The Synthesis of a New Class of Potential Inhibitors for Glycoside Hydrolases†

Some attempts toward the synthesis of novel inhibitors of glycosyl transferases are described. More successfully, the synthesis of an activated cyclopropacyclohexene and an amide and an amine of a cyclopropa‐fused pyranose are described. None of these three novel compounds proved to be a significant inhibitor of a retaining α‐glucosidase from barley.     

Design and Synthesis of Exocyclic Cyclitol Aziridines as Potential Mechanism-Based Glycosidase Inactivators

Cyclophellitol aziridines have found wide application as mechanism-based, covalent, and irreversible inhibitors of retaining glycosidases. These compounds, like their parent compound, cyclophellitol (a natural product retaining β-glucosidase inactivator), make use of the mechanism of action of retaining glycosidases, which process their substrate through the formation of a transient covalent intermediate. In contrast, inverting glycosidases, the other main family of glycosyl hydrolases, do not employ such a covalent intermediate, and, as a consequence, useful scaffolds for mechanism-based inhibitor design have yet to be discovered. In this work, we explore chemistries that allow for the construction of cyclitol aziridines with the aziridine electrophile attached in an exocyclic fashion, more distal from the anomeric carbon – thus putatively closer to an inverting glycosidase active site nucleophile. The developed chemistries have allowed for the synthesis of a focused library of differently N-substituted, α-and β-glucopyranose configured cyclitol aziridines for future evaluation as inhibitors or inactivators of α-and β-glucosidases alike.     

Exploitation of Catalytic Dyads by Short Peptide-Based Nanotubes for Enantioselective Covalent Catalysis

Extant enzymes with precisely arranged multiple residues in their three-dimensional binding pockets are capable of exhibiting remarkable stereoselectivity towards a racemic mixture of substrates. However, how early protein folds that possibly featured short peptide fragments facilitated enantioselective catalytic transformations important for the emergence of homochirality still remains an intriguing open question. Herein, enantioselective hydrolysis was shown by short peptide-based nanotubes that could exploit multiple solvent-exposed residues to create chiral binding grooves to covalently interact and subsequently hydrolyse one enantiomer preferentially from a racemic pool. Single or double-site chiral mutations led to opposite but diminished and even complete loss of enantioselectivities, suggesting the critical roles of the binding enthalpies from the precise localization of the active site residues, despite the short sequence lengths. This work underpins the enantioselective catalytic prowess of short peptide-based folds and argues their possible role in the emergence of homochiral chemical inventory.     

A Fixable Fluorescence-Quenched Substrate for Quantitation of Lysosomal Glucocerebrosidase Activity in Both Live and Fixed Cells

Fluorogenic substrates are emerging tools that enable studying enzymatic processes within their native cellular environments. However, fluorogenic substrates that function within live cells are generally incompatible with cellular fixation, preventing their tandem application with fundamental cell biology methods such as immunocytochemistry. Here we report a simple approach to enable the chemical fixation of a dark-to-light substrate, LysoFix-GBA, which enables quantification of glucocerebrosidase (GCase) activity in both live and fixed cells. LysoFix-GBA enables measuring responses to both chemical and genetic perturbations to lysosomal GCase activity. Further, LysoFix-GBA permits simple multiplexed co-localization studies of GCase activity with subcellular protein markers. This tool will aid studying the role of GCase activity in Parkinson's Disease, creating new therapeutic approaches targeting the GCase pathway. This approach also lays the foundation for an approach to create fixable substrates for other lysosomal enzymes.     

Synthesis of Phenyl or 4-Nitrophenyl Methyl β-Ketophosphonate Esters

Seven new aryl methyl g -ketophosphonate esters were synthesized. The hydrolytic rates of the compounds under physiological conditions were studied. Most of the compounds are effective inhibitors of acetylcholinesterase. The enzyme recovers on the 10-50 h time scale from its adducts with two of the inhibitors.     

Synthesis of 2‐Azabicyclo[3.2.2]nonane‐Derived Monosaccharide Mimics and Their Evaluation as Glycosidase Inhibitors

The racemic 2‐azabicyclo[3.2.2]nonanes 5 and 18 were synthesized and tested as β‐glycosidase inhibitors. The intramolecular Diels–Alder reaction of the masked o‐benzoquinone generated from 2‐(allyloxy)phenol ( 6 ) gave the α‐keto acetal 7 which was reduced with SmI₂ to the hydroxy ketone 8 . Dihydroxylation, isopropylidenation (→ 12 ), and Beckmann rearrangement provided lactam 15 . N‐Benzylation of this lactam, reduction to the amine 17 , and deprotection provided the amino triol 19 which was debenzylated to the secondary amine 5 . Both 5 and 19 proved weak inhibitors of snail β‐mannosidase (IC₅₀ > 10 mM ), Caldocellum saccharolyticum β‐glucosidase (IC₅₀ > 10 mM ), sweet almond β‐glucosidase (IC₅₀ > 10 mM ), yeast α‐glucosidase ( 5 : IC₅₀ > 10 mM ; 19 : IC₅₀ = 1.2 mM ), and Jack bean α‐mannosidase (no inhibition detected).     

