Enhancement in the excitonic spontaneous emission rates for Si nanocrystal multi-layers covered with thin films of Au, Ag, and Al.

The enhancement in the spontaneous emission rate (SER) for Ag, Au, and Al films on multilayer Si nanocrystals (SiNCs) was probed with time-resolved cathodoluminescence (CL). The SiNCs were grown on Si(100) using plasma enhanced chemical vapor deposition. Electron-hole pairs were generated in the metal-covered SiNCs by injecting a pulsed high-energy electron beam through the thin metal films, which is found to be an ideal method of excitation for plasmonic quantum heterostructures and nanostructures that are opaque to laser or light excitation. Spatially, spectrally, and temporally resolved CL was used to measure the excitonic lifetime of the SiNCs in metal-covered and bare regions of the same samples. The observed enhancement in the SER for the metal-covered SiNCs, relative to the SER for the bare sample, is attributed to a coupling of the SiNC excitons with surface plasmon polaritons (SPPs) of the thin metal films. A maximum SER enhancement of ∼2.0, 1.4 and 1.2 was observed for the Ag, Au, and Al films, respectively, at a temperature of 55 K. The three chosen plasmonic metals of Ag, Au, and Al facilitate an interesting comparison of the exciton-SPP coupling for metal films that exhibit varying differences between the surface plasmon energy, ω(sp), and the SiNC excitonic emission energy. A modeling of the temperature dependence of the Purcell enhancement factor, Fp, was performed and included the temperature dependence of the dielectric properties of the metals.

Observations of exciton-surface plasmon polariton coupling and exciton-phonon coupling in InGaN/GaN quantum wells covered with Au, Ag, and Al films.

The coupling of excitons to surface plasmon polaritons (SPPs) and longitudinal optical (LO) phonons in Au-, Ag-, and Al-coated InxGa1-xN/GaN multiple and single quantum wells (SQWs) was studied with time-resolved cathodoluminescence (CL) and CL wavelength imaging techniques. Excitons were generated in the metal-coated SQWs by injecting a pulsed high-energy electron beam through the thin metal films, which is found to be an ideal method of excitation for plasmonic quantum heterostructures and nanostructures which are opaque to laser/light excitation. The Purcell enhancement factor (Fp) at low temperatures was obtained by the direct measurement of changes in the carrier lifetime caused by the SQW exciton-SPP coupling. The deposition of thin films of Al, Ag, and Au on an InGaN/GaN QW enabled a comparison of exciton-SPP coupling for energy ranges in which the surface plasmon energy is greater than, approximately equal to, and less than the QW excitonic transition energy. We investigated the temperature dependence of the Huang-Rhys factors for exciton-to-LO phonon coupling for the metal-covered and bare samples. CL imaging and spectroscopy with variable excitation densities are used to examine the spatial correlations between CL emission intensity, carrier lifetime, QW excitonic emission energy, and the Huang-Rhys factor, all of which are strongly influenced by local fluctuations in the In composition and formation of InN-rich centers.

Facile degradative lactonization of Gln-Arg and Gln-Phe hydroxyethylene dipeptide derivatives.

We have found that hydroxyrthylene (HE) dipeptide analogs of Gln-Arg and Gln-Phe are usually susceptible to acid catalyzed lactonization. The synthesis of substrate-based transition state analog inhibitors of botulinum neurotoxin metalloprotease inhibitors that contain the Gln-Arg or the Gln-Phe HE units is complicated by this facile degradative lactonization.

Synthesis of substrates and inhibitors of botulinum neurotoxin type A metalloprotease.

Botulinum neurotoxin (BoNT) metalloproteases and related proteases are the most selective proteases known. X-ray crystal structures suggest that the active sites of the native enzymes exist in catalytically incompetent forms that must be activated by substrate binding. In order to characterize the postulated substrate-induced conformational changes for enzyme activation, we synthesized a series of transition-state analog inhibitors in which the dipeptide cleavage site is replaced by tetrahedral intermediate analogs within the minimal substrate peptide sequence. In this paper, we report our efforts to design inhibitors of BoNT/A metalloprotease. We confirm that an effective substrate sequence for BoNT/A metalloprotease is a 17-mer peptide corresponding to residues 187-203 of SNAP-25. A more stable substrate, Nle202SNAP-25 [187-203] was synthesized in order to develop an assay for proteolytic activity of BoNT/A metalloprotease that can be used to monitor time-dependent inhibition. Alpha-thiol amide analogs of Gln-197 were incorporated via solid-phase peptide synthesis into both 17-mer minimal peptide substrate sequences. The synthesis, characterization and inhibition kinetics for the alpha-thiol amide analogs of holotoxin A substrate are described. These substrate-derived inhibitors were shown to be submicromolar inhibitors of BoNT/A catalytic activity.

