Enamine N-oxides: Design, Synthesis, and Function in Bioorthogonal Reactions.

Enamine N-oxides act as a chemical linchpin bridging two bioorthogonal associative and dissociative reactions. This article describes the design of enamine N-oxides; their synthesis through the retro-Cope elimination reaction; the use of solvent, hyperconjugation, strain, and rehybridization effects to achieve bioorthogonal reactivity; and their rapid reductive cleavage with diboron reagents. The coordinated assembly and disassembly of the enamine N-oxide motif constitutes a powerful chemical operation that enables the attachment and detachment of small molecules from biomacromolecules in a biological setting.

Theoretical Design and Synthesis of Caged Compounds Using X-Ray-Triggered Azo Bond Cleavage.

Caged compounds are frequently used in life science research. However, the light used to activate them is commonly absorbed and scattered by biological materials, limiting their use to basic research in cells or small animals. In contrast, hard X-rays exhibit high bio-permeability due to the difficulty of interacting with biological molecules. With the main goal of developing X-ray activatable caged compounds, azo compounds are designed and synthesized with a positive charge and long π-conjugated system to increase the reaction efficiency with hydrated electrons. The azo bonds in the designed compounds are selectively cleaved by X-ray, and the fluorescent substance Diethyl Rhodamine is released. Based on the results of experiments and quantum chemical calculations, azo bond cleavage is assumed to occur via a two-step process: a two-electron reduction of the azo bond followed by N─N bond cleavage. Cellular experiments also demonstrate that the azo bonds can be cleaved intracellularly. Thus, caged compounds that can be activated by an azo bond cleavage reaction promoted by X-ray are successfully generated.

An Oxidant-Free and Mild Strategy for Quinazolin-4(3H)-One Synthesis via CuAAC/Ring Cleavage Reaction.

An oxidant-free and highly efficient synthesis of phenolic quinazolin-4(3H)-ones was achieved by simply stirring a mixture of 2-aminobenzamides, sulfonyl azides, and terminal alkynes. The intermediate N-sulfonylketenimine underwent two nucleophilic additions and the sulfonyl group eliminated through the power of aromatization. The natural product 2-(4-hydroxybenzyl)quinazolin-4(3H)-one can be synthesized on a large scale under mild conditions with this method.

Caulobacter segnis Dioxygenase CsO2: A Practical Biocatalyst for Stilbenoid Ozonolysis.

Ozonolysis is a useful as well as dangerous reaction for performing alkene cleavage. On the other hand, enzymes are considered a more sustainable and safer alternative. Among them, Caulobacter segnis dioxygenase (CsO2) known so far for its ability to catalyze the coenzyme-free oxidation of vinylguaiacol into vanillin, was selected and its substrate scope evaluated towards diverse natural and synthetic stilbenoids. Under optimized conditions, CsO2 catalyzed the oxidative cleavage of the C=C double bonds of various trans-stilbenes, providing that a hydroxyl moiety was necessary in para-position of the phenyl group (e. g., resveratrol and its derivatives) for the reaction to take place, which was confirmed by modelling studies. The reactions occurred rapidly (0.5-3 h) with high conversions (95-99 %) and without formation of by-products. The resveratrol biotransformation was carried out on 50-mL scale thus confirming the feasibility of the biocatalytic system as a preparative method.

A Study of the Mechanisms and Characteristics of Fluorescence Enhancement for the Detection of Formononetin and Ononin.

In this work, the origins for the spectral difference between two isoflavones, formononetin (F) and ononin (FG), are revealed via a comparison study of the fluorescence molecular structure. The fluorescence enhancement of FG in hot alkaline conditions is reported for the first time. For F, there is almost no fluorescence under acidic conditions, but when the pH is >4.8, its fluorescence begins to increase due to the deprotonation of 7-OH. Under a pH between 9.3 and 12.0, the anionic form of F produces a strong and stable fluorescence. The fluorescence quantum yield (Yf) of F is measured to be 0.042. FG shows only weak fluorescence in aqueous solutions under a wide range of pH until it is placed in hot alkaline solutions, which is attributed to the cleavage reaction of the γ-pyrone ring in FG. The Yf of FG is determined to be 0.020. Based on the fluorescence sensitization methods of F and FG, the quantitative analysis and detection of two substances can be realized. The limit of the detections for F and FG are 2.60 ng·mL-1 and 9.30 ng·mL-1, respectively. The linear detection ranges of F and FG are 11.7~1860 ng·mL-1 and 14.6~2920 ng·mL-1, respectively. Although the structural relationship between F and FG is glycoside and aglycone, under hot alkaline conditions, the final products after the cleavage and hydrolysis reactions are essentially different. The different fluorescence characteristics between F and FG pave a way for further identification and a quantitative analysis of the corresponding components in Chinese herbal medicine.

