Catalytic Antibodies

The vast binding repertoire of the immune system has been exploited for the generation of tailor-made biological catalysts. A number of strategies have been developed to generate catalytic antibodies that carry out a wide range of reactions with exquisite specificities. The generation and characterization of these novel catalysts is not only providing new insights into the nature of biological recognition and catalysis, but may also lead to novel catalysts for applications in chemistry, biology, and medicine.     

Catalytic Antibodies: A New Class of Transition-State Analogues Used to Elicit Hydrolytic Antibodies

The design and generation of selective catalysts is an important aim of chemists and biologists. A number of successful strategies have emerged, including the synthesis and derivatization of synthetic hosts, the chemical modification and site-directed mutagenesis of enzymes, and the attenuation of natural enzyme activities in organic solvents. Since 1986 several laboratories have exploited the immune system to generate selective catalysts capable of catalyzing a wide range of chemical transformations. These include acyl transfer, β-elimination, carbon—carbon bond-forming, carbon—carbon bond-cleaving, porphyrin metalation, peroxidation, and redox reactions. The variety and number of transformations catalyzed by antibodies in this short period of time is testament to the versatility and power of the method in generating selective catalysts for applications in chemistry, biology, and medicine. Here we report the use of a new class of uncharged transition-state analogues for generating antibodies capable of catalyzing ester and carbonate hydrolysis. These antibodies are compared to those raised against tetrahedral phosphate and phosphonate transition-state analogues.     

Catalytic hydrolysis of phosphate diester （BNPP） and plasmid DNA by mononuclear macrocyclic polyamine metal complexes

The activities of the catalytic hydrolysis of phosphate diester(BNPP)[bis(p-nitrophenyl)phosphate diester]and plasmid DNA (pUC18)by mononuclear macrocyclic polyamine metal complexes have been investigated in this paper.The results showed that the highest activity in hydrolysis of BNPP was obtained with 1e-Zn(Ⅱ)complex(composed of lipophilic group)as catalyst.The hydrolysis rate enhancement is up to 3.64×10~4 fold.These metal complexes could effectively promote the cleavage of plasmid DNA(pUC18)at physiol...     

Investigated aerobic oxidation of aminochlorophenol catalyzed by phthalocyanine complexes

The synthesis of 9-Octadecenamide substituted Fe(II) phthalocyanine (ODAFePc) and Ni(II) phthalocyanine (ODANiPc) complexes from Fe(II) tetracarboxylic acid phthalocyanine (FeTcPc) and Ni(II) tetracarboxylic acid phthalocyanine (NiTcPc) with 9-Octadecenamide. These complexes have high molecular weight and soluble in organic solvents. The complexes have been confirmed by FTIR, Mass spectroscopy, UV–Visible X-ray diffraction, and thermogravimetric analysis. The synthesized complexes exhibit excellent stability and are catalytically active in 2-amino-4-chlorophenol (ACP) oxidation. The new method was used for the determination of the oxidation of phenol by applying different experimental parameters like concentration, catalytic quantity, temperature, and pH to get a good yield and catalytic activity of ODAFePc and ODANiPc were studied. ACP was oxidized by dissolved oxygen with ODAFePc and ODANiPc as a catalyst and immediately combined with 4-aminoantipyrine (4-AAP) to form a pink dye and was determined by appearance of absorption intensity at 580 ​nm. All the experimental results show that ODAFePc and ODANiPc complexes exhibit good catalytic behavior on oxidation of 2-amino-4-chlorophenol.     

A biocatalytic route to enantioenriched, sulfanyl aldol products

The aldol products derived from sulfur- or selenium containing acceptors were prepared by kinetic resolution in the presence of antibody 84G3 with enantiomeric excesses ranging from 56 to 70%. Much higher level of enantioselectivity was obtained (enantiomeric excesses all superior to 96%) for sulfanyl aldol products derived from thiomethoxyacetone with three different acceptors.     

Design and synthesis of a Mn(III)-porphyrin steroid conjugate used as a new cleavable affinity label: on the road to semi-synthetic catalytic antibodies

We here report the synthesis of a novel porphyrin-steroid conjugate which was designed for the site-specific incorporation of a non-natural heme cofactor at the binding site of an anti-estradiol antibody, in order to get a semi-synthetic catalytic antibody with a monooxygenase-like activity. The general strategy involved a coupling reaction between a testosterone modified by an arm bearing a cleavable disulfide bridge and a meso-tetraarylporphyrin bearing two successive meso-ortho-substituted-phenyl rings, α,α-5,10-bis-[{o-(2-carboxyethyl)carboxamido}phenyl]-15,20-diphenyl-porphyrin. The final porphyrin-steroid conjugate was successfully purified and fully characterized, and was subsequently metalated with manganese acetate. The metalloporphyrin moiety will be used to be coupled with the antibody to generate a new biocatalyst with monooxygenase-like activities.     