Solid-phase synthesis of aspartic peptidase inhibitors: 3-alkoxy-4-aryl piperidines.

[reaction: see text]. The 3-alkoxy-4-aryl piperidines are non-peptide peptidomimetic inhibitors of several aspartic peptidases. The solid-phase functionalization of 3,4-disubstituted piperidine scaffolds using a traceless linker strategy is described. Synthesis of diverse analogues based on this scaffold provides the potential to generate selective inhibitors of this important class of peptidase.

Aspartic protease inhibitors: expedient synthesis of 2-substituted statines.

[reaction: see text]. General stereocontrolled synthesis of all four (2,3)-stereoisomers of 2-substituted statines is described. The 2,3-syn and 2,3-anti isomers were synthesized via beta-ketoester reduction and aldol reactions, respectively. Peptides containing 2-substituted statines inhibit porcine pepsin with nanomolar IC50 values.

Aspartic protease inhibitors designed from computer-generated templates bind as predicted.

[reaction: see text] Novel tripeptide-derived peptidomimetics 1, 7ab, and 8ab, inspired by templates generated by the structure-generating program GrowMol, were synthesized, shown to inhibit Rhizopus chinensis pepsin, and found by X-ray crystallography to bind to the enzyme in the GrowMol-predicted mode. Repetitive evaluation of the computer-generated templates for synthetic feasibility and optimal enzyme interactions led to the designed compounds.

Design and synthesis of unsymmetrical peptidyl urea inhibitors of aspartic peptidases.

[structure: see text] The design, synthesis, and enzyme inhibition of a new class of aspartic peptidase inhibitors is described. Unsymmetrical ureas were designed from computer-generated structures. Using mechanism-based and substrate-based design techniques, potent pepsin inhibitors were developed and the binding mode was established. Two X-ray crystal structures of enzyme-bound inhibitors revealed a new binding mode that is closely related to the computer-generated binding mode.

From peptides to non-peptide peptidomimetics: design and synthesis of new piperidine inhibitors of aspartic peptidases.

[reaction: see text] The 3-alkoxy-4-arylpiperidine inhibitors of aspartic peptidases are shown to be a new type of non-peptide peptidomimetic inhibitor. These piperidines can be designed from peptide-derived inhibitors by use of a structure-generating program but only after the enzyme active site conformation has been modified in a mechanistically related fashion. New enantioselective syntheses of 3-alkoxy-4-arylpiperidine analogues are described.

New methods for side-chain protection of cysteine.

[reaction: see text]. Cysteine sulfhydryl protection with either the Fmoc or the Fm group was accomplished in one step and in high yield using commercially available FmocCl or FmocOSu, respectively. Mechanisms for the Fmoc to Fm transformations are discussed. Additionally, Fmoc-Cys(Fmoc)-OH (7) was synthesized and used in amide bond forming reactions. The S-Fmoc group is cleaved selectively from peptides containing the N-Fmoc group.

Synthesis of a tripeptide derivative containing the Phe-Arg hydroxyethylene dipeptide isostere.

The protected hydroxyethylene dipeptide isostere of Phe-Arg and the tripeptide derivative 1 were synthesized as components of potential peptidase inhibitors. Key to the success of these syntheses is selective rhodium-catalyzed hydroboration in the presence of a readily reduced lactone. A convenient one-pot conversion of azides to diprotected guanidines was developed on the basis of the Staudinger reaction.

Purification and crystallization of rhizopuspepsin: the use of nickel chelation chromatography to select for catalytically active species.