Unraveling the Thermal Oxidation Products and Peroxidation Mechanisms of Different Chemical Structures of Lipids: An Example of Molecules Containing Oleic Acid.

Lipid structures affect lipid oxidation, causing differences in types and contents of volatiles and nonvolatiles in various foods. In this study, the oxidation differences of monoacylglycerol (MAG), triacylglycerol (TAG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) with oleoyl residues and oleic acid (FA) during thermal treatment were investigated. Volatiles and nonvolatiles were monitored by gas chromatography-mass spectrometry and ultrahigh-performance liquid chromatography-Q-Exactive HF-X Orbitrap Mass Spectrometer, respectively. The results showed that the structures of MAG and TAG could delay the chain initiation reaction. The polar heads of PC and PE remarkably influenced the oxidation rate and the formation of the oxidation products probably due to the hydrogen bonds formed with free radicals. Among the volatile oxidation products, aldehydes, acids, and furans with eight or nine carbon atoms accounted for the majority in FA, MAG, TAG, and PC samples, but PE samples mainly generated ketones with nine or 10 carbon atoms. The formation of nonvolatile products in TAG samples possessed significant stage-specific changes. Fatty acid esters of hydroxy fatty acids were only produced in the free fatty acid oxidation model. The activity of chemical bonds participating in the truncation reaction decreased to both sides from the double bond position.

Programed Thermoresponsive Polymers with Cleavage-Induced Phase Transition.

A new programed upper critical solution temperature-type thermoresponsive polymer was developed using water-soluble anionic polymer conjugates derived from polyallylamine and phthalic acid with cleavage-induced phase transition property. Intrinsic charge inversion from anion to cation of the polymer side chain is induced through a side chain cleavage reaction in acidic aqueous media. With the progress of side chain cleavage under fixed external conditions, the polymer conjugates express a thermoresponsive property, followed by shifting a phase boundary due to the change in polymer composition. When the phase transition boundary eventually reached the examined temperature, phase transition occurs under fixed external conditions. Such new insight obtained in this study opens up the new concept of time-programed stimuli-responsive polymer possessing a cleavage-induced phase transition.

Organomediated cleavage of benzoyl group enables an efficient synthesis of 1-(6-nitropyridin-2-yl)thiourea and its application for developing 18F-labeled PET tracers.

A novel organomediated cleavage of benzoyl group using ethane-1,2-diamine and acetic acid under neutral condition enables an efficient synthesis of 1-(6-nitropyridin-2-yl)thiourea, which previously has been challenging to prepare by conventional methods. The successful synthesis of 1-(6-nitropyridin-2-yl)thiourea as a synthon permits development of a variety of 18F labeled heterocycles as PET imaging ligands such as N-(pyridin-2-yl)thiazol-2-amine derivatives. The utility of this synthon is demonstrated with the synthesis of a 18F-labeled PET tracer for studying prion disease. In vitro autoradiography using this PET tracer on sagittal rat brain slices showed highest accumulation of radioactivity in the hippocampus, cortex, and striatum, in accordance with reported immunostaining of PrPc in rat brain.

Construction of a Reduction-responsive DNA Microsphere using a Reduction-cleavable Spacer based on a Nitrobenzene Scaffold.

Here, we describe the design and synthesis of a new reduction-cleavable spacer (RCS) based on a nitrobenzene scaffold for constructing reduction-responsive oligonucleotides according to standard phosphoramidite chemistry. In addition, we demonstrate that the introduction of the RCS in the middle of an oligonucleotide (30 nt) enables the construction of a self-assembled microsphere capable of exhibiting a reduction-responsive disassembly.

A lable-free SPR biosensor based on one peptide sequence with three recognition sites for O-GlcNAc transferase detection.

