How To Form a Phosphate Anhydride Linkage in Nucleotide Derivatives

The fundamental roles of nucleoside triphosphates and nucleotide cofactors such as NAD⁺ in biochemistry are well known. In recent decades, continuing research has revealed the key role of 5′‐capped RNA and 5′,5′‐dinucleoside polyphosphates in the regulation of vitally important physiological processes. Last but not least, the commercial potential of nucleoside triphosphate synthesis can hardly be overestimated. Nevertheless, despite decades of investigation and the obvious topicality of the research on the chemical synthesis of the nucleotide compounds containing phosphate anhydride linkages, none of the existing procedures can be considered an up‐to‐date “gold standard”. However, there are a number of fruitful synthetic approaches to forming phosphate anhydride linkages in satisfactory yield. These are summarized in this concise review, organized by the type of active phosphorous intermediate and reagents used.     

Ultramild Protein‐Mediated Click Chemistry Creates Efficient Oligonucleotide Probes for Targeting and Detecting Nucleic Acids

Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)‐binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine‐based fluorescent probes in vitro and rationalize our results by electronic structure calculations.     

Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7‐Substituted 7‐Deazaguanine Nucleobases

Previous studies of polymerase synthesis of base‐modified DNAs and their cleavage by restriction enzymes have mostly related only to 5‐substituted pyrimidine and 7‐substituted 7‐deazaadenine nucleotides. Here we report the synthesis of a series of 7‐substituted 7‐deazaguanine 2′‐deoxyribonucleoside 5′‐O‐triphosphates (dG^RTPs), their use as substrates for polymerase synthesis of modified DNA and the influence of the modification on their cleavage by type II restriction endonucleases (REs). The dG^RTPs were generally good substrates for polymerases but the PCR products could not be visualised on agarose gels by intercalator staining, due to fluorescence quenching. The presence of 7‐substituted 7‐deazaguanine residues in recognition sequences of REs in most cases completely blocked the cleavage.     

Design and synthesis of caged fluorescent nucleotides and application to live-cell RNA imaging

A binary photocontrolled nucleic acid probe that contains a nucleotide modified with one photolabile nitrobenzyl unit and two hybridization-sensitive thiazole orange units has been designed for area-specific fluorescence imaging of RNA in a cell. The synthesized probe emitted very weak fluorescence regardless of the presence of the complementary RNA, whereas it showed hybridization-sensitive fluorescence emission at 532 nm after photoirradiation at 360 or 405 nm for uncaging. Fluorescence suppression of the caged probe was attributed to a decrease in the duplex-formation ability. Caged fluorescent nucleotides with other emission wavelengths (622 and 724 nm) were also synthesized in this study; they were uncaged by 360 nm irradiation, and emitted fluorescence in the presence of the complementary RNA. Such probes were applied to area-specific RNA imaging in a cell. Only probes in the defined irradiation area were activated by uncaging irradiation, and subnuclear mRNA diffusion in a living cell was monitored.     

Triptolide directly inhibits dCTP pyrophosphatase

Triptolide is a potent natural product, with documented antiproliferative, immunosuppressive, anti-inflammatory, antifertility, and antipolycystic kidney disease effects. Despite a wealth of knowledge about the biology of this compound, direct intracellular target proteins have remained elusive. We synthesized a biotinylated photoaffinity derivative of triptolide, and used it to identify dCTP pyrophosphatase 1 (DCTPP1) as a triptolide-interacting protein. Free triptolide interacts directly with recombinant DCTPP1, and inhibits the enzymatic activity of this protein. Triptolide is thus the first dCTP pyrophosphatase inhibitor identified, and DCTPP1 is a biophysically validated target of triptolide.     

Flesh Quality in Snapper, Pagrcrs auratus, Affected by Capture Stress

Muscle metabolites in resting, tank acclimated snapper, Pagrus auratus, were monitored for 72 hr postmortem and compared with values from exercised or commercially caught fish. The physiological status of the live animal was quantified by plasma cortisol and blood chemistry. Cortisol levels were lowest in unstressed controls (6.8 ± 2.1 ng mL^?1) while exercised laboratory fish had highest levels (67.7 ± 11.2 ng mL^?1). Control fish maintained a constant K-value for 72 hr in chilled storage; all other groups had significant increases. Onset of rigor development and muscle ATP depletion was delayed in unstressed fish and was more rapid in line-captured than exercised fish. Commercial users minimizing stress would maintain high flesh quality.     

漂洗过程中白鲢鱼糜风味物质变化的分析

以白鲢为原料,研究了漂洗过程中白鲢鱼糜的呈味核苷酸类物质和游离氨基酸含量的变化,通过电子鼻及气相质谱联用仪(GC-MS)分析了其挥发性成分的变化。结果表明:一次漂洗和二次漂洗后鱼糜中IMP含量显著性减少,三次漂洗后AMP含量显著性减少;漂洗过程中一次、二次和三次漂洗后游离氨基酸总量和鲜、甜味氨基酸总量均显著性减少;漂洗过程对白鲢鱼糜的气味有所影响,电子鼻主成分分析(PCA)结果显示二次漂洗和脱水后的两组鱼糜气味相似,其它各组间均能得到有效区分;通过GC-MS分析,未漂洗、一次漂洗、二次漂洗、三次漂洗、脱水后的白鲢鱼糜样品分别检测确认30、27、21、15、18种挥发性物质,主要为己醛、庚醛、壬醛、1-己醇、1-辛烯-3-醇等羰基化合物和醇类。     

外源核苷酸的生物学特性及在乳代品中的作用

介绍了外源核苷酸作为一种活性物质，对胃肠道的功能、肝脏生长与发育、脂质代谢、动物体免疫系统等具有重要影响。在人和动物体内合成的内源核苷酸不能完全满足机体所需要的某些情况下，如机体迅速生长、受到免疫挑战、饥饿或局部缺血等特殊生理变化时期，食品中添加外源核苷酸能改善动物体的生理机能；且将外源核苷酸加入到婴儿乳代品中，能促进婴幼儿的生长和发育。外源核苷酸的诸多特点使其在临床治疗、医疗保健、动物营养等方面具有广阔的应用价值和市场前景。