Search

Measuring the entropic properties of polymers such as proteins is critical to accurate prediction of their functional properties. However, the measurement of configurational entropy is possible only by low throughput techniques such as calorimetry, NMR and CD spectroscopy. Moreover, to our knowledge no system exists that allows molecular selection/enrichment based on the molecules configurational entropy. We tested the ability of the scalable tripartite GFP system to offer fine resolution of differences in configurational entropy in molecules and to isolate molecules based on their configurational entropy. The system was able to both finely resolve molecules with different configurational entropies, as well as capture them for isolation. We were able to tune the sensitivity of the system by using different mutations of the protein components. Lastly, we were able to apply the system to polypeptoid molecules and posit that the system may be applied to any other hydrophilic polymer of up to 10^3 repeating units.

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibited PLpro with kinact/KI = 10,000 M− 1 s− 1, achieved sub-µM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and did not inhibit a panel of human deubiquitinases at > 30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validated our design strategy and established the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

Vaccines based on proteins and peptides may be safer and more broad-spectrum than other approaches Physicochemical property consensus (PCPcon) alphavirus antigens from the B-domain of the E2 envelope protein were designed and synthesized recombinantly. Those based on individual species (eastern or Venezuelan equine encephalitis (EEEVcon, VEEVcon), or chikungunya (CHIKVcon) viruses generated species-specific antibodies. Peptides designed to surface exposed areas of the E2-A-domain were added to the inocula to provide neutralizing antibodies against CHIKV. EVCcon, based on the three different alphavirus species, combined with E2-A-domain peptides from AllAV, a PCPcon of 24 diverse alphavirus, generated broad spectrum antibodies. The abs in the sera bound and neutralized diverse alphaviruses with less than 35% amino acid identity to each other. These included VEEV and its relative Mucambo virus, EEEV and the related Madariaga virus, and CHIKV strain 181/25. Further understanding of the role of coordinated mutations in the envelope proteins may yield a single, protein and peptide vaccine against all alphaviruses.

We report a critical advance in the generation and characterization of peptoid hetero-oligomers. A library of sub-monomers with amine and carboxylate side-chains are combined in different sequences using microwave-assisted synthesis. Their sequence-structure propensity is confirmed by circular dichroism, and conformer subtypes are enumerated by NMR. Biasing the ψ-angle backbone to trans (180°) in Monte Carlo modelling favors i to i + 3 naphthyl-naphthyl stacking, and matches experimental ensemble distributions. Taken together, high-yield synthesis of heterooligomers and NMR with structure prediction enables rapid determination of sequences that induce secondary structural propensities for predictive design of hydrophilic peptidomimetic foldamers and their future libraries.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (Mpro also known as 3CLpro) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the Mpro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the Mpro as potential COVID-19 therapeutics for further testing and possibly clinical trials.

Vaccines based on proteins and peptides may be safer and if calculated based on many sequences, more broad-spectrum than those designed based on single strains. Physicochemical Property Consensus (PCPsubcon/sub) alphavirus (AV) antigens from the B-domain of the E2 envelope protein were designed, synthesized recombinantly and shown to be immunogenic (i.e. sera after inoculation detected the antigen in dotspots and ELISA). Antibodies in sera after inoculation with B-region antigens based on individual AV species (eastern or Venezuelan equine encephalitis (EEEVsubcon/sub, VEEVsubcon/sub), or chikungunya (CHIKVsubcon/sub) bound only their cognate protein, while those designed against multiple species (Mosaiksubcon/suband EVCsubcon/sub) recognized all three serotype specific antigens. The VEEVsubcon/suband EEEVsubcon/subsera only showed antiviral activity against their related strains (in plaque reduction neutralization assays (PRNTsub50/80/sub). Peptides designed to surface exposed areas of the E2-A-domain of CHIKVsubcon/subwere added to CHIKVsubcon/subinocula to provide anti-CHIKV antibodies. EVCsubcon/sub, based on three different alphavirus species, combined with E2-A-domain peptides from AllAVsubcon/sub, a PCPcon of 24 diverse AV, generated broad spectrum, antiviral antibodies against VEEV, EEEV and CHIKV, AV with less than 35% amino acid identity to each other (gt;65% diversity). This is a promising start to a molecularly defined vaccine against all AV. Further study with these antigens can illuminate what areas are most important for a robust immune response, resistant to mutations in rapidly evolving viruses. The validated computational methods can also be used to design broad spectrum antigens against many other pathogen families.

