Acyclic ribooxacarbenium ion mimics as transition state analogues of human and malarial purine nucleoside phosphorylases

Transition state analogues of PNP, the Immucillins and DADMe-Immucillins, were designed to match transition state features of bovine and human PNPs, respectively. A third generation of inhibitors has been designed that contain an acyclic iminoalcohol to replace the cyclic mimic of the ribooxacarbenium ion at the transition states of PNPs. The best third generation inhibitor is equivalent to the best inhibitors found in the previous transition state analogues.     

Synthesis of 5'-methylthio coformycins: specific inhibitors for malarial adenosine deaminase

Transition state theory suggests that enzymatic rate acceleration (kcat/knon) is related to the stabilization of the transition state for a given reaction. Chemically stable analogues of a transition state complex are predicted to convert catalytic energy into binding energy. Because transition state stabilization is a function of catalytic efficiency, differences in substrate specificity can be exploited in the design of tight-binding transition state analogue inhibitors. Coformycin and 2'-deoxycoformycin are natural product transition state analogue inhibitors of adenosine deaminases (ADAs). These compounds mimic the tetrahedral geometry of the ADA transition state and bind with picomolar dissociation constants to enzymes from bovine, human, and protozoan sources. The purine salvage pathway in malaria parasites is unique in that Plasmodium falciparum ADA (PfADA) catalyzes the deamination of both adenosine and 5'-methylthioadenosine. In contrast, neither human adenosine deaminase (HsADA) nor the bovine enzyme (BtADA) can deaminate 5'-methylthioadenosine. 5'-Methylthiocoformycin and 5'-methylthio-2'-deoxycoformycin were synthesized to be specific transition state mimics of the P. falciparum enzyme. These analogues inhibited PfADA with dissociation constants of 430 and 790 pM, respectively. Remarkably, they gave no detectable inhibition of the human and bovine enzymes. Adenosine deamination is involved in the essential pathway of purine salvage in P. falciparum, and prior studies have shown that inhibition of purine salvage results in parasite death. Inhibitors of HsADA are known to be toxic to humans, and the availability of parasite-specific ADA inhibitors may prevent this side-effect. The potent and P. falciparum-specific inhibitors described here have potential for development as antimalarials without inhibition of host ADA.     

Structural Insight into the Binding of Hypoxanthine with Purine Nucleoside Phosphorylase from Streptococcus mutants

Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway and an attractive target for drug design. The crystal structure of Streptococcus mutants purine nucleoside phosphorylase(Smu PNP) has been solved by molecular replacement at 1.80  resolution and refined to R factors of 19.9%/23.7%(Rcryst/Rfree) . Sequence alignment and structural comparison show that Smu PNP has more similarity with PNPs isolated from human and malarial sources than the bacterial PNPs. The structure complexed with hypoxanthine(HPA) and sulfate ion was solved at 2.24  resolution and refined to R factors of 21.6%/24.1%(Rcryst/Rfree) . It is interesting to note that the resulting electron density indicated the product,HPA,presents in the active site although inosine was included in the crystallization mixture with Smu PNP. Asn233 and Glu191 are the important residues for ligand binding and recognition. Comparison with PNPs from different species gives detailed information about binding of small molecules on the active site,which is important for the studies of enzymatic mechanism and rational design of specific inhibitors for PNPs.     

Multifunctional polymeric nanoparticles from diverse bioactive agents

We present a rational approach for assembling diverse bioactive agents, such as DNA, proteins, and drug molecules, into core-shell multifunctional polymeric nanoparticles (PNPs) that can be internalized in human breast cancer cells. Using ring-opening metathesis polymerization (ROMP), block copolymers containing small-molecule drug segments (>50% w/w) and tosylated hexaethylene glycol segments were prepared and assembled into PNPs that allowed for the surface conjugation of single-stranded DNA sequences and/or tumor-targeting antibodies. The resulting antibody-functionalized particles were readily uptaken by breast cancer cells that overexpressed the corresponding antigens.     

