核苷酸生产技术现状及展望

核苷酸的生产方法主要有化学合成法、RNA酶解法、微生物发酵法以及生物催化法。探讨了这些方法的原理和发展及其在工业化生产中的优劣势。化学合成法的路线长、立体选择性差,所用试剂昂贵并有一定毒性,生产成本较高;酶解法能一次得到4种核苷酸的混合物且收率较高,是目前我国核苷酸工业生产所用的主要技术,但其后提取难度大,产品纯度不高;微生物发酵法难以解决细胞通透性的问题;生物催化法是发酵法的延伸,菌体培养和酶催化反应分两步进行,有效地解决了细胞通透性问题,并可以通过偶联不同的基因工程菌株生产多种复杂核苷酸、核苷糖乃至寡聚糖,这在核苷酸工业、医药及糖化学、糖生物学合成工业中是极其重要的一个环节。     

7-(Dialkylamino)coumarin-4-yl]methyl-Caged Compounds as Ultrafast and Effective Long-Wavelength Phototriggers of 8-Bromo-Substituted Cyclic Nucleotides

[7-(Dimethylamino)coumarin-4-yl]methyl (DMACM) and [7-(diethylamino)coumarin-4-yl]methyl (DEACM) esters of 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) and 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) are described as novel caged compounds for 8-bromo-substituted cyclic nucleotides. Synthesis is accomplished by treatment of the free acids of the cyclic nucleotides with the corresponding 7(dialkylamino)-substituted 4(diazomethyl)coumarins. Irradiation of the DMACM- and DEACM-caged cyclic nucleotides with UV light stimulates the release of the cyclic nucleotides within roughly a nanosecond. The new caged compounds are resistant to hydrolysis in aqueous buffers and exhibit long-wavelength absorption properties with maxima at 400 nm, high extinction coefficients, and high quantum yields (0.15-0.31). Their favorable properties render these compounds the most efficient and rapid phototriggers of 8-bromo-substituted cyclic nucleotides known. The usefulness of the compounds for physiological studies under nondamaging light conditions was examined in HEK293 cells expressing the alpha subunit of the cyclic-nucleotide-gated (CNG) channel of cone photoreceptors (CNGA3) and of olfactory neurons (CNGA2) by using confocal laser scanning microscopy and the patch clamp technique.     

Shrinkable Nanotubes for Duplex Formation of Short Nucleotides

Molecular monolayer nanotubes produced by self‐assembly of an amphiphile modified with a 2‐nitrobenzyl group as a photoresponsive unit are able to encapsulate dinucleotides via electrostatic attraction. Upon photoirradiation, the 18 nm inner diameter of the nanotubes shrinks to less than 2 nm as a result of photochemical cleavage of the 2‐nitrobenzyl group in the amphiphile. This shrinking of the nanotube channels leads to a propulsive release of the dinucleotides into the bulk solution and simultaneously accelerates formation of the dinucleotide duplexes. The larger nanotube channels without photoirradiation merely release each dinucleotide into the bulk solution, indicating that the squeezing via transportation in the narrow nanotube channels is necessary for duplex formation. In addition to the size effect, water with a lower polarity confined within the narrow nanotube channels helps to stabilize the energetically unfavorable hydrogen‐bonded base pair between the dinucleotides. This system should enable researchers to perform biological reactions that occur only in specific environments and conditions in living organisms.     

Extracellular Nucleotides Affect the Proangiogenic Behavior of Fibroblasts,Keratinocytes, and Endothelial Cells

Chronic wound healing is currently a severe problem due to its incidence and associated complications. Intensive research is underway on substances that retain their biological activity in the wound microenvironment and stimulate the formation of new blood vessels critical for tissue regeneration. This group includes synthetic compounds with proangiogenic activity. Previously, we identified phosphorothioate analogs of nucleoside 5′-O-monophosphates as multifunctional ligands of P2Y6 and P2Y14 receptors. The effects of a series of unmodified and phosphorothioate nucleotide analogs on the secretion of VEGF from keratinocytes and fibroblasts, as well as their influence on the viability and proliferation of keratinocytes, fibroblasts, and endothelial cells were analyzed. In addition, the expression profiles of genes encoding nucleotide receptors in tested cell models were also investigated. In this study, we defined thymidine 5′-O-monophosphorothioate (TMPS) as a positive regulator of angiogenesis. Preliminary analyses confirmed the proangiogenic potency of TMPS in vivo.     