A new method to obtain pure zymogen-derived peptidases is presented. The key strategy is to install a polyhistidine peptide tag on the N-terminus of the propeptide sequence of a zymogen. After expression, purification, and folding of the protein, autocatalytic posttranslational cleavage and filtration through a nickel affinity column gives pure, functional peptidase. This method takes advantage of the nickel affinity chromatography system that removes both zymogen peptide and nonfunctional folded peptidase without the need to use external enzymes to remove, often incompletely, the resulting fusion peptide. This technique was used to prepare the aspartic peptidase rhizopuspepsin. His-tagged rhizopuspepsinogen was expressed, and the desired protein was isolated as inclusion bodies and refolded. The proenzyme was purified by normal methods and then the relatively pure proenzyme was activated via intramolecular proteolysis at low pH. The propeptide and any inactive rhizopuspepsinogen were removed via affinity chromatography. This procedure yields a highly active rhizopuspepsin in 99% purity, which was demonstrated by PAGE, protein sequencing, and X-ray crystallography (1.5 A) of the isolated peptidase. A new fluorescent assay system is introduced for rhizopuspepsin, utilizing the substrate KPVSY(4-NO(3)-F)RL. The kinetics constants were K(m) = 3.4 microM +/- 0.31 and k(cat) = 55 +/- 1.0 s(-1).

Escherichia coli methionine aminopeptidase: implications of crystallographic analyses of the native, mutant, and inhibited enzymes for the mechanism of catalysis.

By improving the expression and purification of Escherichia coli methionine aminopeptidase (eMetAP) and using slightly different crystallization conditions, the resolution of the parent structure was extended from 2.4 to 1.9 A resolution. This has permitted visualization of the coordination geometry and solvent structure of the active-site dinuclear metal center. One solvent molecule (likely a mu-hydroxide) bridges the trigonal bipyramidal (Co1) and octahedral (Co2) cobalt ions. A second solvent (possibly a hydroxide ion) is bound terminally to Co2. A monovalent cation binding site was also identified about 13 A away from the metal center at an interface between the two subdomains of the protein. The first structure of a substrate-like inhibitor, (3R)-amino-(2S)-hydroxyheptanoyl-L-Ala-L-Leu-L-Val-L-Phe-OMe, bound to a methionine aminopeptidase, has also been determined. This inhibitor coordinates the metal center through four interactions as follows: (i) ligation of the N-terminal (3R)-nitrogen to Co2, (ii, iii) bridging coordination of the (2S)-hydroxyl group, and (iv) terminal ligation to Co1 by the keto oxygen of the pseudo-peptide linkage. Inhibitor binding occurs with the displacement of two solvent ligands and the expansion of the coordination sphere of Co1. In addition to the tetradentate, bis-chelate metal coordination, the substrate analogue forms hydrogen bonds with His79 and His178, two conserved residues within the active site of all MetAPs. To evaluate their importance in catalysis His79 and His178 were replaced with alanine. Both substitutions, but especially that of His79, reduce activity. The structure of the His79Ala apoenzyme and the comparison of its electronic absorption spectra with other variants suggest that the loss in activity is not due to a conformational change or a defective metal center. Two different reaction mechanisms are proposed and are compared to those of related enzymes. These results also suggest that inhibitors analogous to that reported here may be useful in preventing angiogenesis in cancer and in the treatment of microbial and fungal infections.

Novel cyclic tripeptides and substituted aromatic amino acids via ruthenium-activated S(N)Ar reactions.

[formula: see text] Chlorophenylalanines eta 6-complexed to ruthenium undergo SNAr reactions with a variety of nucleophiles to form substituted phenylalanines exemplified by 4b. Extension of these reactions to intramolecular ruthenium-activated SNAr cyclizations led to three novel cyclic tripeptide systems (exemplified by 17 and 20).

Two syntheses of the 16- and 17-membered DEF ring systems of chloropeptin and complestatin.

[formula: see text] Two syntheses of a model system of the DEF ring system of complestatin and chloropeptin are described. The key step in both of these syntheses involves the formation of the biaryl linkage using a palladium-catalyzed Suzuki cross-coupling reaction and a catalytic enantioselective ene reaction to form the 6-bromo-D-tryptophan. Additionally, ring contraction of the 17-membered DEF ring system of complestatin generates the 16-membered DEF ring system of chloropeptin in a biomimetic fashion.

Biological activities of non-immunosuppressive cyclosporin analogs.

Recent discoveries that non-immunosuppressive analogs of cyclosporin (Cs) A have unanticipated and potentially useful biological activities have rekindled research interest in this class of molecules. This review outlines these discoveries and focuses on their possible clinical utilities, as well as their role as tools to study biochemical processes.