Nucleophilic proteolytic antibodies

Proteolytic antibodies appear to utilizecatalytic mechanisms akin to nonantibody serine proteases, assessed from mutagenesis and protease-inhibitor studies. The catalytic efficiency derives substantially from the ability to recognize the ground state with high affinity. Because the proteolytic activity is germline-encoded, catalysts with specificity for virtually any target polypeptide could potentially be developed by applying appropriate immunogens and selection strategies. Analysis of transition-state stabilizing interactions suggests that chemical reactivity ofactive-site serine residues is an important contributor to catalysis. A prototype antigen analog capable of reacting covalently with nucleophilic serine residues permitted enrichment of the catalysts from a phage-displayed lupus light-chain library. Further mechanistic developments in understanding proteolytic antibodies may lead to the isolation of catalysts suitable for passive immunotherapy of major diseases, and elicitation of catalytic immunity as a component of prophylactic vaccination.     

Synthesis, crystal structures and catalytic abilities of new macrocyclic bis-pyridineamido Mn and Fe complexes

A new macrocyclic ligand H₂L (H₂L = 1,2-[bis(6′-pyridine-2′-carboxamido)-ethane]benzene) has been designed and synthesized by condensation of pyridine-carboxylic acid and diamine. Its Mn(III) and Fe(III) complexes, [Mn(L)Cl (DMF)] and [Fe(L)Cl], were prepared and respectively characterized by IR, UV-Vis, ESI-Mass, elemental analysis and X-ray single crystal diffraction. The catalytic abilities of them were examined, and up to 95% yield was achieved in epoxidation of styrene. The preliminary investigation of catalytic mechanism by manganese complex was carried out, suggesting the involvement of Mn(V) oxo species during catalysis.     

Dynamic imaging of single DNA-protein interactions using atomic force microscopy

Atomic force microscopy (AFM) imaging of static DNA-protein complexes, in air and in liquid, can be used to directly obtain quantitative and qualitative information on the structure of different complexes. For example, DNA length, the location of preferential binding sites for proteins and bending of DNA as a result of the complexation can all be measured. Recording consecutive AFM images of DNA and protein molecules under conditions that they are still able to move and interact, or dynamic AFM imaging, however, can reveal information on the dynamic aspects of the interactions between these molecules. Here, an overview is given of the technical challenges that need to be considered for successful dynamic AFM imaging studies of individual DNA-protein interactions. Necessary technical improvements to the AFM set-up and the development of new sample preparation methods are described in this paper.     

艾滋病（AIDS）病毒抗体检测方法研究进展

对艾滋病(A IDS) 病毒抗体检测方法的研究进展作了较系统的评述。从抗原试剂的构成及样品的来源等方面对各种检测方法进行归纳分类。论述了病毒抗体检测方法研究发展的三个阶段, 分析比较了各种艾滋病毒抗体检测方法的优缺点。给出125 篇参考文献。     

Facile synthesis and reactions of some olefin-substituted acetylacetonate complexes

Synthesis of transition metal complexes of olefin-substituted acetylacetones has been effected by reaction of nonaqueous solutions of substituted acetylacetones with the metal salt in the presence of Zn or Mg. 3-Allylacetylacetonates of Be(II), Cu(II), Ni(II), Fe(III), Co(II), Pd(II) and Zn(II) have thus been synthesized in quantitative yields. 3-Propylideneacetylacetone, which exists mainly in the keto form, afforded the corresponding 3-propenylacetylacetonates in which 3-propylideneacetylacetone has rearranged to 3-propenylacetylacetone which has a greater probability of existing in the enol form, thus making its reaction with metal ions favorable. Metal–metal exchange reactions of transition metal salts with olefin-substituted Zn or Mg acetylacetonates provide yet another route for the synthesis of transition metal olefin-substituted acetylacetonates. The homogeneous catalytic hydrogenation of the olefin moiety in olefin-substituted metal acetylacetonates could be achieved by a Pt/Sn catalyst.     

DNA-hydrolyzing autoantibodies

Catalysis by antibodies could be a frequent phenomenon if the immune system generates a sufficiently diverse number of antibodyactive sites, some of which may possess catalytic activity. A catalytic antibody can be expected to do more damage than one that simply binds antigen. The best biochemical marker of systemic lupus erythematosus (SLE) is presence of autoantibodies to DNA. In the present article, we describe the DNA-hydrolyzing activity of DNA-binding autoantibodies purified from SLE patients. The substrates employed were supercoiled plasmid, radiolabeled plasmid fragments, and oligonucleotides. Hydrolysis of DNA by the antibodies was indicated by the appearance of fragments visualized by ethidium bromide staining of agarose gels or autoradiography of polyacrylamide gels. Changes in linear dichroism values were also indicative of DNA hydrolysis. The antibody activity was purified by protein A-sepharose chromatography, high-performance liquid chromatography gel filtration, and DNA-affinity chromatography. Scrupulous control studies were done to demonstrate that DNA-hydrolyzing activity really belongs to the antibodies. Purified Fab fragments showed hydrolyzing activity, whereas the Fc fragment was inactive. The specificity of DNA cleavage was investigated, and the rate parameters of hydrolysis by antibodies and conventional nucleases were compared.     

