烟酰型辅酶NAD(P)+和NAD(P)H再生的研究进展

大部分氧化还原酶的催化反应需要烟酰型辅酶NAD(P) 和NAD(P)H作为氧化剂或还原剂参与,由于氧化还原酶应用广泛而辅酶价格昂贵,使得辅酶再生逐渐成为研究热点.综述了近年来NAD(P) 和NAD(P)H酶法再生、电化学法及光化学法再生的研究进展,并介绍了各再生技术的应用和开发状况.     

细胞内NAD(P)H水平分析技术的研究进展

细胞内NAD(P)H水平直接控制着细胞的衰老、节律、癌变、死亡等重大生命过程,NAD(P)H水平的研究是生命过程中新的研究热点之一.本文介绍了NAD(P)H的结构、特性及检测方法,重点探讨了近年来国内外NAD(P)H水平的检测,并对其研究现状进行了综述.     

辅酶NAD(P)H模型分子的研究进展

辅酶NAD(P)H在生物体内起着重要的调节作用, 已引起了有机化学工作者极大的兴趣, 尤其是在还原反应的立体选择性上, 人们已经开展了大量的研究工作. 讨论了NAD(P)H模型分子进行立体专一性还原反应的影响因素, 并对NAD(P)H模型分子的研究工作做了总结.     

Photoelectrocyclization as an Activation Mechanism for Organelle‐Specific Live‐Cell Imaging Probes

Photoactivatable fluorophores are useful tools in live‐cell imaging owing to their potential for precise spatial and temporal control. In this report, a new photoactivatable organelle‐specific live‐cell imaging probe based on a 6π electrocyclization/oxidation mechanism is described. It is shown that this new probe is water‐soluble, non‐cytotoxic, cell‐permeable, and useful for mitochondrial imaging. The probe displays large Stokes shifts in both pre‐activated and activated forms, allowing simultaneous use with common dyes and fluorescent proteins. Sequential single‐cell activation experiments in dense cellular environments demonstrate high spatial precision and utility in single‐ or multi‐cell labeling experiments.     

Enzyme-Compatible Core-Shell Nanoreactor for in Situ H2-Driven NAD(P)H Regeneration

The regeneration of the reduced form cofactor NAD(P)H is essential for the extra-cellular application of bio-reduction, which necessitates not only the development of efficient artificial NAD(P)H regeneration catalytic system but also its well compatibility with the cascade enzymatic reduction system. In this work, we reported the preparation of a metal nanoparticle (NP) and metal complex integrated core-shell nanoreactor for H₂-driven NAD(P)H regeneration through the immobilization of a Rh complex on Ni/TiO₂ surface via a bipyridine contained 3D porous organic polymer (POP). In comparison with the corresponding single component metal NPs and the immobilized Rh complex, the integrated catalyst presented simultaneously enhanced activity and selectivity in NAD(P)H regeneration thanks to the rapid spillover of activated H species from metal NPs to Rh complex. In addition, the size-sieving effect of POP precluded the direct interaction of enzyme and Rh complex confined in the pores, enabling the success coupling of core-shell nanoreactor and aldehyde ketone reductase (AKR) for chemoenzymatic reduction of acetophenone to (R)-1-phenylethan-1-ol. This work provides a strategy for the rational manipulation of multicomponent cooperation catalysis.     

烟酰胺辅酶模型对N－芳基芴亚胺的酸催化还原反应的研究

报道了５种Ｎ－芳基芴亚胺在酸性条件下被烟酰胺辅酶模型（Ｈａｎｔｚｓｃｈ酯，ＢＮＡＨ）还原的反应。结果表明：亚胺的结构、酸的强度以及溶剂的不同均会影响亚胺的还原效率，本文结合反应的结构效应、溶剂效应和同位素效应，对其可能的酸催化氢负离子转移机理进行了讨论。     

Pre‐targeted Imaging of Protease Activity through In Situ Assembly of Nanoparticles

The pre‐targeted imaging of enzyme activity has not been reported, likely owing to the lack of a mechanism to retain the injected substrate in the first step for subsequent labeling. Herein, we report the use of two bioorthogonal reactions—the condensation reaction of aromatic nitriles and aminothiols and the inverse‐electron demand Diels–Alder reaction between tetrazine and trans‐cyclooctene (TCO)—to develop a novel strategy for pre‐targeted imaging of the activity of proteases. The substrate probe ( TCO‐C‐SNAT4 ) can be selectively activated by an enzyme target (e.g. caspase‐3/7), which triggers macrocyclization and subsequent in situ self‐assembly into nanoaggregates retained at the target site. The tetrazine‐imaging tag conjugate labels TCO in the nanoaggregates to generate selective signal retention for imaging in vitro, in cells, and in mice. Owing to the decoupling of enzyme activation and imaging tag immobilization, TCO‐C‐SNAT4 can be repeatedly injected to generate and accumulate more TCO‐nanoaggregates for click labeling.     

