Fluorescent P-Hydroxyphosphole for Peptide Labeling through P-N Bond Formation

Synthesis of fluorescent P-hydroxybinaphtylphosphole-oxide or -sulfide was achieved by trapping a binaphtyl dianion with methyl dichlorophosphite or P-(N,N-diethylamino)dichlorophosphine, followed by oxidation or sulfuration of the P-center. After saponification or acid hydrolysis, the P-hydroxyphospholes were coupled to peptides using the coupling agent BOP, under the conditions required for the synthesis in solution or on a solid support. This new method was illustrated by the labeling of the JMV2959, a potent antagonist of the Growth Hormone Secretagogue Receptor type 1a (GHS−R1a). The labeled conjugates were used to characterize GHSR ligands by competition assays, based on Fluorescence Resonance Energy Transfer (FRET). Such P-hydroxyphosphole-oxide or -sulfide constitute a promising new class of compact fluorophores with large Stokes shift, for labeling biomolecules by grafting through the phosphorus atom.     

Inverse Polyamidoamine (i-PAMAM) Dendrimer Antimicrobials

Here we redesigned the branches of polyamidoamine (PAMAM) dendrimers by moving the amide carbonyl group on the other side of the amide nitrogen atom, transforming the β-alaninyl-amidoethylamine branch, which easily undergoes retro-Michael reactions and renders PAMAMs intrinsically unstable, into a more stable glycyl-amidopropylamine branch. The resulting inverse PAMAM (i-PAMAM) dendrimers have the same carbon framework as PAMAMs and only differ by the position of the carbonyl group. In contrast to PAMAMs which are prepared in solution and are difficult to purify, we synthesize i-PAMAMs using high-temperature solid-phase peptide synthesis by iterative coupling and deprotection of the commercially available N,N-bis(N′-Fmoc-3-aminopropyl)glycine and purify them preparative reverse phase HPLC. Our i-PAMAM dendrimers show no detectable degradation over time. We demonstrate this new class of dendrimers with the synthesis of antimicrobial dendrimers with potent yet non-membrane disruptive activities against both Gram-negative and Gram-positive bacteria.     

Luminescence switch-on detection of protein tyrosine kinase-7 using a G-quadruplex-selective probe

A series of luminescent iridium(iii) complexes were synthesised and evaluated for their ability to act as luminescent G-quadruplex-selective probes. The iridium(iii) complex 9 [Ir(pbi)₂(5,5-dmbpy)]PF₆ (where pbi = 2-phenyl-1H-benzo[d]imidazole; 5,5-dmbpy = 5,5′-dimethyl-2,2′-bipyridine) exhibited high luminescence for G-quadruplex DNA compared to dsDNA and ssDNA, and was employed to construct a G-quadruplex-based assay for protein tyrosine kinase-7 (PTK7) in aqueous solution. PTK7 is an important biomarker for a range of leukemias and solid tumors. In the presence of PTK7, the specific binding of the sgc8 aptamer sequence triggers a structural transition and releases the G-quadruplex-forming sequence. The formation of the nascent G-quadruplex structure is then detected by the G-quadruplex-selective iridium(iii) complex with an enhanced luminescent response. Moreover, the application of the assay for detecting PTK7 in cellular debris and membrane protein extract was demonstrated. To our knowledge, this is the first G-quadruplex-based assay for PTK7.     

