Rapid microwave-assisted CNBr cleavage of bead-bound peptides

Large libraries of peptides, cyclic peptides, and other molecules are standard tools for the discovery of drugs, molecular probes, and affinity reagents. In particular, one-bead-one-compound (OBOC) libraries,(1) prepared by the split-and-mix method,(2) provide access to a broad chemical space with a minimum of reagents. Once such a library has been screened against the target of interest, the chemical identity of the library elements on the hit beads is identified. For peptide libraries and their variants, mass spectrometry (MS) based peptide sequencing provides the most rapid method for such analysis. OBOC libraries are constructed in a number of ways to facilitate MS analysis,(3-5) but one common feature is that the peptide must be cleaved from the bead prior to being introduced into the mass spectrometer. While a number of chemical(6) and photochemical(7) cleavage strategies have been developed, the most common strategy is to incorporate a CNBr-cleavable methionine-linker group at the C-terminus of the peptide.(8) CNBr cleavage has also been widely used in proteomics to cleave proteins.(9) With such chemistry, up to 100 beads from an OBOC peptide library can be sequenced in a 24 h period.(10) A large fraction of that time, however, is devoted to the CNBr cleavage step. Standard literature protocols describe CNBr cleavage as requiring between 12 and 24 h, using 20−30 μL of 0.25 M CNBr in 70% aqueous formic acid at room temperature.(11) Although the CNBr cleavage time may be reduced to 2−4 h at elevated temperatures (47 °C), significant side-products may be generated.(12) All reports that we have found that describe CNBr cleavage chemistry from single beads have used the same conditions as for proteomics, although the two chemical processes are not necessarily equivalent

Synthesis and bioactivity of a conjugate composed of green tea catechins and hyaluronic acid

(-)-Epigallocatechin-3-gallate (EGCG) is a green tea polyphenol that has several biological activities, including anti-cancer activity and anti-inflammation. Hyaluronic acid (HA) is a naturally-occurring polysaccharide that is widely used as a biomaterial for drug delivery and tissue engineering due to its viscoelastic, biocompatible and biodegradable properties. By conjugating HA with EGCG, the resulting HA-EGCG conjugate is expected to exhibit not only the inherent properties of HA but also the bioactivities of EGCG. Toward this end, we report the synthesis of an amine-functionalized EGCG as an intermediate compound for conjugation to HA. EGCG was reacted with 2,2-diethoxyethylamine (DA) under acidic conditions, forming ethylamine-bridged EGCG dimers. The EGCG dimers were composed of four isomers, which were characterized by HPLC, high-resolution mass spectrometry and NMR spectroscopy. The amine-functionalized EGCG dimers were conjugated to hyaluronic acid (HA) through the formation of amide bonds. HA-EGCG conjugates demonstrated several bioactivities which were not present in unmodified HA, including resistance to hyaluronidase-mediated degradation, inhibition of cell growth and scavenging of radicals. The potential applications of HA-EGCG conjugates are discussed

Accurate MALDI-TOF/TOF Sequencing of One-Bead−One-Compound Peptide Libraries with Application to the Identification of Multiligand Protein Affinity Agents Using in Situ Click Chemistry Screening

Combinatorial one-bead−one-compound (OBOC) peptide libraries are widely used for affinity screening, and the sequencing of peptides from hit beads is a key step in the process. For rapid sequencing, CNBr cleavage of the peptides from the beads, followed by de novo sequencing by MALDI-TOF/TOF, is explored. We report on a semiautomated sequencing algorithm and validate it through comparison against Edman degradation sequencing. The initial 44% sequencing success rate of the standard de novo sequencing software was improved to nearly 100%. The sequencing algorithm incorporates existing knowledge of amino acid chemistry and a new strategy for differentiating isobaric amino acids. We tested the algorithm by using MALDI-TOF/TOF to identify a peptide biligand affinity agent against the protein bovine carbonic anhydrase II, starting from comprehensive one-bead−one-compound peptide libraries comprised of non-natural and artificial amino acid components and using the strategy of in situ click/OBOC library screening

Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1

We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to preinhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties

