Cyclic Allene Approach to the Manzamine Alkaloid Keramaphidin B.

We report an approach to the core of the manzamine alkaloid keramaphidin B that relies on the strain-promoted cycloaddition of an azacyclic allene with a pyrone trapping partner. The cycloaddition is tolerant of nitrile and primary amide functional groups and can be complemented with a subsequent retro-Diels-Alder step. These efforts demonstrate that strained cyclic allenes can be used to build significant structural complexity and should encourage further studies of these fleeting intermediates.

Evaluating the Effect of Dye-Dye Interactions of Xanthene-Based Fluorophores in the Fluorosequencing of Peptides.

A peptide sequencing scheme utilizing fluorescence microscopy and Edman degradation to determine the amino acid position in fluorophore-labeled peptides was recently reported, referred to as fluorosequencing. It was observed that multiple fluorophores covalently linked to a peptide scaffold resulted in a decrease in the anticipated fluorescence output and worsened the single-molecule fluorescence analysis. In this study, we report an improvement in the photophysical properties of fluorophore-labeled peptides by incorporating long and flexible (PEG)10 linkers at the peptide attachment points. Long linkers to the fluorophores were installed using copper-catalyzed azide-alkyne cycloaddition conditions. The photophysical properties of these peptides were analyzed in solution and immobilized on a microscope slide at the single-molecule level under peptide fluorosequencing conditions. Solution-phase fluorescence analysis showed improvements in both quantum yield and fluorescence lifetime with the long linkers. While on the solid support, photometry measurements showed significant increases in fluorescence brightness and 20 to 60% improvements in the ability to determine the amino acid position with fluorosequencing. This spatial distancing strategy demonstrates improvements in the peptide sequencing platform and provides a general approach for improving the photophysical properties in fluorophore-labeled macromolecules.

Capture and Release of Protein-Nanoparticle Conjugates by Reversible Covalent Molecular Linkers.

A hybrid approach to covalently detachable molecules for nanoparticle capture and release from several custom-functionalized surfaces is described. This new surface chemistry capability provides a means for reversible binding of functionalized nanoparticles without relying on costly nucleic acid-based complexation. A new surface linker motif was devised wherein custom molecules were synthesized with components for surface anchoring, cleavage, and target capture through biotin-streptavidin binding. All capture-and-release chemistry is performed using physiological conditions (aqueous, pH 7). Covalent cleavage of linker molecules was achieved through incorporation of a tunable orthogonal reversible covalent (TORC) hydrazone functional group which underwent exchange with a competitive hydrazide aided by an aniline catalyst. The influence of the linker architecture on hydrazone exchange and nanoparticle release was probed by altering the distance between hydrazone and biotin groups using different length PEG spacers. Cleavable linkers were used to functionalize microwells, magnetic separation beads, and gold-coated glass surfaces. Upon functionalization, all surface types bound streptavidin and conjugated nanoparticles regardless of the linker structure. Conversely, the extent of hydrazone exchange as well as release of nanoparticles were influenced both by the hydrazone surface density and the linker molecular structure.

Chemically Triggered Synthesis, Remodeling, and Degradation of Soft Materials.

Polymer topology dictates dynamic and mechanical properties of materials. For most polymers, topology is a static characteristic. In this article, we present a strategy to chemically trigger dynamic topology changes in polymers in response to a specific chemical stimulus. Starting with a dimerized PEG and hydrophobic linear materials, a lightly cross-linked polymer, and a cross-linked hydrogel, transformations into an amphiphilic linear polymer, lightly cross-linked and linear random copolymers, a cross-linked polymer, and three different hydrogel matrices were achieved via two controllable cross-linking reactions: reversible conjugate additions and thiol-disulfide exchange. Significantly, all the polymers, before or after topological changes, can be triggered to degrade into thiol- or amine-terminated small molecules. The controllable transformations of polymeric morphologies and their degradation herald a new generation of smart materials.

Synthesis of Carboxy ATTO 647N Using Redox Cycling for Xanthone Access.

A synthesis of the carbopyronine dye Carboxy ATTO 647N from simple materials is reported. This route proceeds in 11 forward steps from 3-bromoaniline with the key xanthone intermediate formed using a new oxidation methodology. The step utilizes an oxidation cycle with base, water, iodine, and more than doubles the yield of the standard permanganate oxidation methodology, accessing gram-scale quantities of this late-stage product. From this, Carboxy ATTO 647N was prepared in only four additional steps. This facile route to a complex fluorophore is expected to enable further studies in fluorescence imaging.

Platinum(IV)-Ferrocene Conjugates and Their Cyclodextrin Host-Guest Complexes.