Peptidomimetics—Tailored Enzyme Inhibitors

Peptides and proteins (there is no clear boundary between the two classes of compounds) are absolutely essential components of organisms in many ways. While proteins have biocatalytic functions and are important components of tissues, peptides play an important role in the organism as hormones, neurotransmitters, and neuromodulators. Peptides and their analogues have long been used in medicinal chemistry as therapeutic agents for pathological conditions generally characterized by a disruption of the interplay between messenger molecules or enzyme substrates and their targets, the receptors and enzymes. For various biochemical and biophysical reasons there is an increasing tendency towards the use of chemical “Trojan horses” known as peptidomimetics. The chances that such agents are active generally increase with the magnitude of the “deceptive effect”, in other words in proportion to the degree of conversion of a peptide into a non-peptide. Rational design has become a catchphrase which is at present applied frequently to the development of peptidomimetics. New computer programs are invaluable tools in such design processes. However, in spite of the many advances already made, we are still far from the final goal, the de novo design of peptidomimetics. Rational design is nonetheless advancing rapidly, and it is already clear that developments in the area of peptidomimetics have given a great boost to peptide chemistry as a whole. This can be expected to continue, so that in future peptide chemistry may be characterized by a type of symbiotic alliance between peptides and non-peptides.     

Novel Approaches To Design Glycan-Based Antibacterial Inhibitors

The interactions between bacterial lectins and carbohydrates on the host cell surface can mediate bacterial adhesion, invasion, and immune evasion. Multivalency plays a key role in these binding events. However, additional molecular mechanisms greatly impact multivalent binding recognition. To develop specific and effective bacterial inhibitors, a deeper understanding of the complex underlying mechanisms of bacterial adhesion processes is necessary. By interfering with bacterial adhesion, synthetic multivalent glycoconjugates do not only have the potential to improve or replace antibiotic treatments, but also represent useful tools to study carbohydrate-pathogen interactions. In this review, we highlight a few recent advances in the synthesis and application of synthetic glycan-based scaffolds to uncover the nature of glycan-bacteria interactions and to design efficient bacterial inhibitors.     

荧光光度法二氢叶酸还原酶抑制剂筛选模型的建立和应用

本文应用二氢叶酸(DHFA)和辅酶Ⅱ(NADPH)的荧光性质,建立了二氢叶酸还原酶(DHFR)活力测定的反应体系,并对酸度等反应条件进行了优化。在该体系中,用双倒数作图法分别测定了二氢叶酸还原酶对二氢叶酸和辅酶Ⅱ的表观米氏常数。将所建立的体系应用于氨甲喋呤,甲氧苄氨嘧啶等已知抑制剂的抑制率的测定,结果满意。     

秸秆稀酸水解液中抑制物的研究

采用气相色谱/质谱(GC/MS)法对秸秆稀酸水解液中主要抑制物进行分析与鉴定,得到主要抑制物为乙酸、糠醛、乙酰丙酸和5-羟甲基糠醛.用气相色谱法定量乙酸、糠醛、乙酰丙酸和5-羟甲基糠醛的线性范围分别为0.07～0.51、0.05～0.351、0.11～0.85、0.08～0.63 mg/mL,相关系数大于0.995.对秸秆稀酸水解液进行测定的结果表明,不同温度下木糖和葡萄糖降解有着不同变化规律,为秸秆稀酸水解工艺的改进提供了参考.     

Discriminant Analysis of Selected Organics Evaluated as Corrosion Inhibitors in 0.5 M HCL

Abstract

Discriminant analysis can be used to (1) find criteria to separate observations into groups and (2) to optimally assign a new observation to its correct group. It is rather exploratory in nature especially when causal relationships are not known. Twenty four selected organics were tested as corrosion inhibitors and both physical and structural features were encoded as criteria for discrimination. In addition, a narrower set of twelve nitrogen containing compounds was also studied. In both cases the carbon chain length was an important uncoded criterion. The physical factors such as boiling point, molecular weight, pKa, and molecular area were not as important as some of the structural parameters.     

蛋白-脂质体复合物毛细管电泳筛选单胺氧化酶抑制剂

基于酶与底物间的相互作用,建立了蛋白-脂质体复合物毛细管电泳筛选单胺氧化酶(MAO)抑制剂的新方法.分别将不同浓度的4种N-炔丙基胺类化合物添加至含有蛋白-脂质体复合物的毛细管电泳缓冲液中,抑制MAO活性.考察MAO底物犬尿胺(Kyn)在含有不同浓度N-炔丙基胺类化合物缓冲液中的迁移时间比率(RMTR).结果表明,化合物N-炔丙基-N-甲基-R-2-庚胺(R-2-HMP)和N-炔丙基-R-2-庚胺(R-2-HPA)能够明显抑制MAO活性,导致MAO与Kyn的相互作用减弱,Kyn的RMTR值随着R-2-HMP和R-2-HPA的增加呈现出明显增加的趋势.化合物N,N-二炔丙基-R-2-己胺和N,N-二炔丙基-R-2-辛胺对MAO活性抑制不明显,Kyn的RMTR随这两种浓度增加变化不大.此结果与柱外孵育测定化合物活性结果一致.与传统MAO筛选剂筛选方法相比,本方法快速,成本低,酶消耗量少且不受分离电压等于扰因素的影响.     