Abnormal O-linked N-acetylglucosamine (O-GlcNAc) concentrations have been associated with many diseases, but the lack of accurate detection method limited O-GlcNAc to be used as a biomarker in clinical diagnosis. Then O-GlcNAc transferase (OGT) has drawn researchers' attention as it closed related to the level of O-GlcNAc and be considered to be a promising new target for diseases diagnosis. Nevertheless, the existing OGT detection methods are either need labeling or the sensitity can not meet the needs of clinic testing. Herein, a label-free and sensitive SPR biosensor was developed for accurate detection of OGT based on a multi-functional peptide. The designed peptide contains three recognition sites, one is the cleavage site of protease K, one is the O-GlcNAcylated site by OGT, and another is six histidine which be used as the signal report probe to recognize Ni2+. The immobilized peptide would be cleavaged by proteinase K, then the His-tag residue part will leave the surface of Au film, resulting less His-tag could bind to Ni2+ and a small SPR signal would be record. If the peptide is O-GlcNAcylated by OGT, the cleaving reaction would be limited due to the adjacent site of O-GlcNAcylation. Then more His-tag can be left on the Au film and a bigger SPR signal could be record, this signal is associated with the concentration of OGT. Utilizing the change of the peptide configuration as a signal report probe for OGT detection not only avoids labeling of peptide, but also makes the method more sensitive. The determination linear range of OGT is from 2.00 × 10-13 to 5.00 × 10-8 M with a detection limit of 1.19 × 10-13 M, and the separation of two enzyme reactions ensured the high selectivity of the method. Finally, the sensing system was successfully used for OGT detection in blood samples with satisfied recovery. In summary, the label-free SPR platform for accurate detection of OGT in real samples is helpful to promote OGT serve as a biomarker for early clinical diagnosis of O-GlcNAc related diseases.

A Novel Gold Nanoprobe for a Simple Electrochemiluminescence Determination of a Prostate-specific Antigen Based on a Peptide Cleavage Reaction.

A novel gold nanoprobe for a sensitive and simple determination of a prostate-specific antigen (PSA) was designed on the basis of homogeneous detection and a peptide cleavage reaction. The gold nanoprobe (AuNPs-peptide-Ru1) consisted of a specific peptide tagged with a ruthenium(II) complex (Ru1) and gold nanoparticles (AuNPs) conjugated with the peptide via the strong Au-S bond between the AuNPs surface and the thiol group of the peptide. The electrochemiluminescence (ECL) enzymatic-cleavage-reaction-based bioanalytic system based on homogeneous detection has overcome shortcomings from a complicated fabrication process of traditional electrodes. In the presence of the target PSA, it specifically cleaved the peptide of the AuNPs-peptide-Ru1, and the ECL signal substance (Ru1) part dissociated from AuNPs-peptide-Ru1. This resulted in an increase in the ECL intensity. The ECL biosensor could detect PSA concentrations in the range from 1.0 × 10-12 to 1.0 × 10-9 g/mL, the detection limit was 4.0 × 10-13 g/mL. The assay with the advantages of a simple method for PSA was selective and fast. It is superior to the immunoassay, and is a promising strategy to develop biosensors based on enzymatic cleavage including electrochemistry and optics.

Label-Free Sensing of Human 8-Oxoguanine DNA Glycosylase Activity with a Nanopore.

Human 8-oxoguanine DNA glycosylase (hOGG1) plays a significant role in maintaining the genomic integrity of living organisms for its capability of repairing DNA lesions. Accurate detection of hOGG1 activity would greatly facilitate the screening and early diagnosis of diseases. In this work, we report a nanopore-based sensing strategy to probe the hOGG1 activity by employing the enzyme-catalytic cleavage reaction of DNA substrate. The hOGG1 specifically catalyzed the removal of the 8-hydroxyguanine (8-oxoG) and cleaved the DNA substrates immobilized on magnetic beads, thereby releasing the output DNA which would quantitatively produce the signature current events when subjected to α-hemolysin (α-HL) nanopore test. The approach enables the sensitive detection of hOGG1 activity without the need of any labeling or signal amplification route. Furthermore, the method can be applied to assay the inhibition of hOGG1 and evaluate the activity of endogenous hOGG1 in crude cell extracts. Importantly, since DNAs with specific sequences are the catalytic substrates of a wide variety of enzymes, the proposed strategy should be universally applicable for probing the activities of different types of enzymes with nanopore sensors.

Mycothiol synthesis by an anomerization reaction through endocyclic cleavage.

Mycothiol is found in Gram-positive bacteria, where it helps in maintaining a reducing intracellular environment and it plays an important role in protecting the cell from toxic chemicals. The inhibition of the mycothiol biosynthesis is considered as a treatment for tuberculosis. Mycothiol contains an α-aminoglycoside, which is difficult to prepare stereoselectively by a conventional glycosylation reaction. In this study, mycothiol was synthesized by an anomerization reaction from an easily prepared ß-aminoglycoside through endocyclic cleavage.

[Application of inteins in building protein affinity purification system].

Intein is a part of polypeptide in the premature protein with the capability of self-splicing, which is widely applied in protein purification, protein conjuction, cyclopeptide preparation, protein labeling and biosensor. In this review, we summarized the development of intein used in protein purification, discussed intein-mediated chromatographic and non-chromatographic purification systems, and summarized the researches in manipulating intein cleavage reaction. This work is to provide clues for improvement of intein-mediated protein purification.