In situ click chemistry: from small molecule discovery to synthetic antibodies

Advances in the fields of proteomics, molecular imaging, and therapeutics are closely linked to the availability of affinity reagents that selectively recognize their biological targets. Here we present a review of Iterative Peptide In Situ Click Chemistry (IPISC), a novel screening technology for designing peptide multiligands with high affinity and specificity. This technology builds upon in situ click chemistry, a kinetic target-guided synthesis approach where the protein target catalyzes the conjugation of two small molecules, typically through the azide–alkyne Huisgen cycloaddition. Integrating this methodology with solid phase peptide libraries enables the assembly of linear and branched peptide multiligands we refer to as Protein Catalyzed Capture Agents (PCC Agents). The resulting structures can be thought of as analogous to the antigen recognition site of antibodies and serve as antibody replacements in biochemical and cell-based applications. In this review, we discuss the recent progress in ligand design through IPISC and related approaches, focusing on the improvements in affinity and specificity as multiligands are assembled by target-catalyzed peptide conjugation. We compare the IPISC process to small molecule in situ click chemistry with particular emphasis on the advantages and technical challenges of constructing antibody-like PCC Agents

Giant thermal hysteresis in Verwey transition of single domain Fe3O4 nanoparticles

Most interesting phenomena of condensed matter physics originate from interactions among different degrees of freedom, making it a very intriguing yet challenging question how certain ground states emerge from only a limited number of atoms in assembly. This is especially the case for strongly correlated electron systems with overwhelming complexity. The Verwey transition of Fe3O4 is a classic example of this category, of which the origin is still elusive 80 years after the first report. Here we report, for the first time, that the Verwey transition of Fe3O4 nanoparticles exhibits size-dependent thermal hysteresis in magnetization, 57Fe NMR, and XRD measurements. The hysteresis width passes a maximum of 11 K when the size is 120 nm while dropping to only 1 K for the bulk sample. This behavior is very similar to that of magnetic coercivity and the critical sizes of the hysteresis and the magnetic single domain are identical. We interpret it as a manifestation of charge ordering and spin ordering correlation in a single domain. This work paves a new way of undertaking researches in the vibrant field of strongly correlated electron physics combined with nanoscience.Comment: 13 pages, 4 figure

Synthesis and Thermoelectric Characterization of Lead Telluride Hollow Nanofibers

Lead telluride (PbTe) nanofibers were fabricated by galvanic displacement of electrospun cobalt nanofibers where their composition and morphology were altered by adjusting the electrolyte composition and diameter of sacrificial cobalt nanofibers. By employing Co instead of Ni as the sacrificial material, residue-free PbTe nanofibers were synthesized. The Pb content of the PbTe nanofibers was slightly affected by the Pb2+ concentration in the electrolyte, while the average outer diameter increased with Pb2+ concentration. The surface morphology of PbTe nanofibers was strongly dependent on the diameter of sacrificial nanofibers where it altered from smooth to rough surface as the Pb2+ concentration increased. Some of thermoelectric properties [i.e., thermopower (S) and electrical conductivity(σ)] were systematically measured as a function of temperature. Energy barrier height (Eb) was found to be one of the key factors affecting the thermoelectric properties–that is, higher energy barrier heights increased the Seebeck coefficient, but lowered the electrical conductivity

Simultaneous Cr(VI) reduction and methylene blue removal by \u3ci\u3eBacillus\u3c/i\u3e sp. JH2-2 isolated from mining site soil

A Bacillus sp. strain (JH2-2), isolated from soil at an abandoned mine site, reduced Cr(VI) to Cr(III) under aerobic conditions. At pH 7, the strain reduced essentially all of the Cr(VI) in M9 minimal medium to Cr(III) at initial concentrations ≤50 mg Cr(VI) L−1 within 100 h. The X-ray diffraction pattern of the Cr(III) precipitate matched chromium (III) hydrogen phosphate (CrH2P3O10∙2H2O). The JH2-2 strain showed high tolerance to other heavy metal (loid)s, with minimal inhibitory concentrations in liquid medium of (mg L−1): As (500), Cd (100), Cu (350), Ni (300), Zn (200), and Pb (1800). JH2-2 also promoted decolorization of methylene blue dye. Decolorization was faster in the presence of 10 mg Cr(VI) L−1 than in the absence of Cr(VI). A lag in decolorization at 30 and 50 mg Cr(VI) L−1 is likely due to initial toxicity and inhibition of bacterial growth. The chemistry of MB is complicated by its reduction to colorless leucomethylene blue, which can reoxidize to MB. However, aeration of the solution did not restore measurable MB, supporting removal of the dye via biosorption. Results indicate the bioremediation potential of Bacillus sp. JH2-2 for simultaneous Cr(VI) reduction and methylene blue removal from contaminated water