Synthesis and Pharmacological Evaluation of Enantiopure N-Substituted Ortho-c Oxide-Bridged 5-Phenylmorphans.

The design of enantiopure stereoisomers of N-2-phenylcyclopropylmethyl-substituted ortho-c oxide-bridged phenylmorphans, the E and Z isomers of an N-cinnamyl moiety, and N-propyl enantiomers were based on combining the most potent oxide-bridged phenylmorphan (the ortho-c isomer) with the most potent N-substituent that we previously found with a 5-(3-hydroxy)phenylmorphan (i.e., N-2-phenylcyclopropyl methyl moieties, N-cinnamyl, and N-propyl substituents). The synthesis of the eight enantiopure N-2-phenylcyclopropylmethyl ortho-c oxide-bridged phenylmorphans and six additional enantiomers of the N-substituted ortho-c oxide-bridged phenylmorphans (N-E and Z-cinnamyl compounds, and N-propyl compounds) was accomplished. The synthesis started from common intermediates (3R,6aS,11aS)-10-methoxy-1,3,4,5,6,11a-hexahydro-2H-3,6a-methano-benzofuro[2,3-c]azocine (+)-6 and its enantiomer, (3S, 6aR, 11aR)-(-)-6, respectively. The enantiomers of ±-6 were obtained through salt formation with (S)-(+)- and (R)-(-)-p-methylmandelic acid, and the absolute configuration of the (R)-(-)-p-methylmandelate salt of (3S, 6aR, 11aR)-(-)-6 was determined by single-crystal X-ray analysis. The enantiomeric secondary amines were reacted with N-(2-phenylcyclopropyl)methyl derivatives, 2-(E)-cinnamyl bromide, and (Z)-3-phenylacrylic acid. These products led to all of the desired N-derivatives of the ortho-c oxide-bridged phenylmorphans. Their opioid receptor binding affinity was measured. The compounds with MOR affinity < 50 nM were examined for their functional activity in the forskolin-induced cAMP accumulation assay. Only the enantiomer of the N-phenethyl ortho-c oxide-bridged phenylmorphan ((-)-1), and only the (3S,6aR,11aR)-2-(((1S,2S)-2-phenylcyclopropyl)methyl)-1,3,4,5,6,11a-hexahydro-2H-3,6a-methanobenzofuro[2,3-c]azocin-10-ol isomer ((+)-17), and the N-phenylpropyl derivative ((-)-25) had opioid binding affinity < 50 nM. Both (-)-1 and (-)-25 were partial agonists in the cAMP assay, with the former showing high potency and low efficacy, and the latter with lower potency and less efficacy. Most interesting was the N-2-phenylcyclopropylmethyl (3S,6aR,11aR)-2-(1S,2S)-enantiomer ((+)-17). That compound had good MOR binding affinity (Ki = 11.9 nM) and was found to have naltrexone-like potency as a MOR antagonist (IC50 = 6.92 nM).

Quantum Dot-Peptide-Fullerene Bioconjugates for Visualization of in Vitro and in Vivo Cellular Membrane Potential.