A Three‐Photon Active Organic Fluorophore for Deep Tissue Ratiometric Imaging of Intracellular Divalent Zinc

Deep tissue bioimaging with three‐photon (3P) excitation using near‐infrared (NIR) light in the second IR window (1.0–1.4 μm) could provide high resolution images with an improved signal‐to‐noise ratio. Herein, we report a photostable and nontoxic 3P excitable donor‐π‐acceptor system (GMP) having 3P cross‐section (σ₃) of 1.78×10^?80 cm⁶ s² photon^?2 and action cross‐section (σ₃η₃) of 2.31×10^?81 cm⁶ s² photon^?2, which provides ratiometric fluorescence response with divalent zinc ions in aqueous conditions. The probe signals the Zn²⁺ binding at 530 and 600 nm, respectively, upon 1150 nm excitation with enhanced σ₃ of 1.85×10^?80 cm⁶ s² photon^?2 and σ₃η₃ of 3.33×10^?81 cm⁶ s² photon^?2. The application of this probe is demonstrated for ratiometric 3P imaging of Zn²⁺ in vitro using HuH‐7 cell lines. Furthermore, the Zn²⁺ concentration in rat hippocampal slices was imaged at 1150 nm excitation after incubation with GMP, illustrating its potential as a 3P ratiometric probe for deep tissue Zn²⁺ ion imaging.     

Cysteine‐Mediated Intracellular Building of Luciferin to Enhance Probe Retention and Fluorescence Turn‐On

The development of sensitive and selective small molecular probes that enable real‐time detection of endogenous cysteine (Cys) has become an attractive topic because of the essential roles played by Cys in controlling the cellular nitrogen balance and in maintaining biological redox homeostasis. Herein, we report a Cys‐specific probe, 2‐cyanobenzothiazol‐6‐yl acrylate (CBTOA), that shows not only fluorescence turn‐on for sensitive detection of endogenous Cys but also enhanced probe retention inside cells for real‐time monitoring of Cys levels upon external stimulation. Cys‐mediated intracellular formation of luciferin from CBTOA was the key strategy leading to this new type of fluorogenic probe. CBTOA showed fast response to Cys in living cells and liver tissue slices with high sensitivity and selectivity. By using CBTOA as a real‐time probe, we were able to monitor the change in Cys levels in living HeLa cells under ROS‐induced oxidative stress as well as in human mesenchymal stem cells during adipogenic differentiation.     

NAD(P)H模型引发的还原环化——2,2-双取代1,2-二氢茚的合成

辅酶NAD(P)H在生物氧化还原反应中起着重要作用[1].1-苄基-1,4-二氢尼古丁酰胺(BNAH)作为其模型物,被广泛用于物理有机和生物化学的研究之中[2].虽然绝大多数的研究都集中于还原反应机理方面[3,4],BNAH作为还原剂在有机合成中的应用也是值得注意的.我们曾用BNAH还原2-溴-1-苯亚乙基丙二腈及其类似物合成取代环丙烷[5～7],方法简便.五元环结构广泛存在于萜类和甾体等天然产物中.对于茚等苯并五元环结构的合成已有许多方法[8～11]. 其中,2,2-双取代1,2-二氢茚(1)(吸电子取代基)是用邻-二溴甲基苯与丙二腈等活泼亚甲基化合物在DMSO中,NaH存在下双分子缩合制备的[12].     

Water‐Soluble Triarylborane Chromophores for One‐ and Two‐Photon Excited Fluorescence Imaging of Mitochondria in Cells

Three water‐soluble tetracationic quadrupolar chromophores comprising two three‐coordinate boron π‐acceptor groups bridged by thiophene‐containing moieties were synthesised for biological imaging applications. Compound 3 containing the bulkier 5‐(3,5‐Me₂C₆H₂)‐2,2′‐(C₄H₂S)₂‐5′‐(3,5‐Me₂C₆H₂) bridge is stable over a long period of time, exhibits a high fluorescence quantum yield and strong one‐ and two‐photon absorption (TPA), and has a TPA cross section of 268 GM at 800 nm in water. Confocal laser scanning fluorescence microscopy studies in live cells indicated localisation of the chromophore at the mitochondria; moreover, cytotoxicity measurements proved biocompatibility. Thus, chromophore 3 has excellent potential for one‐ and two‐photon‐excited fluorescence imaging of mitochondrial function in cells.     

Biomimetic Asymmetric Reduction of Quinazolinones with Chiral and Regenerable NAD(P)H Models

A facile approach to chiral dihydroquinazolinone derivatives has been described via biomimetic asymmetric reduction of quinazolinones with chiral and regenerable NAD(P)H models. The utility of this method was demonstrated by a concise synthesis of the bromodomain protein divalent inhibitor.     