聚合物纳米光诊疗剂的制备与应用新进展

共轭聚合物纳米颗粒是由π-共轭有机聚合物组成的尺寸在1~100nm范围内的新型有机纳米材料。与传统的有机小分子、半导体量子点和无机纳米材料相比,聚合物纳米颗粒具有光学性质特殊、结构多样、表面易修饰和生物相容性好等优点,因而被广泛应用于生物成像、传感与检测、载药和治疗等领域。本文主要围绕聚合物纳米颗粒的制备方法、性质结构和生物相容性等方面,重点介绍了聚合物纳米颗粒作为光诊疗剂在荧光成像、光声成像,以及光动力和光热治疗领域的研究进展,并对聚合物纳米颗粒的发展前景和未来面临的挑战进行了探讨。     

Modular synthesis of functional polymer nanoparticles from a versatile platform based on poly(pentafluorophenylmethacrylate)

Control of functionalities in polymer nanoparticles (PNPs) is important for their application. Thus, there is an interest for well‐defined nanoparticle platforms to which desired functions could readily, and in modular fashion, be imparted. Herein, we report an amine‐reactive PNP platform for the modular synthesis of functional PNPs from poly(pentafluorophenylmethacrylate) (poly(PFPMA)) through a simple substitution/nanoprecipitation/photo‐crosslinking strategy. Substitution of amine containing coumarin into poly(PFPMA) allows for the achievement of structural stability of nanoprecipitated particles through photo‐crosslinking after nanoprecipitation, making it possible to carry out subsequent chemical transformations in organic solvents if needed. We demonstrate that various small molecules and an amine‐terminated polymer could be used to modify the crosslinked PNPs to endow them with various functions including fluorescence and responsiveness to temperature changes. The functional PNPs were characterized with variable temperature dynamic light scattering (DLS), UV–vis, and photoluminescence (PL) spectroscopy, and transmission electron microscopy (TEM). © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1895–1901     

Polymer Zwitterions for Stabilization of CsPbBr3 Perovskite Nanoparticles and Nanocomposite Films

Functional polymers with sulfobetaine or phosphorylcholine zwitterions as pendent groups are demonstrated as both ligands and host matrices for CsPbBr₃ perovskite nanoparticles (PNPs). These polymers produce nanocomposite films with excellent NP dispersion, optical transparency, and impressive resistance to NP degradation upon exposure to water. Multidentate interactions of the zwitterion‐containing copolymers with the PNPs induce dispersed or weakly aggregated nanocomposite morphologies, depending on the extent of zwitterionic functionality in the polymer. Incorporating additional functionality into the polymers, such as benzophenone pendent groups, yields lithographically patternable films, while time‐resolved photoluminescence measurements provide insight into the electronic impact of PNPs in zwitterionic polymer matrices.     

单个等离子体纳米颗粒在生化分析和生物成像中的应用

等离子体纳米颗粒（PNPs）因其独特的物理、化学、光学和生物学特性而被广泛地应用于材料科学、生物学和医药学等研究领域。PNPs的光学性质是可以通过改变其组成、形状和大小来进行调控的,所以利用可控合成的方式能够筛选出适合的光散射探针。在单分子水平上实时研究PNPs的动态行为对于理解细胞及活体组织的生命活动机制、制备功能型纳米材料和开发新型化学生物传感器等有着重要的意义。基于传统的暗场显微镜（DFM）,通过对光源、检测器及其它光学元件的择优组装和调试,我们开发出了一系列具有高灵敏度、高时空分辨率和高通量的等离子体光散射成像技术,并将其应用于单分子检测、多颗粒传感、单细胞成像以及生物过程示踪等领域。基于具有光学各向异性的PNPs,我们还研制出了活细胞三维扫描成像系统和超连续激光光片成像与高速毛细管电泳联用系统,推进了单分子光谱方面的研究。本文将总结近十年来本课题组在PNP单颗粒分析及成像中的工作,并为该领域未来的发展提出一些新的思路。     

Temperature‐induced conformational transition of bovine serum albumin in neutral aqueous solution by reversed‐phase liquid chromatography