An optimal method of DNA silver staining in polyacrylamide gels

A silver staining technique has widely been used to detect DNA fragments with high sensitivity on polyacrylamide gels. The conventional procedure of the silver staining is tedious, which takes about 40-60 min and needs five or six kinds of chemicals and four kinds of solutions. Although our previous improved method reduced several steps, it still needed six kinds of chemicals. The objective of this study was to improve further the existing procedures and develop an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure could be completed with only four chemicals and two solutions within 20 min. The steps of ethanol, acetic acid, and nitric acid precession before silver impregnation have been eliminated and the minimal AgNO3 dose has been used in this up-to-date method. The polyacrylamide gel of the DNA silver staining displayed a golden yellow and transparent background with high sensitivity. The minimum 0.44 and 3.5 ng of DNA amount could be detected in denaturing and nondenaturing polyacrylamide gel, respectively. This result indicated that our optimal method can save time and cost, and still keep a high sensitivity for DNA staining in polyacrylamide gels.     

Silver staining of DNA in polyacrylamide gels

Nucleic acids can be detected at the picogram level using a quick and simple silver staining method (2). Using very thin polyesterbacked polyacrylamide gels, a further simplified protocol was compared to other widely used silver staining procedures. The improved protocol described here was the most sensitive, the fastest to perform, and had relatively few steps and reagents. This method also produced the least number of staining artifacts and offered images of high contrast.     

Efficient and sensitive method of DNA silver staining in polyacrylamide gels

DNA silver staining is widely used to detect DNA fragment in polyacrylamide gel with high sensitivity. Conventional procedures of the silver staining involve several steps, which take about 40 min to 2 h in total. To improve the efficiency of DNA silver staining, a more efficient protocol is developed in this study. The procedure comprises only four steps including impregnating, rinsing, developing, and stopping, and could be completed within 20 min. Nitric acid and ethanol in the silver-impregnation step of the new procedure eliminates the need for prior treatment of gels with a fixing solution and following rinse prior to impregnation with silver. The procedure has high sensitivity and long storage lifetime. The minimum detectable mass of DNA is 0.44 and 3.5 ng in denaturing and nondenaturing polyacrylamide gel, respectively.     

Novel functional activities of anti-dna autoantibodies from sera of patients with lymphoproliferative and autoimmune diseases

DNA-hydrolyzing activity of IgG autoantibodies from sera of patients with various types of lymphoproliferative diseases was investigated. The association of DNA-hydrolyzing activity with the antibody (Ab) fraction has been proved by newly developed affinity-capture assay. Study of abzyme incidence in blood tumors and systemic lupus erythematosis (SLE) revealed linkage of anti-DNA Ab catalysts to mature B-cell tumors, and increased probability of DNA-abzymes formation on the background of autoimmune manifestations. These data suggest possible similarity between mechanisms of abzyme formation in SLE and B-cell lymphomas. A new mechanism of formation of DNA-specific catalytic Abs has been proposed based on the increased crossreactivity of polyclonal DNA-abzymes to DNA-depleted nuclear matrix proteins. The possibility of the abzyme production as Ab to the energetically destabilized ground state of the antigen has been discussed. Preliminary results were obtained that indicate the complement-independent cytotoxicity of anti-DNA autoantibodies isolated from blood of patients with SLE and chronic lymphocytic leukemia.     

Alkene epoxidation catalysed by binuclear manganese complexes

Binuclear manganese(II) complexes with macrocyclic ligands have been synthesized by template Schiff base condensation of diethylenetriamine and pentane-2,4-dione or 1,3-diphenyl-propane-1,3-dione. Catalytic epoxidation of simple olefins with hydrogen peroxide and t-BHP were studied using the above manganese complexes in the presence of a base. The influence of reaction temperature, the additive methanol and the cocatalyst had been investigated. The major products of the oxidations were the epoxides. The new manganese complexes showed significant catalytic activities for the epoxidation of alkenes using hydrogen peroxide as oxidant and ammonium acetate as cocatalyst.     

Immunologically driven antibodies chemical engineering: design and synthesis of a hapten aimed at nerve agent hydrolysis

In this letter, we describe the design and the synthesis of an organophosphorus hapten aimed at a mixed biotechnological-chemical strategy for the mild decontamination of exceedingly toxic nerve agent VX. Hapten will be used to raise and select monoclonal antibodies (mAbs) able to bind both the nerve agent, and an oxime, derived from pyridinaldoxime, able to specifically hydrolyze its P-S bond. In order to significantly increase the hydrolysis reaction rate, the controlled respective positioning of the oxime and of the substrate will be achieved through the immunization, and in a second step, the oxime will be bound to the mAb in a reactive position towards the reactive thiophosphate functionality of the substrate.     

Convergence of Catalytic Antibody and Terpene Cyclase Mechanisms: Polyene Cyclization Directed by Carbocation–π Interactions

A tandem cationic cyclization cascade similar to a terpenoid cyclase reaction is catalyzed by antibody HA5-19A4. The 2.7-Å resolution crystal structure of the Fab–hapten complex reveals convergence with the same catalytic strategy employed by a terpenoid cyclase: both serve as templates that enforce the productive conformation of a flexible polyene substrate, and both stabilize carbocation intermediates through cation–π interactions with multiple aromatic residues in their active sites.