Reported here are new platinum(IV) (Pt(IV)) complexes bearing ferrocene (Fc) moieties. These systems differ from one another only by the nature of the functional group (ester vs amide) connecting the linker to the Fc subunits. This minor structural variation (one atom difference) leads to major differences in solubility, stability, and antiproliferative activity against lung (A549) cancer cells. The host-guest chemistry of these complexes was investigated in an aqueous medium in the presence of ß-cyclodextrins (ß-CD), either free or in the form of a covalently linked Fc-Pt-ß-CD hybrid. An inclusion complex between Fc and ß-CD is formed in aqueous media, presumably as a result of hydrophobic interactions involving the Fc and the inner ß-CD cavity. Consequently, it proved possible to use a ß-CD-based strategy to purify the Pt-Fc conjugates in this study under aqueous conditions (by means of C18 silica gel columns). The use of a ß-CD adjuvant also allowed dimethyl sulfoxide (DMSO) to be avoided as an organic cosolvent in cell studies. The amide version reported here (2) proved to be more soluble, more stable, and more active than the ester analogue (11) in A549 cells. The use of a ß-CD functionalized with a fluorescent probe allowed intracellular Pt-Fc localization to be visualized by confocal fluorescence microscopy.

Improved Xanthone Synthesis, Stepwise Chemical Redox Cycling.

A base-catalyzed direct oxidation of rhodamine, carborhodamine, and siliconrhodamine pyronines to the corresponding xanthones is studied. This methodology utilizes addition of water to split pyronines into xanthone and reduced xanthene, the latter of which is returned to pyronine by oxidation with iodine. The transformation is general, working on the three most recalcitrant versions of N, N, N', N'-tetramethylpyronines in good to excellent yields.

Highly parallel single-molecule identification of proteins in zeptomole-scale mixtures.

The identification and quantification of proteins lags behind DNA-sequencing methods in scale, sensitivity, and dynamic range. Here, we show that sparse amino acid-sequence information can be obtained for individual protein molecules for thousands to millions of molecules in parallel. We demonstrate selective fluorescence labeling of cysteine and lysine residues in peptide samples, immobilization of labeled peptides on a glass surface, and imaging by total internal reflection microscopy to monitor decreases in each molecule's fluorescence after consecutive rounds of Edman degradation. The obtained sparse fluorescent sequence of each molecule was then assigned to its parent protein in a reference database. We tested the method on synthetic and naturally derived peptide molecules in zeptomole-scale quantities. We also fluorescently labeled phosphoserines and achieved single-molecule positional readout of the phosphorylated sites. We measured >93% efficiencies for dye labeling, survival, and cleavage; further improvements should enable studies of increasingly complex proteomic mixtures, with the high sensitivity and digital quantification offered by single-molecule sequencing.

Synthesis and biological evaluation of pyrido[2,3-d]pyrimidine-2,4-dione derivatives as eEF-2K inhibitors.

A small molecule library of pyrido[2,3-d]pyrimidine-2,4-dione derivatives 6-16 was synthesized from 6-amino-1,3-disubstituted uracils 18, characterized, and screened for inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K). To understand the binding pocket of eEF-2K, structural modifications of the pyrido[2,3-d]pyrimidine were made at three regions (R(1), R(2), and R(3)). A homology model of eEF-2K was created, and compound 6 (A-484954, Abbott laboratories) was docked in the catalytic domain of eEF-2K. Compounds 6 (IC50=420nM) and 9 (IC50=930nM) are found to be better molecules in this preliminary series of pyrido[2,3-d]pyrimidine analogs. eEF-2K activity in MDA-MB-231 breast cancer cells is significantly reduced by compound 6, to a lesser extent by compound 9, and is unaffected by compound 12. Similar inhibitory results are observed when eEF-2K activity is stimulated by 2-deoxy-d-glucose (2-DOG) treatment, suggesting that compounds 6 and 9 are able to inhibit AMPK-mediated activation of eEF-2K to a notable extent. The results of this work will shed light on the further design and optimization of novel pyrido[2,3-d]pyrimidine analogs as eEF-2K inhibitors.

Decaffeination and measurement of caffeine content by addicted Escherichia coli with a refactored N-demethylation operon from Pseudomonas putida CBB5.

The widespread use of caffeine (1,3,7-trimethylxanthine) and other methylxanthines in beverages and pharmaceuticals has led to significant environmental pollution. We have developed a portable caffeine degradation operon by refactoring the alkylxanthine degradation (Alx) gene cluster from Pseudomonas putida CBB5 to function in Escherichia coli. In the process, we discovered that adding a glutathione S-transferase from Janthinobacterium sp. Marseille was necessary to achieve N 7 -demethylation activity. E. coli cells with the synthetic operon degrade caffeine to the guanine precursor, xanthine. Cells deficient in de novo guanine biosynthesis that contain the refactored operon are ″addicted″ to caffeine: their growth density is limited by the availability of caffeine or other xanthines. We show that the addicted strain can be used as a biosensor to measure the caffeine content of common beverages. The synthetic N-demethylation operon could be useful for reclaiming nutrient-rich byproducts of coffee bean processing and for the cost-effective bioproduction of methylxanthine drugs.