The Computer-Aided Design,Synthesis, and Activity Prediction of New Leucine Aminopeptidase Inhibitors

The design, stereo-, and enantioselective synthesis and activity prediction of aminophosphonic acids as new leucine aminopeptidase inhibitors will be discussed.     

过亚硝酸根诱导的DNA损伤及其抑制剂的研究

过亚硝酸根（ONOO^-）具有细胞毒性,能够修饰DNA碱基、造成DNA单链断裂。本文采用HPLC-ECD以及琼脂糖凝胶电泳法分别研究了ONOO^-诱导的DNA碱基修饰以及DNA链断裂。结果表明,在一定ONOO^-浓度范围内,8-OHdG的生成量以及DNA链断裂程度与其浓度有依赖关系。同时还分别研究了槲皮素、黄酮、α-萘基黄酮、咖啡酸以及含有巯基的谷胱甘肽、硫辛酸等天然抗氧化抑制剂对ONOO^-诱导DNA损伤的抑制效果。结果表明,除黄酮、α-萘基黄酮外,其它物质都有明显的抑制作用,其中抑制效果最显著的是槲皮素。     

二酮酸类HIV-1整合酶抑制剂的定量构效关系

应用遗传函数分析法(GFA)和分子场分析法(MFA)对一系列二酮酸类整合酶抑制剂分别进行了二维和三维定量构效关系研究, 并对随机选择的5个化合物组成的测试集进行了预测, 外在预测的r2pred值分别达到0.987和0.759, 表明模型具有良好的预测能力, 同时利用药效团分析的方法, 验证了QSAR (quantitave structure-activity relationship)模型, 并概括了疏水作用对抑制剂活性的重要影响. 研究结果表明, 电性描述符(Apol)对活性有重要影响, 意味着抑制剂与金属离子的螯合作用, 同时空间和结构因素特别是疏水作用也对活性有重要作用. 利用这些规律进行了分子设计, 在理论上获得了一些具有较高抑制剂活性的新的二酮酸类衍生物, 并期待实验证实.     

Quinazoline Antifolate Thymidylate Synthase Inhibitors: Replacement of Glutamic Acid by Aminophosphonic Acids

The synthesis of six analogues of the potent thymidylate synthase (TS) inhibitor N -[4-[ N -[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinoyl)-methyl]- N -prop-2-ynylamino]benzoyl]- L -glutamic acid 2 is described in which the glutamic acid residue has been replaced by DL -aminophosphonic acids. New antifolates were tested as inhibitors of TS isolated from mouse L1210 leukemic cells as well as inhibitors of growth mouse leukemic L5178Y cells. In general these modifications result in compounds that are considerably less potent than 2 as TS inhibitors with K i 's 0.17-1.10 w M. Very poor solubility in water limited their proper assay of growth cells inhibition.     

Structure Based Library Approach to Photosynthesis Inhibitors:The Solid Phase Synthesis of Hydro-Uracils and Thiouracils

Combinatorialchemistryhasemergedasanefficienttoolforchedriststosynthesize"comPoundslibraries"fortheraPididentificationandoPtforizationofnewleads-lSolidphaseorganicsynthesisprovidesaraPidmeansforpreparationofcomPoundslibraries,andhasbeenusedfortheconstrUctionofoligomericcomPounds,'andsmallorganicmoleculessuchasbenzodiazepine,'9-lactams,'andquinolones.'ThemajortargetfortheinhibitionofphotosystemII(PSII)istheDlprotein,apolyPePtidewithamolecularweightof32kDa.'Wehavedevelopedmodelsoftheher-b…     

Chemical Proteomic Discovery of Isotype-Selective Covalent Inhibitors of the RNA Methyltransferase NSUN2

5-Methylcytosine (m⁵C) is an RNA modification prevalent on tRNAs, where it can protect tRNAs from endonucleolytic cleavage to maintain protein synthesis. The NSUN family (NSUN1-7 in humans) of RNA methyltransferases are capable of installing the methyl group onto the C⁵ position of cytosines in RNA. NSUNs are implicated in a wide range of (patho)physiological processes, but selective and cell-active inhibitors of these enzymes are lacking. Here, we use cysteine-directed activity-based protein profiling (ABPP) to discover azetidine acrylamides that act as stereoselective covalent inhibitors of human NSUN2. Despite targeting a conserved catalytic cysteine in the NSUN family, the NSUN2 inhibitors show negligible cross-reactivity with other human NSUNs and exhibit good proteome-wide selectivity. We verify that the azetidine acrylamides inhibit the catalytic activity of recombinant NSUN2, but not NSUN6, and demonstrate that these compounds stereoselectively disrupt NSUN2-tRNA interactions in cancer cells, leading to a global reduction in tRNA m⁵C content. Our findings thus highlight the potential to create isotype-selective and cell-active inhibitors of NSUN2 with covalent chemistry targeting a conserved catalytic cysteine.