DNA G-四链体的结构完整性对催化性质的影响

DNA酶中的G-四链体-血红素(G4-hemin)DNA酶结构具有较高的设计性和化学稳定性,因此格外受研究者关注.G-平面作为辅酶因子hemin的结合位点,不仅提供大π平面与hemin结合,而且其平面上的G碱基还可以充当近端配位基团与hemin进行配位.因此,研究G-平面完整性在G4-DNA酶体系中的作用具有重要意义.本文设计了一系列含有空位的G4(G-vacancy,GV)及G-三链体,通过“鸟嘌呤类似物插入”策略实现G-平面完整性以及DNA酶催化活性的恢复.结果表明,末端G-平面完整性是G4-DNA酶具有催化活性的必要条件,且其能够充当近端配位基团与末端碱基协同激活G4-DNA酶.考虑到hemin会选择性地结合于G4的3’-端平面,本文以含有3’-端空位的G4以及G-三链体为模型进行DNA酶的构建.结果表明,相较于末端完整的G4-hemin DNA酶,末端不完整的G4结构所形成的DNA酶催化活性很低.为了进一步验证该平面完整性的重要性,本文提出了“鸟嘌呤衍生物插入”策略,即将鸟嘌呤衍生物(无环鸟苷和鸟苷)插入G-空位以恢复G-平面的完整性.通过圆二色光谱和紫外熔解实验,发现末端平面完整性的缺失会使圆二色特征峰信号和G4结构热稳定性下降,而鸟嘌呤碱基类似物的加入则可以使特征峰信号以及热稳定性得到一定程度的恢复,表明鸟嘌呤碱基类似物的加入确实使G-平面完整性得到恢复.与此同时,随着鸟嘌呤碱基类似物浓度的增加,G4-hemin DNA酶活性逐渐增强,最终恢复至与完整G4一样的活性.在以G-三链体为模型的实验中,本文通过另一条富G序列与G-三链体进行结合,形成复合的(3+1)型G4结构,最终实现了DNA酶活性的恢复.同时,在3’-G-平面末端增加了激活碱基(dA或dTC),结果表明,即使G-平面不完整,末端碱基依旧能够激活DNA酶,但酶活性整体弱于完整G4时的活性.同样,“鸟嘌呤衍生物插入”策略可以使酶活性得到恢复.本文系列实验充分说明了末端碱基可与G-平面形成协同作用,与hemin的铁中心共同形成六配位关系,加速催化中间体生成,进而增强催化活性.有趣的是,通过设计Holliday junction结构研究发现,“鸟嘌呤衍生物插入”策略仅适用于平行G4结构.G-空位的存在不仅降低了G4结构的稳定性,而且降低了其与hemin间的亲和力,二者均是造成G4-DNA酶催化能力下降的主要因素.总之,本文证明了3’-端G-平面的完整性是G4-DNA酶实现其催化能力必不可少的因素,对理解末端G-平面在G4-DNA酶中的作用具有重要的参考意义.     

Microprojectile velocities in a gas driven launcher

Microprojectile methods of genetic engineering sometimes employ a macroprojectile which launches the microprojectiles when it hits a stopping plate. A study has been made of the relationship between the velocities of the macroprojectile and the microprojectile it launches. Macroprojectile velocities were measured using a beam blanking method and the microprojectile velocities were measured with a laser Doppler anemometer. Particles of tungsten, titanium dioxide and silicon carbide were investigated as microprojectiles. The microprojectile velocity was found to depend not only on the macroprojectile velocity but also on the microprojectile size and material, the bond strength with the macroprojectile and the gas flow that develops ahead of the macroprojectile. On average the microprojectile launch velocity was similar to the macroprojectile velocity. However, some tungsten particles had velocities twice this value. Experimental evidence suggests that these higher velocities were caused by early break-way of particles from the macroprojectile followed by further acceleration by aerodynamic drag arising from the gas flow ahead of the macroprojectile.We are especially grateful for the advice of Dr. M. Gharib on the use of the HSI color system. John Lyons provided expert help in the laboratory and materials for and advice on photography. We thank the staff of the SIO Hydraulics Laboratory for making the wind-wave channel available for our use, and the staff of UCSD library for enabling us to use the Barneyscanner photometer-digitizer. We thank an anonymous reviewer who pointed out a numerical error and improved the clarity of the text.     

Deduction as Reduction, from a Categorical Point of View

Deduction systems and graph transformation systems are compared within a common categorical framework. This comparison results in a proposal for a new deduction method in diagrammatic logics, allowing the deletion of intermediate lemmas.