We report the development of a quantum dot (QD)-peptide-fullerene (C60) electron transfer (ET)-based nanobioconjugate for the visualization of membrane potential in living cells. The bioconjugate is composed of (1) a central QD electron donor, (2) a membrane-inserting peptidyl linker, and (3) a C60 electron acceptor. The photoexcited QD donor engages in ET with the C60 acceptor, resulting in quenching of QD photoluminescence (PL) that tracks positively with the number of C60 moieties arrayed around the QD. The nature of the QD-capping ligand also modulates the quenching efficiency; a neutral ligand coating facilitates greater QD quenching than a negatively charged carboxylated ligand. Steady-state photophysical characterization confirms an ET-driven process between the donor-acceptor pair. When introduced to cells, the amphiphilic QD-peptide-C60 bioconjugate labels the plasma membrane by insertion of the peptide-C60 portion into the hydrophobic bilayer, while the hydrophilic QD sits on the exofacial side of the membrane. Depolarization of cellular membrane potential augments the ET process, which is manifested as further quenching of QD PL. We demonstrate in HeLa cells, PC12 cells, and primary cortical neurons significant QD PL quenching (ΔF/F0 of 2-20% depending on the QD-C60 separation distance) in response to membrane depolarization with KCl. Further, we show the ability to use the QD-peptide-C60 probe in combination with conventional voltage-sensitive dyes (VSDs) for simultaneous two-channel imaging of membrane potential. In in vivo imaging of cortical electrical stimulation, the optical response of the optimal QD-peptide-C60 configuration exhibits temporal responsivity to electrical stimulation similar to that of VSDs. Notably, however, the QD-peptide-C60 construct displays 20- to 40-fold greater ΔF/F0 than VSDs. The tractable nature of the QD-peptide-C60 system offers the advantages of ease of assembly, large ΔF/F0, enhanced photostability, and high throughput without the need for complicated organic synthesis or genetic engineering, respectively, that is required of traditional VSDs and fluorescent protein constructs.

Multi-channeled single chain variable fragment (scFv) based microfluidic device for explosives detection.

The development of explosives detection technologies has increased significantly over the years as environmental and national security agencies implement tighter pollution control measures and methods for improving homeland security. 2, 4, 6-Trinitrotoluene (TNT), known primarily as a component in munitions, has been targeted for both its toxicity and carcinogenic properties that if present at high concentrations can be a detriment to both humans, marine and plant ecosystems. Enabling end users with environmental detection and monitoring systems capable of providing real-time, qualitative and quantitative chemical analysis of these toxic compounds would be extremely beneficial. Reported herein is the development of a multi-channeled microfluidic device immobilized with single chain fragment variable (scFv) recombinant proteins specific for the explosive, TNT. Fluorescence displacement immunoassays performed under constant flow demonstrated trace level sensitivity and specificity for TNT. The utility of three multi-channeled devices immobilized with either (1) scFv recombinant protein, (2) biotinylated-scFv (bt-scFv) and (3) monoclonal anti-TNT (whole IgG molecule) were investigated and compared. Fluorescence dose response curves, crossreactivity measurements and limits of detection (LOD) for TNT were determined. Fluorescence displacement immunoassays for TNT in natural seawater demonstrated detection limits at sub-parts-per-billion levels (0.5 ppb) utilizing the microfluidic device with immobilized bt-scFv.

PABA/NO lead optimization: Improved targeting of cytotoxicity to glutathione S-transferase P1-overexpressing cancer cells.

PABA/NO [O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate] is a nitric oxide (NO)-releasing arylating agent designed to be selectively activated by reaction with glutathione (GSH) on catalysis by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancer cells. PABA/NO has proven active in several cancer models in vitro and in vivo, but its tendency to be metabolized via a variety of pathways, some that generate inactive metabolites and hydrolysis products, limits its potential as a drug. Here we show that a simple replacement of cyano for nitro at the 4 position to give compound 4b ('p-cyano-PABA/NO') has the dual effect of slowing the undesired side reactions while enhancing the proportion of NO release and arylating activity on catalysis by GSTP1. Compound 4b showed increased resistance to hydrolysis and uncatalyzed reaction with GSH, along with a more favorable product distribution in the presence of GSTP1. It also showed significant proapoptotic activity. The data suggest p-cyano-PABA/NO to be a more promising prodrug than PABA/NO, with better selectivity toward cancer cells.

Efficacy, but not antibody titer or affinity, of a heroin hapten conjugate vaccine correlates with increasing hapten densities on tetanus toxoid, but not on CRM197 carriers.