Analysis of Modified Nucleotide Aptamer Library Generated by Thermophilic DNA Polymerases

One of the pivotal steps in aptamer selection is the amplification of target-specific oligonucleotides by thermophilic DNA polymerases; it can be a challenging task if nucleic acids possessing modified nucleotides are to be amplified. Hence, the identification of compatible DNA polymerase and modified nucleotide pairs is necessary for effective selection of aptamers with unnatural nucleotides. We present an in-depth study of using 5-indolyl-AA-dUTP (TAdUTP) to generate oligonucleotide libraries for aptamer selection. We found that, among the eight studied DNA polymerases, only Vent(exo-) and KOD XL are capable of adapting TAdUTP, and that replacing dTTP did not have a significant effect on the productivity of KOD XL. We demonstrated that water-in-oil emulsion PCR is suitable for the generation of aptamer libraries of modified nucleotides. Finally, high-throughput sequence analysis showed that neither the error rate nor the PCR bias was significantly affected by using TAdUTP. In summary, we propose that KOD XL and TAdUTP could be effectively used for aptamer selection without distorting the sequence space of random oligonucleotide libraries.     

反相高效液相色谱法测定中国对虾肉中的呈味核苷酸

核苷酸5＇-CMP、5＇-UMP、5＇-GMP、5＇-IMP、5＇-AMP 等是食品重要的鲜味物质。采用C18 高效液相色谱柱,以甲醇与0.05mol/L KH2PO4 溶液1:1(V/V)为流动相,梯度洗脱,能将其很好地分离。DAD 检测器在260nm处检测,各呈味核苷酸的测定标准曲线在0.00～0.10mg/ml 的范围内呈很好的线性关系,R2 分别在0.9902～1.0000之间。对中国对虾肉中的呈味核苷酸进行测定,结果表明：加标回收率在99.96%～100.34% 之间,变异系数在0.04%～0.4% 之间。方法的精密度和准确度均较高,可应用于食品中呈味核苷酸的分析检测。     

Uncoupling Protein 1 Does Not Produce Heat without Activation

Mitochondrial uncoupling protein 1 (UCP1) is the crucial mechanistic component of heat production in classical brown fat and the newly identified beige or brite fat. Thermogenesis inevitably comes at a high energetic cost and brown fat, ultimately, is an energy-wasting organ. A constrained strategy that minimizes brown fat activity unless obligate will have been favored during natural selection to safeguard metabolic thriftiness. Accordingly, UCP1 is constitutively inhibited and is inherently not leaky without activation. It follows that increasing brown adipocyte number or UCP1 abundance genetically or pharmacologically does not lead to an automatic increase in thermogenesis or subsequent metabolic consequences in the absence of a plausible route of concomitant activation. Despite its apparent obviousness, this tenet is frequently ignored. Consequently, incorrect conclusions are often drawn from increased BAT or brite/beige depot mass, e.g., predicting or causally linking beneficial metabolic effects. Here, we highlight the inherently inactive nature of UCP1, with a particular emphasis on the molecular brakes and releases of UCP1 activation under physiological conditions. These controls of UCP1 activity represent potential targets of therapeutic interventions to unlock constraints and efficiently harness the energy-expending potential of brown fat to prevent and treat obesity and associated metabolic disorders.     

Chemical Synthesis and Enzymatic Testing of CMP‐Sialic Acid Derivatives

The cycloSal approach has been used in the past for the synthesis of a range of phosphorylated bioconjugates. In those reports, cycloSal nucleotides were allowed to react with different phosphate nucleophiles. With glycopyranosyl phosphates as nucleophiles, diphosphate‐linked sugar nucleotides were formed. Here, cycloSal‐nucleotides were used to prepare monophosphate‐linked sugar nucleotides successfully in high anomeric purity and high chemical yield. The method was successfully used for the synthesis of three nucleotide glycopyranoses as model compounds. The method was then applied to the syntheses of CMP‐N‐acetyl‐neuraminic acids (CMP‐Neu5NAc) and of four derivatives with different modifications at their amino functions (N‐propanoyl, N‐butanoyl, N‐pentanoyl and N‐cyclopropylcarbonyl). The compounds were used for initial enzymatic studies with a bacterial polysialyltransferase (polyST). Surprisingly, the enzyme showed marked differences in terms of utilisation of the four derivatives. The N‐propanoyl, N‐butanoyl, and N‐pentanoyl derivatives were efficiently used in a first transfer with a fluorescently labelled trisialo‐acceptor. However, elongation of the resulting tetrasialo‐acceptors worsened progressively with the size of the N‐acyl chain. The N‐pentanoyl derivative allowed a single transfer, leading to a capped tetramer. The N‐cyclopropylcarbonyl derivative was not transferred.