Substrates and inhibitors of human T-cell leukemia virus type I protease.

HTLV-I is an oncogenic retrovirus that is associated with adult T-cell leukemia. HTLV-I protease and HTLV-I protease fused to a deca-histidine containing leader peptide (His-protease) have been cloned, expressed, and purified. The refolded proteases were active and exhibited nearly identical enzymatic activities. To begin to characterize the specificity of HTLV-I, we measured protease cleavage of peptide substrates and inhibition by protease inhibitors. HTLV-I protease cleavage of a peptide representing the HTLV-I retroviral processing site P19/24 (APQVLPVMHPHG) yielded Km and kcat values of 470 microM and 0.184 s-1 while cleavage of a peptide representing the processing site P24/15 (KTKVLVVQPK) yielded Km and kcat values of 310 microM and 0.0060 s-1. When the P1' proline of P19/24 was replaced with p-nitro-phenylalanine (Nph), the ability of HTLV-I protease to cleave the substrate (APQVLNphVMHPL) was improved. Inhibition of HTLV-I protease and His-protease by a series of protease inhibitors was also tested. It was found that the Ki values for inhibition of HTLV-I protease and His-protease by a series of pepsin inhibitors ranged from 7 nM to 10 microM, while the Ki values of a series of HIV-1 protease inhibitors ranged from 6 nM to 127 microM. In comparison, the Ki values for inhibition of pepsin by the pepsin inhibitors ranged from 0.72 to 19.2 nM, and the Ki values for inhibition of HIV-1 protease by the HIV protease inhibitors ranged from 0.24 nM to 1.0 microM. The data suggested that the substrate binding site of HTLV-I protease is different from the substrate binding sites of pepsin and HIV-1 protease, and that currently employed HIV-1 protease inhibitors would not be effective for the treatment of HTLV-I infections.

Peptidomimetic design.

Major discoveries have been made of new type-I and type-III peptidomimetic inhibitors of peptide-derived systems. Innovative reversible inhibitors of cysteine proteases and renin, and additional examples of peptidomimetic inhibitors of interleukin-1 beta-converting enzyme, neutral endopeptidase, herpes simplex virus protease, thrombin, HIV protease, Ras farnesyltransferase, the RGD motif, Factor Xa and various aspartic proteases have been discovered.

ICD restudy: results and potential benefit from routine predischarge and 2-month evaluation.

Evaluation of ICD function can now be performed noninvasively with intravenous sedation. To determine the value of follow-up electrophysiological studies for ICD implants, we performed a retrospective review of predischarge and 2-month ICD re studies, identifying critical problems uncovered. Of the 123 patients implanted, 122 had a predischarge study, 105 had both predischarge and elective 2-month follow-up studies, and 1 patient expired prior to restudy. Patients who underwent 2-month studies for nonelective indications (e.g., frequent shocks) were excluded from analysis. Programming changes were made in 62% of the predischarge studies (n = 122) and 70% of the elective 2-month studies (n = 105). The average number of programming changes per study was 1.3 for predischarge testing and 1.1 for 2-month testing. The most common changes at predischarge testing were adjustment of the tachyarrhythmia rate cutoff (35%) and at 2-month study, reprogramming of bradycardia pacing parameters (41%). Of the patients who underwent both predischarge and 2-month testing, 91% had programming changes in at least one of their re studies. Of 227 re studies performed, 18 studies in 14 patients yielded 24 critical findings which included: DFT increases to > or = 25 J (n = 13); sensing abnormalities of induced ventricular arrhythmia (n = 6); dislodged lead (n = 2); and serious pacemaker interactions (n = 3). Six of these critical cases (5% of total patients) required reoperation. The data suggests that routine ICD restudy is a valuable tool for management of the ICD patient. Additionally, ICD restudy is likely to increase the diagnostic yield of clinically silent critical system problems that could result in device failure.

Synthesis of novel cyclic protease inhibitors using Grubbs olefin metathesis.

The unusual amino acid bishomoallylglycine was synthesized and used to form cyclic P3-P1 tripeptide inhibitors via a Grubbs olefin metathesis method. These compounds show micro- to nanomolar inhibition of Rhizopus chinensis pepsin and represent a new class of simplified aspartic protease inhibitors lacking P' residues.