Vaccines against drugs of abuse have induced antibodies in animals that blocked the biological effects of the drug by sequestering the drug in the blood and preventing it from crossing the blood-brain barrier. Drugs of abuse are too small to induce antibodies and, therefore, require conjugation of drug hapten analogs to a carrier protein. The efficacy of these conjugate vaccines depends on several factors including hapten design, coupling strategy, hapten density, carrier protein selection, and vaccine adjuvant. Previously, we have shown that 1 (MorHap), a heroin/morphine hapten, conjugated to tetanus toxoid (TT) and mixed with liposomes containing monophosphoryl lipid A [L(MPLA)] as adjuvant, partially blocked the antinociceptive effects of heroin in mice. Herein, we extended those findings, demonstrating greatly improved vaccine induced antinociceptive effects up to 3% mean maximal potential effect (%MPE). This was obtained by evaluating the effects of vaccine efficacy of hapten 1 vaccine conjugates with varying hapten densities using two different commonly used carrier proteins, TT and cross-reactive material 197 (CRM197). Immunization of mice with these conjugates mixed with L(MPLA) induced very high anti-1 IgG peak levels of 400-1500 µg/mL that bound to both heroin and its metabolites, 6-acetylmorphine and morphine. Except for the lowest hapten density for each carrier, the antibody titers and affinity were independent of hapten density. The TT carrier based vaccines induced long-lived inhibition of heroin-induced antinociception that correlated with increasing hapten density. The best formulation contained TT with the highest hapten density of ≥30 haptens/TT molecule and induced %MPE of approximately 3% after heroin challenge. In contrast, the best formulation using CRM197 was with intermediate 1 densities (10-15 haptens/CRM197 molecule), but the %MPE was approximately 13%. In addition, the chemical synthesis of 1, the optimization of the conjugation method, and the methods for the accurate quantification of hapten density are described.

Delivery and tracking of quantum dot peptide bioconjugates in an intact developing avian brain.

Luminescent semiconductor ∼9.5 nm nanoparticles (quantum dots: QDs) have intrinsic physiochemical and optical properties which enable us to begin to understand the mechanisms of nanoparticle mediated chemical/drug delivery. Here, we demonstrate the ability of CdSe/ZnS core/shell QDs surface functionalized with a zwitterionic compact ligand to deliver a cell-penetrating lipopeptide to the developing chick embryo brain without any apparent toxicity. Functionalized QDs were conjugated to the palmitoylated peptide WGDap(Palmitoyl)VKIKKP9GGH6, previously shown to uniquely facilitate endosomal escape, and microinjected into the embryonic chick spinal cord canal at embryo day 4 (E4). We were subsequently able to follow the labeling of spinal cord extension into the ventricles, migratory neuroblasts, maturing brain cells, and complex structures such as the choroid plexus. QD intensity extended throughout the brain, and peaked between E8 and E11 when fluorescence was concentrated in the choroid plexus before declining to hatching (E21/P0). We observed no abnormalities in embryonic patterning or embryo survival, and mRNA in situ hybridization confirmed that, at key developmental stages, the expression pattern of genes associated with different brain cell types (brain lipid binding protein, Sox-2, proteolipid protein and Class III-ß-Tubulin) all showed a normal labeling pattern and intensity. Our findings suggest that we can use chemically modified QDs to identify and track neural stem cells as they migrate, that the choroid plexus clears these injected QDs/nanoparticles from the brain after E15, and that they can deliver drugs and peptides to the developing brain.

Design of chemically stable, potent, and efficacious MDM2 inhibitors that exploit the retro-mannich ring-opening-cyclization reaction mechanism in spiro-oxindoles.