The temperature‐induced conformational transition of bovine serum albumin (BSA) in neutral aqueous solution was studied using intrinsic fluorescence emission spectrum, reversed‐phase liquid chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the conformation transition thermodynamic parameters were determined in the temperature range 12–50 °C. The results showed that, in the temperature range 12–20 °C, BSA only existed in a single conformation state A, while in the temperature range 22–50 °C, it existed in two different conformation states: A and B. The percentage of conformation state A decreased while that of conformation state B increased with the increase in temperatures, and when temperature approached 50 °C conformation state B accounted for approximately 25% of all conformation states of BSA. In the conformational transition of BSA from conformation state A to conformation state B, the positive enthalpy change, entropy change and free energy changes demonstrated that the conformational transition was endothermic, nonspontaneous and mainly entropy‐driven. © 2013 The Authors. Biomedical Chromatography published by John Wiley & Sons, Ltd.     

Transition state structure of E. coli tRNA-specific adenosine deaminase

Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single tRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNA(Arg2) was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1'-(3)H], [5'-(3)H2], [1'-(14)C], [6-(13)C], [6-(15)N], [6-(13)C, 6-(15)N] and [1-(15)N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-(13)C]-, [6-(15)N]-, [1-(15)N]-, [1'-(3)H]-, [5'-(3)H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late SNAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-(15)N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 A in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 A, respectively. The large inverse [5'-(3)H] KIE of -3.7% and modest normal [1'-(3)H] KIE of 1.4% indicate that significant ribosyl 5'-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition state characterization of other RNA editing enzymes.     

Comparative DSC study of human and bovine serum albumin

The thermal denaturation process of bovine and human both fatty acid containing and fatty acid free albumins in aqueous solution was studied by use of differential scanning calorimetry. Human serum albumins were found to be more stable than their bovine counterparts. Fatty acid free albumins were characterized as generally less stable, more susceptible to aggregation, their unfolding endothermic transition was less cooperative and with the smaller degree of reversibility. Deconvolution analysis with using a non-two-state model with two component transitions showed essential differences in the thermodynamic parameters between all studied albumins, particularly regarding the high-temperature component transition.     

Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-GlcNAcase: mechanistic and structural insights into inhibitor selectivity and transition state poise

O-GlcNAcase catalyzes the cleavage of beta-O-linked 2-acetamido-2-deoxy-beta-d-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Two potent inhibitors of this enzyme are O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2'-methyl-alpha-d-glucopyranoso[2,1-d]-Delta2'-thiazoline (NAG-thiazoline). Derivatives of these inhibitors differ in their selectivity for human O-GlcNAcase over the functionally related human lysosomal beta-hexosamindases, with PUGNAc derivatives showing modest selectivities and NAG-thiazoline derivatives showing high selectivities. The molecular basis for this difference in selectivities is addressed as is how well these inhibitors mimic the O-GlcNAcase-stabilized transition state (TS). Using a series of substrates, ground state (GS) inhibitors, and transition state mimics having analogous structural variations, we describe linear free energy relationships of log(KM/kcat) versus log(KI) for PUGNAc and NAG-thiazoline. These relationships suggest that PUGNAc is a poor transition state analogue, while NAG-thiazoline is revealed as a transition state mimic. Comparative X-ray crystallographic analyses of enzyme-inhibitor complexes reveal subtle molecular differences accounting for the differences in selectivities between these two inhibitors and illustrate key molecular interactions. Computational modeling of species along the reaction coordinate, as well as PUGNAc and NAG-thiazoline, provide insight into the features of NAG-thiazoline that resemble the transition state and reveal where PUGNAc fails to capture significant binding energy. These studies also point to late transition state poise for the O-GlcNAcase catalyzed reaction with significant nucleophilic participation and little involvement of the leaving group. The potency of NAG-thiazoline, its transition state mimicry, and its lack of traditional transition state-like design features suggest that potent rationally designed glycosidase inhibitors can be developed that exploit variation in transition state poise.     

