso-pentylmethacrylamide- co-4-t-butyloxylvinylphenylcarbonate) [poly(iPMA-t-BVPC39)] and a photoacid generator (PAG), tri(2,3-dibrompropyl)iso-cyanvrate (TDBPIC). In the two-component system, the acid generated by the photoacid generator (PAG) catalyzes the deprotection reaction of poly(iPMA-t-BVPC39) to re- move the tert-butoxycarbonyl group (t-BOC) in the exposed region during the post exposure baking process, thus rendering the exposure region soluble to alkali aqueous to form a positive tone. Phhot- olithographic properties of the LB films have been evaluated. The patterns can be resolved with a resolution of 1 μm line width by UV irradiation,followed by development with an alkali solution. Quantum yield is determined to be 0.065 and catalytic chain length is also calculated to be 2460. The LB films used above can be used to generate etched gold patterns on a glass substrate using an aqueous iodide, ammonium iodide in alcohol /water，as the etchant. The etch resistance of such LB films is sufficiently good, allowing patterning of a gold film suitable for photomask fabrication.

Catalytic Chain Transfer Mediated Autopolymerization of Divinylbenzene: Toward Facile Synthesis of High Alkene Functional Group Density Hyperbranched Materials

A facile and highly reproducible autopolymerization method, mediated by catalytic chain transfer, for the synthesis of hyperbranched materials with high alkene functional group density is reported....

Stochastic Effect of Acid Catalytic Chain Reaction in Chemically Amplified Extreme Ultraviolet Resists

The stochastic effect of incident photons is a significant concern in nanometer-scale fabrication using highly sensitive resists because the fluctuation of the line edges of resist patterns should be suppressed to guarantee normal operation of fabricated devices. However, what matters the most in the line edge roughness (LER) formation is not photon fluctuation but protected unit fluctuation. In this study, the changes in the stochastic effect of acid catalytic chain reaction were investigated using the Monte Carlo method. It was theoretically confirmed that the reason why LER decreases with the increase in exposure dose and/or acid generator concentration is not the reduction of the fluctuations of concentrations of photons (shot noise), acids, or protected units but the increase in chemical gradient.

Stochastic Effect of Acid Catalytic Chain Reaction in Chemically Amplified Extreme Ultraviolet Resists

Stochastic Effect of Acid Catalytic Chain Reaction in Chemically Amplified Extreme Ultraviolet Resists

Comparison of designed and randomly generated catalysts for simple chemical reactions.

There has been recent success in designing enzymes for simple chemical reactions using a two-step protocol. In the first step, a geometric matching algorithm is used to identify naturally occurring protein scaffolds at which predefined idealized active sites can be realized. In the second step, the residues surrounding the transition state model are optimized to increase transition state binding affinity and to bolster the primary catalytic side chains. To improve the design methodology, we investigated how the set of solutions identified by the design calculations relate to the overall set of solutions for two different chemical reactions. Using a TIM barrel scaffold in which catalytically active Kemp eliminase and retroaldolase designs were obtained previously, we carried out activity screens of random libraries made to be compositionally similar to active designs. A small number of active catalysts were found in screens of 10³ variants for each of the two reactions, which differ from the computational designs in that they reuse charged residues already present in the native scaffold. The results suggest that computational design considerably increases the frequency of catalyst generation for active sites involving newly introduced catalytic residues, highlighting the importance of interaction cooperativity in enzyme active sites.

End-Functional Styrene–Maleic Anhydride Copolymers via Catalytic Chain Transfer Polymerization

Styrene–maleic anhydride copolymers have been successfully synthesized using catalytic chain transfer polymerization employing the low spin [bis(difluoroboryl)dimethylglyoximato]cobalt(II) (COBF) complex. By partially replacing styrene with α-methylstyrene (while maintaining the amount of maleic anhydride at 50 mol %) over a range of ratios, it was shown that the rate of reaction and molar mass decreases with increasing α-methylstyrene content. The polymers were characterized using MALDI–ToF–MS and 1H–13C gHMQC NMR to determine the end groups, which in the presence of α-methylstyrene was an α-methylstyrene unit with a vinylic functionality. For styrene–maleic anhydride copolymers, the end group was determined to be predominantly maleic anhydride with a vinylic functionality. Considering the fact that in a styrene–maleic anhydride copolymerization the propagating radicals are predominantly of a styrenic nature, this was a very surprising result, suggesting that the maleic anhydride radicals undergo a chain...