Inhibition of the MDM2-p53 protein-protein interaction is being actively pursued as a new anticancer therapeutic strategy, and spiro-oxindoles have been designed as a class of potent and efficacious small-molecule inhibitors of this interaction (MDM2 inhibitors). Our previous study showed that some of our first-generation spiro-oxindoles undergo a reversible ring-opening-cyclization reaction that, from a single compound in protic solution, results in an equilibrium mixture of four diastereoisomers. By exploiting the ring-opening-cyclization reaction mechanism, we have designed and synthesized a series of second-generation spiro-oxindoles with symmetrical pyrrolidine C2 substitution. These compounds undergo a rapid and irreversible conversion to a single, stable diastereoisomer. Our study has yielded compound 31 (MI-1061), which binds to MDM2 with Ki = 0.16 nM, shows excellent chemical stability, and achieves tumor regression in the SJSA-1 xenograft tumor model in mice.

Synthesis and immunological effects of heroin vaccines.

Three haptens have been synthesized with linkers for attachment to carrier macromolecules at either the piperidino-nitrogen or via an introduced 3-amino group. Two of the haptens, with a 2-oxopropyl functionality at either C6, or at both the C3 and C6 positions on the 4,5-epoxymorphinan framework, as well as the third hapten (DiAmHap) with diamido moieties at both the C3 and C6 positions, should be much more stable in solution, or in vivo in a vaccine, than a hapten with an ester in one of those positions, as found in many heroin-based haptens. A "classical" opioid synthetic scheme enabled the formation of a 3-amino-4,5-epoxymorphinan which could not be obtained using palladium chemistry. Our vaccines are aimed at the reduction of the abuse of heroin and, as well, at the reduction of the effects of its predominant metabolites, 6-acetylmorphine and morphine. One of the haptens, DiAmHap, has given interesting results in a heroin vaccine and is clearly more suited for the purpose than the other two haptens.

Competition between Förster resonance energy transfer and electron transfer in stoichiometrically assembled semiconductor quantum dot-fullerene conjugates.

Understanding how semiconductor quantum dots (QDs) engage in photoinduced energy transfer with carbon allotropes is necessary for enhanced performance in solar cells and other optoelectronic devices along with the potential to create new types of (bio)sensors. Here, we systematically investigate energy transfer interactions between C60 fullerenes and four different QDs, composed of CdSe/ZnS (type I) and CdSe/CdS/ZnS (quasi type II), with emission maxima ranging from 530 to 630 nm. C60-pyrrolidine tris-acid was first coupled to the N-terminus of a hexahistidine-terminated peptide via carbodiimide chemistry to yield a C60-labeled peptide (pepC60). This peptide provided the critical means to achieve ratiometric self-assembly of the QD-(pepC60) nanoheterostructures by exploiting metal affinity coordination to the QD surface. Controlled QD-(pepC60)N bioconjugates were prepared by discretely increasing the ratio (N) of pepC60 assembled per QD in mixtures of dimethyl sulfoxide and buffer; this mixed organic/aqueous approach helped alleviate issues of C60 solubility. An extensive set of control experiments were initially performed to verify the specific and ratiometric nature of QD-(pepC60)N assembly. Photoinitiated energy transfer in these hybrid organic-inorganic systems was then interrogated using steady-state and time-resolved fluorescence along with ultrafast transient absorption spectroscopy. Coordination of pepC60 to the QD results in QD PL quenching that directly tracks with the number of peptides displayed around the QD. A detailed photophysical analysis suggests a competition between electron transfer and Förster resonance energy transfer from the QD to the C60 that is dependent upon a complex interplay of pepC60 ratio per QD, the presence of underlying spectral overlap, and contributions from QD size. These results highlight several important factors that must be considered when designing QD-donor/C60-acceptor systems for potential optoelectronic and biosensing applications.

Diastereomeric spirooxindoles as highly potent and efficacious MDM2 inhibitors.