Distribution and Transition of Native and Completely Unfolded Conformations in the Unfolding of Bovine Heart Cytochrome c Induced by Urea and Guanidine Hydrochloride

The unfolding of bovine heart cytochrome c induced by urea and guanidine hydrochloride was first studied through intrinsic fluorescence emission spectra and fluorescence phase diagram and the results showed that both of them separately followed a two‐state model. As the simplest sample of the unfolding of protein molecules induced by denaturants, an equation was presented to show the effect of the denaturant concentrations in denaturation solution on the residual activity ratios of bovine heart cytochrome c in their two‐state unfolding. There are two characteristic unfolding parameters K and m in this equation. The former is the thermodynamic equilibrium constant of the unfolding of bovine heart cytochrome c induced by denaturants, the latter is the number of denaturant molecules associated with a bovine heart cytochrome c molecule during the unfolding procedure, and through them the distribution and transition of native and completely unfolded bovine heart cytochrome c conformations under different concentrations of urea or guanidine hydrochloride in denaturation solution can be accurately described.     

Polymeric Nanoparticles Active against Dual-Species Bacterial Biofilms

Biofilm infections are a global public health threat, necessitating new treatment strategies. Biofilm formation also contributes to the development and spread of multidrug-resistant (MDR) bacterial strains. Biofilm-associated chronic infections typically involve colonization by more than one bacterial species. The co-existence of multiple species of bacteria in biofilms exacerbates therapeutic challenges and can render traditional antibiotics ineffective. Polymeric nanoparticles offer alternative antimicrobial approaches to antibiotics, owing to their tunable physico-chemical properties. Here, we report the efficacy of poly(oxanorborneneimide) (PONI)-based antimicrobial polymeric nanoparticles (PNPs) against multi-species bacterial biofilms. PNPs showed good dual-species biofilm penetration profiles as confirmed by confocal laser scanning microscopy. Broad-spectrum antimicrobial activity was observed, with reduction in both bacterial viability and overall biofilm mass. Further, PNPs displayed minimal fibroblast toxicity and high antimicrobial activity in an in vitro co-culture model comprising fibroblast cells and dual-species biofilms of Escherichia coli and Pseudomonas aeruginosa. This study highlights a potential clinical application of the presented polymeric platform.     

硅胶功能化的N-丙哌嗪固载钯纳米粒子作为有效的多相催化剂用于氰化反应(英文)

An efficient heterogeneous Pd catalytic system has been developed, based on immobilization of Pd nanoparticles (PNPs) on a silica-bonded N-propylpiperazine (SBNPP) substrate. The SBNPP substrate effectively stabilizes the PNPs and improves their stability against aggregation. The catalytic activity of this catalyst was investigated in the cyanation of aryl halides with K4[Fe(CN)6 ] as the cyanide source. The catalyst could be recycled several times without appreciable loss of catalytic activity.     

Transition-state variation in human, bovine, and Plasmodium falciparum adenosine deaminases

Adenosine deaminases (ADAs) from human, bovine, and Plasmodium falciparum sources were analyzed by kinetic isotope effects (KIEs) and shown to have distinct but related transition states. Human adenosine deaminase (HsADA) is present in most mammalian cells and is involved in B- and T-cell development. The ADA from Plasmodium falciparum (PfADA) is essential in this purine auxotroph, and its inhibition is expected to have therapeutic effects for malaria. Therefore, ADA is of continuing interest for inhibitor design. Stable structural mimics of ADA transition states are powerful inhibitors. Here we report the transition-state structures of PfADA, HsADA, and bovine ADA (BtADA) solved using competitive kinetic isotope effects (KIE) and density functional calculations. Adenines labeled at [6-13C], [6-15N], [6-13C, 6-15N], and [1-15N] were synthesized and enzymatically coupled with [1'-14C] ribose to give isotopically labeled adenosines as ADA substrates for KIE analysis. [6-13C], [6-15N], and [1-15N]adenosines reported intrinsic KIE values of (1.010, 1.011, 1.009), (1.005, 1.005, 1.002), and (1.004, 1.001, 0.995) for PfADA, HsADA, and BtADA, respectively. The differences in intrinsic KIEs reflect structural alterations in the transition states. The [1-15N] KIEs and computational modeling results indicate that PfADA, HsADA, and BtADA adopt early SNAr transition states, where N1 protonation is partial and the bond order to the attacking hydroxyl nucleophile is nearly complete. The key structural variation among PfADA, HsADA, and BtADA transition states lies in the degree of N1 protonation with the decreased bond lengths of 1.92, 1.55, and 1.28 A, respectively. Thus, PfADA has the earliest and BtADA has the most developed transition state. This conclusion is consistent with the 20-36-fold increase of kcat in comparing PfADA with HsADA and BtADA.     