Anacardic Acid Inhibits the Catalytic Activity of Matrix Metalloproteinase-2 and Matrix Metalloproteinase-9

Cashew nut shell liquid (CNSL) has been used in traditional medicine for the treatment of a wide variety of pathophysiological conditions. To further define the mechanism of CNSL action, we investigated the effect of cashew nut shell extract (CNSE) on two matrix metalloproteinases, MMP-2/gelatinase A and MMP-9/gelatinase B, which are known to have critical roles in several disease states. We observed that the major constituent of CNSE, anacardic acid, markedly inhibited the gelatinase activity of 3T3-L1 cells. Our gelatin zymography studies on these two secreted gelatinases, present in the conditioned media from 3T3-L1 cells, established that anacardic acid directly inhibited the catalytic activities of both MMP-2 and MMP-9. Our docking studies suggested that anacardic acid binds into the MMP-2/9 active site, with the carboxylate group of anacardic acid chelating the catalytic zinc ion and forming a hydrogen bond to a key catalytic glutamate side chain and the C15 aliphatic group being accommodated within the relatively large S1' pocket of these gelatinases. In agreement with the docking results, our fluorescence-based studies on the recombinant MMP-2 catalytic core domain demonstrated that anacardic acid directly inhibits substrate peptide cleavage in a dose-dependent manner, with an IC₅₀ of 11.11 μM. In addition, our gelatinase zymography and fluorescence data confirmed that the cardol-cardanol mixture, salicylic acid, and aspirin, all of which lack key functional groups present in anacardic acid, are much weaker MMP-2/MMP-9 inhibitors. Our results provide the first evidence for inhibition of gelatinase catalytic activity by anacardic acid, providing a novel template for drug discovery and a molecular mechanism potentially involved in CNSL therapeutic action.

Structure of the catalytic chain of Methanococcus jannaschii aspartate transcarbamoylase in a hexagonal crystal form: insights into the path of carbamoyl phosphate to the active site of the enzyme.

Crystals of the catalytic chain of Methanococcus jannaschii aspartate transcarbamoylase (ATCase) grew in the presence of the regulatory chain in the hexagonal space group P6(3)22, with one monomer per asymmetric unit. This is the first time that crystals with only one monomer in the asymmetric unit have been obtained; all known structures of the catalytic subunit contain several crystallographically independent monomers. The symmetry-related chains form the staggered dimer of trimers observed in the other known structures of the catalytic subunit. The central channel of the catalytic subunit contains a sulfate ion and a K(+) ion as well as a glycerol molecule at its entrance. It is possible that itis involved in channeling carbamoyl phosphate (CP) to the active site of the enzyme. A second sulfate ion near Arg164 is near the second CP position in the wild-type Escherichia coli ATCase structure complexed with CP. It is suggested that this position may also be in the path that CP takes when binding to the active site in a partial diffusion process at 310 K. Additional biochemical studies of carbamoylation and the molecular organization of this enzyme in M. jannaschii will provide further insight into these points.

Biological features of human catalytic antibody light chains showing anti‐cancer activity

The authors have reported about the human catalytic antibody light chains possessing catalytic activity, which was mostly encoded in Subgroup II in kappa type. In this study, the highly purified human light chains were submitted to investigate the function of the toxicity against cancer cells.We designed two primer sets and performed a semi‐nested PCR using cDNA prepared from human leukocyte. As the results, eighteen clones of subgroup II germline gene of human kappa light chain were established. The mutants of C220A corresponding to the 18 clones were made. And the wild types and mutants were expressed in E. coli and the light chains recovered were highly purified by two‐step purification system. Out of 18 clones, #1 clone showed the cytotoxicity to both human lung (A548) and human stomach cancer cells (SNU‐1) but not to pancreas cancer cells (PANC‐1). Interestingly, the dimeric form showed the stronger effect than the monomeric form (C220A). The #1 clone could hydrolyze QAR‐MCA synthetic peptide showing the amidase activity. In addition, #1 clone has a unique amino acid sequence compared with that of other clones. Although it is still unclear that the relationship between catalytic activity and cytotoxicity of human light chain (#1 clone) at this moment, the catalytic triad structure of the clone may contribute the anti‐cancer effect.

Catalytic and Biological Features of Human Light Chains Suppressing of Infection of Influenza Virus A Type

By immunizing ground‐state peptides or proteins into mice, the authors have established murine catalytic antibody light chains (antigenases) that could destroy such as HIV‐1 gp41, H. pylori urease, TNFa etc. We proposed a new concept that the antigenase posseses a catalytic‐triad structure composed Ser, His and Asp.Based on above concept, we cloned cDNAs (from a volunteer infected with influenza virus) encoding human antibody kappa light chain belonging to subgroup II of germline genes, because the group encodes a catalytic triad‐like structure with high possibility. The cDNAs were transformed into E. coli and each human light chain was expressed as the protein. The highly purified (>95%) human light chains were firstly submitted to the catalytic activity tests and to in vitro assays for examining the infectivity of influenza virus. Some clones showed the amidase and DNase activity. Surprisingly, #4 clone exhibited a strong activity to digest pBR322 plasmid. And the #4 clone suppressed the infection of influenza virus (H1N1) in vitro assay. In addition, we performed in vivo assays. The administered mice with the influenza virus treated with #4 antigenase enhanced the number of survival mice compared with non‐treated mice. Furthermore, the weigh of the antigenase‐treated mice did not decrease at all, while the antigenase‐not‐treated mice showed the marked decrease of the weigh.