Small-molecule inhibitors that block the MDM2-p53 protein-protein interaction (MDM2 inhibitors) are being intensely pursued as a new therapeutic strategy for cancer treatment. We previously published a series of spirooxindole-containing compounds as a new class of MDM2 small-molecule inhibitors. We report herein a reversible ring-opening-cyclization reaction for some of these spirooxindoles, which affords four diastereomers from a single compound. Our biochemical binding data showed that the stereochemistry in this class of compounds has a major effect on their binding affinities to MDM2, with >100-fold difference between the most potent and the least potent stereoisomers. Our study has led to the identification of a set of highly potent MDM2 inhibitors with a stereochemistry that is different from that of our previously reported compounds. The most potent compound (MI-888) binds to MDM2 with a Ki value of 0.44 nM and achieves complete and long-lasting tumor regression in an animal model of human cancer.

Novel adamantyl cannabinoids as CB1 receptor probes.

In previous studies, compound 1 (AM411), a 3-(1-adamantyl) analogue of the phytocannabinoid (-)-Δ(8)-tetrahydrocannabinol (Δ(8)-THC), was shown to have improved affinity and selectivity for the CB1 receptor. In this work, we further explored the role of the 1-adamantyl group at the C-3 position in a series of tricyclic cannabinoid analogues modified at the 9-northern aliphatic hydroxyl (NAH) position. Of these, 9-hydroxymethyl hexahydrocannabinol 11 (AM4054) exhibited high CB1 affinity and full agonist profile. In the cAMP assay, the 9-hydroxymethyl cannabinol analogue 24 (AM4089) had a partial agonist profile, with high affinity and moderate selectivity for rCB1 over hCB2. In vivo results in rat models of hypothermia and analgesia were congruent with in vitro data. Our in vivo data indicate that 3-(1-adamantyl) substitution, within NAH cannabinergics, imparts improved pharmacological profiles when compared to the corresponding, traditionally used 3-dimethylheptyl analogues and identifies 11 and 24 as potentially useful in vivo CB1 cannabinergic probes.

Optimizing protein coordination to quantum dots with designer peptidyl linkers.

Semiconductor quantum dots (QDs) demonstrate select optical properties that make them of particular use in biological imaging and biosensing. Controlled attachment of biomolecules such as proteins to the QD surface is thus critically necessary for development of these functional nanobiomaterials. QD surface coatings such as poly(ethylene glycol) impart colloidal stability to the QDs, making them usable in physiological environments, but can impede attachment of proteins due to steric interactions. While this problem is being partially addressed through the development of more compact QD ligands, here we present an alternative and complementary approach to this issue by engineering rigid peptidyl linkers that can be appended onto almost all expressed proteins. The linkers are specifically designed to extend a terminal polyhistidine sequence out from the globular protein structure and penetrate the QD ligand coating to enhance binding by metal-affinity driven coordination. α-Helical linkers of two lengths terminating in either a single or triple hexahistidine motif were fused onto a single-domain antibody; these were then self-assembled onto QDs to create a model immunosensor system targeted against the biothreat agent ricin. We utilized this system to systematically evaluate the peptidyl linker design in functional assays using QDs stabilized with four different types of coating ligands including poly(ethylene glycol). We show that increased linker length, but surprisingly not added histidines, can improve protein to QD attachment and sensor performance despite the surface ligand size with both custom and commercial QD preparations. Implications for these findings on the development of QD-based biosensors are discussed.

Proteolytic activity at quantum dot-conjugates: kinetic analysis reveals enhanced enzyme activity and localized interfacial "hopping".

Recent studies show that polyvalent, ligand-modified nanoparticles provide significantly enhanced binding characteristics compared to isolated ligands. Here, we assess the ability of substrate-modified nanoparticles to provide enhanced enzymatic activity. Energy transfer assays allowed quantitative, real-time measurement of proteolytic digestion at polyvalent quantum dot-peptide conjugates. Enzymatic progress curves were analyzed using an integrated Michaelis-Menten (MM) formalism, revealing mechanistic details, including deviations from classic MM-behavior. A "hopping" mode of proteolysis at the nanoparticle was identified, confirming enhanced activity.