Application of inteins in building protein affinity purification system / 生物工程学报

Intein is a part of polypeptide in the premature protein with the capability of self-splicing, which is widely applied in protein purification, protein conjuction, cyclopeptide preparation, protein labeling and biosensor. In this review, we summarized the development of intein used in protein purification, discussed intein-mediated chromatographic and non-chromatographic purification systems, and summarized the researches in manipulating intein cleavage reaction. This work is to provide clues for improvement of intein-mediated protein purification.

Semisynthesis of a post-translationally modified protein by using chemical cleavage and activation of an expressed fusion polypeptide.

Herein, we describe a new semisynthetic strategy of a post-translationally modified protein in which the middle region is glycosylated. We designed a single-plasmid coding for a fusion polypeptide, which can provide both an N-terminal α-thioester and a C-terminal cysteine peptide of a target glycoprotein by using chemical-cleavage and activation methods. The use of these resultant peptide derivatives resulted in the successful synthesis of N-glycosylated-interleukin 13.

Selective conversion of biorefinery lignin into dicarboxylic acids.

The emerging biomass-to-biofuel conversion industry has created an urgent need for identifying new applications for biorefinery lignin. This paper demonstrates a new route to producing dicarboxylic acids from biorefinery lignin through chalcopyrite-catalyzed oxidation in a highly selective process. Up to 95 % selectivity towards stable dicarboxylic acids was obtained for several types of biorefinery lignin and model compounds under mild, environmentally friendly reaction conditions. The findings from this study paved a new avenue to biorefinery lignin conversions and applications.

A novel Au-nanoparticle biosensor for the rapid and simple detection of PSA using a sequence-specific peptide cleavage reaction.

PSA (prostate-specific antigen) is one of the most widely used proteins for the diagnosis of breast and prostate cancer. Of note, PSA displays enzymatic activity for the specific peptide sequence HSSKLQ, which it recognizes and cleaves. In this study, we developed a site-specific enzymatic-cleavage-reaction-based biosensor for the detection of PSA using fluorescein isothiocyanate (FITC)/peptide-conjugated gold (Au) nanoparticle complexes (FPANs). The FPANs do not initially fluoresce in the spectral region associated with the fluorophore, due to the quenching effect of the Au nanoparticles. When PSA was added to a solution containing the FPANs, PSA recognized and cleaved the specific sequence of the peptides attached to the Au nanoparticles. As a result, FITCs were separated from the Au nanoparticles and emitted strong fluorescence in their spectral region. Using this detection method, PSA was successfully detected as a function of concentration (10 pM-100 nM). This approach is superior to the immunoassay with respect to the performance of sensor, which is very rapid, simple, and one-step method for the detection of PSA and other protein markers can be measured for the early detection of several diseases.

Iso-seco-tanapartholides: Isolation, Synthesis and Biological Evaluation.

The isolation, identification and total synthesis of two plant-derived inhibitors of the NF-κB signaling pathway from the iso-seco-tanapartholide family of natural products is described. A key step in the efficient reaction sequence is a late-stage oxidative cleavage reaction that was carried out in the absence of protecting groups to give the natural products directly. A detailed comparison of the synthetic material with samples of the natural products proved informative. Biological studies on synthetic material confirmed that these compounds act late in the NF-κB signaling pathway. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009).

Effects of Divalent Mg2+ and Monovalent Na+ on Cleavage Reactions by Multiribozyme System in vitro / 中国生物化学与分子生物学报

To better understand the cleavage efficiency of muhiribozyme system on its RNA substrate in the presence and absence of divalent magnesium and monovalent sodium ions.we constructed pGEM-Coat'A,pGEM-Coat'A196Rz plasmids and pGEM-MDRl target plasmid.They were applied to transcribe RNAs with SP6/T7 transcription kit.Cleavage reactions were carried out in cell-free system and reaction products were analyzed by electrophoresis on 6% denaturing polyacrylamide gels in TBS buffer.The gels were dried and exposed to X-ray films for autoradiography.The Image J software was employed to analyze the dried gels.The results indicated that the cleavage efficiency of the muhiribozyme was dependent on the concentration of divalent Mg2+.The cleavage products increased with the concentrations of divalent Mg2+ and were Mg2+ concentration and time dependent.No cleavage product was obtained in the presence of lower than 200 mmol/L Na+ alone.On the contrary,monovalent Na+ inhibited the Mg2+ -induced cleavage reaction in Na+ and Mg2+ coexistance.The cleavage rate was significantly lower than that observed with divalent Mg2+ alone.These results suggested that divalent Mg2+ was required for muhiribozyme on substrate cleavage reaction in the physical condition,whereas monovalent Na+ was not.