A Quadruplex‐Based,Label‐Free,and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

We demonstrate a unique quadruplex‐based fluorescence assay for sensitive, facile, real‐time, and label‐free detection of RNase H activity and inhibition by using a G‐quadruplex formation strategy. In our approach, a RNA–DNA substrate was prepared, with the DNA strand designed as a quadruplex‐forming oligomer. Upon cleavage of the RNA strand by RNase H, the released G‐rich DNA strand folds into a quadruplex in the presence of monovalent ions and interacts with a specific G‐quadruplex binder, N‐methyl mesoporphyrin IX (NMM); this gives a dramatic increase in fluorescence and serves as a reporter of the reaction. This novel assay is simple in design, fast in operation, and is more convenient and promising than other methods. It takes less than 30 min to finish and the detection limit is much better or at least comparable to previous reports. No sophisticated experimental techniques or chemical modification for either RNA or DNA are required. The assay can be accomplished by using a common spectrophotometer and obviates possible interference with the kinetic behavior of the catalysts. Our approach offers an ideal system for high‐throughput screening of enzyme inhibitors and demonstrates that the structure of the G‐quadruplex can be used as a functional tool in specific fields in the future.     

Ultra‐fast Cycling for Multiplexed Cellular Fluorescence Imaging

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Herein, we describe an ultra‐fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz)/trans‐cyclooctene (TCO)‐mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t_1/2 <1 s) than predicted. This allowed multi‐cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.     

Photoswitching Kinetics and Phase‐Sensitive Detection Add Discriminative Dimensions for Selective Fluorescence Imaging

Non‐invasive separation‐free protocols are attractive for analyzing complex mixtures. To increase selectivity, an analysis under kinetic control, through exploitation of the photochemical reactivity of labeling contrast agents, is described. The simple protocol is applied in optical fluorescence microscopy, where autofluorescence, light scattering, as well as spectral crowding presents limitations. Introduced herein is OPIOM (out‐of‐phase imaging after optical modulation), which exploits the rich kinetic signature of a photoswitching fluorescent probe to increase selectively and quantitatively its contrast. Filtering the specific contribution of the probe only requires phase‐sensitive detection upon matching the photoswitching dynamics of the probe and the intensity and frequency of a modulated monochromatic light excitation. After in vitro validation, we applied OPIOM for selective imaging in mammalian cells and zebrafish, thus opening attractive perspectives for multiplexed observations in biological samples.     

NAD(P)H辅酶模型物结构与反应性关系的研究──3-位酰基的电子效应

合成了一系列3酰胺基氮取代的NAD(P)H模型物,测定了其与5硝基异喹啉正离子的二级反应速率常数,并与模型物的氧化还原电势进行了比较.实验结果表明,模型物3位酰基氧一方面可离域二氢吡啶环上N的电子;另一方面负电性的3位酰基氧在反应过渡态中又可引起分子内和分子间的两种静电作用;3位酰基的电子效应对模型物动力学反应性的影响是这两种效应综合作用的结果.     

A Far‐Red Emitting Fluorescent Chemogenetic Reporter for In Vivo Molecular Imaging

Far‐red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far‐red Fluorescence Activating and absorption Shifting Tag), a 14‐kDa monomeric protein that forms a bright far‐red fluorescent assembly with (4‐hydroxy‐3‐methoxy‐phenyl)allylidene rhodanine (HPAR‐3OM). As HPAR‐3OM is essentially non‐fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR‐3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae, and chicken embryos. Beyond enabling the genetic encoding of far‐red fluorescence, frFAST allowed the design of a far‐red chemogenetic reporter of protein–protein interactions, demonstrating its great potential for the design of innovative far‐red emitting biosensors.     

Analysis of NAD(P) and NAD(P)H cofactors by means of imprinted polymers associated with Au surfaces:: A surface plasmon resonance study

Crosslinked films consisting of the acrylamide-acrylamidophenylboronic acid copolymer that are imprinted with recognition sites for β-nicotinamide adenine dinucleotide (NAD⁺), β-nicotinamide adenine dinucleotide phosphate NADP⁺, and their reduced forms (NAD(P)H), are assembled on Au-coated glass supports. The binding of the oxidized cofactors NAD⁺ or NADP⁺ or the reduced cofactors NADH or NADPH to the respective imprinted sites results in the swelling of the polymer films through the uptake of water. Surface plasmon resonance (SPR) spectroscopy is employed to follow the binding of the different cofactors to the respective imprinted sites. The imprinted recognition sites reveal selectivity towards the association of the imprinted cofactors. The method enables the analysis of the NAD(P)⁺ and NAD(P)H cofactors in the concentration range of 1×10⁻⁶ to 1×10⁻³ M. The cofactor-imprinted films associated with the Au-coated glass supports act as active interfaces for the characterization of biocatalyzed transformations that involve the cofactor-dependent enzymes. This is exemplified with the characterization of the biocatalyzed oxidation of lactate to pyruvate in the presence of NAD⁺ and lactate dehydrogenase using the NADH-imprinted polymer film.