Nonpeptidic and potent small-molecule inhibitors of cIAP-1/2 and XIAP proteins.

A series of compounds were designed and synthesized as antagonists of cIAP-1/2 and XIAP based upon our previously identified lead compound SM-122 (1). The most potent of these (7) binds to XIAP, cIAP-1, and cIAP-2 proteins with K(i) values of 36, <1, and <1.9 nM, respectively. Consistent with its potent binding affinities to IAPs, 7 effectively antagonizes XIAP in a cell-free caspase-9 functional assay, efficiently induces cIAP-1 degradation in cells at concentrations as low as 10 nM, and triggers activation of caspases and PARP cleavage in the MDA-MB-231 breast cancer cell line. Compound 7 potently inhibits cell growth in the MDA-MB-231 cancer cell line with an IC(50) value of 200 nM and is 9 times more potent than compound 1.

Reversal of pancreatitis-induced pain by an orally available, small molecule interleukin-6 receptor antagonist.

Pancreatic pain resulting from chronic inflammation of the pancreas is often intractable and clinically difficult to manage with available analgesics reflecting the need for more effective therapies. The mechanisms underlying pancreatitis pain are not well understood. Here, the possibility that interleukin-6 (IL-6) may promote pancreatitis pain was investigated with TB-2-081 (3-O-formyl-20R,21-epoxyresibufogenin, EBRF), a small molecule IL-6 receptor antagonist that was semi-synthetically derived from natural sources. The potential activity and mechanism of TB-2-081 were investigated following the induction of persistent pancreatitis using dibutyltin dichloride (DBTC) in rats. TB-2-081 displaces the binding of IL-6 to the human recombinant soluble IL-6 receptor with apparent high affinity and inhibits IL-6 mediated cell growth. Systemic or oral, but not intrathecal, administration of TB-2-081 reversed DBTC-induced abdominal hypersensitivity in a dose- and time-dependent manner. IL-6 levels were significantly up-regulated in the dorsal root ganglia (DRG) of rats with pancreatitis on day 6 after DBTC injection. IL-6-enhanced capsaicin-evoked release of calcitonin gene-related peptide from cultured DRG neurons was blocked by TB-2-081. Our data demonstrate that TB-2-081 acts as a systemically available and orally active small molecule IL-6 receptor antagonist. TB-2-081 effectively reduces pancreatitis-induced pain through peripheral mechanisms that are likely due to (a) increased expression of IL-6 in the DRG and (b) IL-6-mediated sensitization of nociceptive neurons. The activity of TB-2-081 implicates an important role for IL-6 in sustaining pancreatitis pain. Strategies targeting IL-6 actions through small molecule antagonists may offer novel approaches to improve the therapy of chronic pancreatitis and other chronic pain states.

Polyvalent display and packing of peptides and proteins on semiconductor quantum dots: predicted versus experimental results.

Quantum dots (QDs) are loaded with a series of peptides and proteins of increasing size, including a <20 residue peptide, myoglobin, mCherry, and maltose binding protein, which together cover a range of masses from <2.2 to approximately 44 kDa. Conjugation to the surface of dihydrolipoic acid-functionalized QDs is facilitated by polyhistidine metal affinity coordination. Increasing ratios of dye-labeled peptides and proteins are self-assembled to the QDs and then the bioconjugates are separated and analyzed using agarose gel electrophoresis. Fluorescent visualization of both conjugated and unbound species allows determination of an experimentally derived maximum loading number. Molecular modeling utilizing crystallographic coordinates or space-filling structures of the peptides and proteins also allow the predicted maximum loadings to the QDs to be estimated. Comparison of the two sets of results provides insight into the nature of the QD surface and reflects the important role played by the nanoparticle's hydrophilic solubilizing surface ligands. It is found that for the larger protein molecules steric hindrance is the major packing constraint. In contrast, for the smaller peptides, the number of available QD binding sites is the principal determinant. These results can contribute towards an overall understanding of how to engineer designer bioconjugates for both QDs and other nanoparticle materials.