金属-血清蛋白的结构研究Ⅲ.Ni2+离子诱导的HSA和BSA的缓慢构象变化

用紫外光谱观察到Ni2+离子与人或牛血清白蛋白相互作用有显著的滞后效应,表明Ni2+离子的结合可以诱导人或牛血清白蛋白发生从对Ni2+离子有较弱亲和力至较强亲和力构象态的缓慢变化(T-R转化);这一构象变化为试样的旋光能力随时间变化进一步证实;测得并讨论了这一构象变化的速度常数和活化参数;推测这一构象变化可能主要发生在蛋白质的IA亚区,并且很可能是一种促使IA亚区变得更加开放的"绞链式运动".     

金属-血清蛋白的结构研究 3: Ni^2^+离子诱导的HSA和BSA的缓慢构象变化

用紫外光谱观察到Ni^2^+离子与人或牛血清白蛋白相互作用有显著的滞后效应, 表明Ni^2^+离子的结合可以诱导人或牛血清白蛋白发生从对Ni^2^+离子有较弱亲和力至较强亲和力构象态的缓慢变化(T-R转化); 这一构象变化为试样的旋光能力随时间变化进一步证实;测得并讨论了这一构象变化的速度常数和活化参数; 推测这一构象变化可能主要发生在蛋白质的IA亚区, 并且很可能是一种促使IA亚区变得更加开放的"绞链式运动"。     

Analysis of local polarity change around Cys34 in bovine serum albumin during N→B transition by a polarity-sensitive fluorescence probe

The change trend of the local environment of Cys34 domain in bovine serum albumin has been studied as a function of pH value by using thiol-specific and polarity-sensitive fluorescent probe 3-(4-chloro-6-p-maleimidylphenoxyl-1,3,5-triazinylamino)-7-dimethylamino-2-methyl-phenazine. The local polarity of the Cys34 domain is found to rise with the increase of pH values, and the corresponding dielectric constant is raised from 12.8 at pH 6.0 to 23.3 at pH 9.1. The result shows that the environment of the Cys34 domain is rather hydrophobic in normal state at pH 6.0 and becomes a little hydrophilic in the course of N→B transition, which may be attributed to the slight unfolding of the protein and thus the increasing of exposure of the previously relatively buried Cys34. In addition, the increased dielectric constant (23.3) is much lower than that (80.1) of water, suggesting that the unfolding of bovine serum albumin does not cause the full exposure of the Cys34 to the aqueous media during the transition.     

Bulk polymer nanoparticles containing a tetrakis(3-hydroxyphenyl)porphyrin for fast and highly selective separation of mercury ions

We report on the synthesis of polymeric nanoparticles (PNPs) containing a tetrakis(3-hydroxyphenyl)porphyrin, and their use for the separation of mercury(II) ion. The PNPs were prepared by bulk polymerization from methacrylic acid (the monomer), ethyleneglycol dimethacrylate (the cross-linker), 2,2′-azobisisobutyronitrile (the radical initiator) and the mercury(II) complex of 5,10,15,20-tetrakis(3-hydroxyphenyl)-porphyrin. The Hg(II) ion was then removed by treatment with dilute hydrochloric acid. The PNPs were characterized by colorimetry, FT-IR spectroscopy, and scanning electron microscopy. The material is capable of binding Hg(II) from analyte samples. Bound Hg(II) ions can be eluted with dilute nitric acid and then quantified by cold vapor AAS. The extraction efficiency, the effects of pH, preconcentration and leaching times, sample volume, and of the nature, concentration and volume of eluent were investigated. The maximum adsorption capacity of the PNPs is 249 mg g^?1, the relative standard deviation of the AAS assay is 2.2 %, and the limit of detection (3σ) is 8 ng.L^?1. The nanoparticles exhibit excellent selectivity for Hg(II) ion over other metal ions and were successfully applied to the selective extraction and determination of Hg(II) ion in spiked water samples.