North- and south-bicyclo[3.1.0]hexene nucleosides: the effect of ring planarity on anti-HIV activity.

The syntheses of new conformationally locked North- and South-bicyclo[3.1.0]hexene nucleosides is reported. The North analogues were synthesized by a convergent approach from the known (1S,2R,5R)-5-[(tert-butyldiphenylsilyloxy)methyl]bicyclo[3.1.0]hex-3-en-2-ol by Mitsunobu coupling with the nucleobases. The South analogues were synthesized from their bicyclo[3.1.0]hexane nucleoside precursors by the selective protection of the primary hydroxy group, conversion of the secondary alcohol into a good leaving group, and base-catalyzed elimination to generate the olefin. The transformation of a bicyclo[3.1.0]hexane nucleoside into a bicyclo[3.1.0]hexene nucleoside flattens the five-membered ring of the bicyclic system and rescues anti-HIV activity for North-D4T, North-D4A, and South-D4C. The relationship between planarity and the anti/syn disposition of the nucleobase that is favored by a particular pseudosugar platform are proposed as key parameters in controlling biological activity.

Multiplex charge-transfer interactions between quantum dots and peptide-bridged ruthenium complexes.

Simultaneous detection of multiple independent fluorescent signals or signal multiplexing has the potential to significantly improve bioassay throughput and to allow visualization of concurrent cellular events. Applications based on signal multiplexing, however, remain hard to achieve in practice due to difficulties in both implementing hardware and the photophysical liabilities associated with available organic dye and protein fluorophores. Here, we used charge-transfer interactions between luminescent semiconductor quantum dots (QDs) and proximal redox complexes to demonstrate controlled quenching of QD photoemission in a multiplexed format. In particular, we show that, because of the ability of the Ru complex to effectively interact with CdSe-ZnS QDs emitting over a broad window of the optical spectrum, higher orders of multiplexed quenching can be achieved in a relatively facile manner. Polyhistidine-appended peptides were site-specifically labeled with a redox-active ruthenium (Ru) phenanthroline complex and self-assembled onto QDs, resulting in controlled quenching of the QD emission. Different QD colors either alone or coupled to Ru-phen-peptide were then mixed together and optically interrogated. Composite spectra collected from mixtures ranging from four up to eight distinct QD colors were deconvoluted, and the individual QD photoluminescence (PL) loss due to charge transfer was quantified. The current multiplexing modality provides a simpler format for exploiting the narrow, size-tunable QD emissions than that offered by resonance energy transfer; for the latter, higher orders of multiplexing are limited by spectral overlap requirements.

Hydrolytic reactivity trends among potential prodrugs of the O2-glycosylated diazeniumdiolate family. Targeting nitric oxide to macrophages for antileishmanial activity.

Glycosylated diazeniumdiolates of structure R 2NN(O)NO-R' (R' = a saccharide residue) are potential prodrugs of the nitric oxide (NO)-releasing but acid-sensitive R 2NN(O)NO (-) ion. Moreover, cleaving the acid-stable glycosides under alkaline conditions provides a convenient protecting group strategy for diazeniumdiolate ions. Here, we report comparative hydrolysis rate data for five representative glycosylated diazeniumdiolates at pH 14, 7.4, and 3.8-4.6 as background for further developing both the protecting group application and the ability to target NO pharmacologically to macrophages harboring intracellular pathogens. Confirming the potential in the latter application, adding R 2NN(O)NO-GlcNAc (where R 2N = diethylamino or pyrrolidin-l-yl and GlcNAc = N-acetylglucosamin-l-yl) to cultures of infected mouse macrophages that were deficient in inducible NO synthase caused rapid death of the intracellular protozoan parasite Leishmania major with no host cell toxicity.

Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates.

Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis-Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD-thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.