Direct-acting antivirals for the treatment of COVID-19, which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), are needed to complement vaccination efforts. The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral proliferation. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophilic “warheads” onto analogs of the noncovalent PLpro inhibitor GRL0617. We show that the most promising PLpro inhibitor is potent and selective, with activity in cell-based antiviral assays rivaling that of the RNA-dependent RNA polymerase inhibitor remdesivir. An X-ray crystal structure of the most promising lead compound bound covalently to PLpro establishes the molecular basis for protease inhibition and selectivity against structurally similar human deubiquitinases. These findings present an opportunity for further development of potent and selective covalent PLpro inhibitors.

We report a critical advance in the generation and characterization of peptoid hetero-oligomers. A library of sub-monomers with amine and carboxylate side-chains are combined in different sequences using microwave-assisted synthesis. Their sequence-structure propensity is confirmed by circular dichroism, and conformer subtypes are enumerated by NMR. Biasing the

The development of efficient catalysts and processes for synthesizing functionalized (olefinic and/or chiral) primary alcohols and fluoral hemiacetals is currently needed. These are valuable buildi...

There is an urgent need for inexpensive, rapid and specific antigen-based assays to test for infection with SARS-CoV-2 and distinguish variants arising as the COVID-19 pandemic spreads. We have identified a small, synthetic protein (JS7), representing a region of maximum variability within the receptor binding domain (RBD), which binds antibodies in sera from nine patients with PCR-verified COVID-19 of varying severity. Antibodies binding to either JS7 or the SARS-CoV-2 recombinant RBD, as well as those that disrupt binding between a fragment of the ACE2 receptor and the RBD, are proportional to disease severity and clinical outcome. Binding to JS7 was inhibited by linear peptides from the RBD interface with ACE2. Variants of JS7, such as N501Y, can be quickly synthesized in a pure form in large quantities by automated methods. JS7 and related synthetic antigens can provide a basis for specific diagnostics for SARS-CoV-2 infections.

There is an urgent need for inexpensive, rapid and specific antigen-based assays to test for vaccine efficacy and detect infection with SARS-CoV-2 and its variants. We have identified a small, synthetic protein (JS7), representing a region of maximum variability within the receptor binding domain (RBD), which binds antibodies in sera from nine patients with PCR-verified COVID-19 of varying severity. Antibodies binding to either JS7 or the SARS-CoV-2 recombinant RBD, as well as those that disrupt binding between a fragment of the ACE2 receptor and the RBD, are proportional to disease severity and clinical outcome. Binding to JS7 was inhibited by linear peptides from the RBD interface with ACE2. Variants of JS7, such as E484 K or N501Y, can be quickly synthesized in a pure form in large quantities by automated methods. JS7 and related synthetic antigens can provide a basis for specific diagnostics for SARS-CoV-2 infections.

Anti-malaria drugs chloroquine and amodiaquine and their metabolites were synthesized to incorporate 13 C and 15 N starting from U-13 C-labeled benzene to give M + 7 isotopomers. Chloroquine and its metabolites were prepared from 7-chloro-1,2,3,4-tetrahydroquinolin-4-one through an aryl substitution with the corresponding amines; and the amodiaquine and its metabolites were prepared from 4,7-dichloroquinoline in a similar fashion.

13C labeled (>M + 4) metabolites of nitrogen and sulfur-based chemical warfare agent metabolites were prepared from readily available and 13C labeled commercial starting materials. The new chemical routes are efficient in the number of chemical steps, can be scaled to afford gram quantities, and occur in good yields on the basis of the 13C label. These labeled compounds are useful as internal standards in mass spectrometry for monitoring chemical warfare agents and their metabolites. Published 2012. This article is a US Government work and is in the public domain in the USA.

A significant challenge in bacterial detection is the identification of viable bacteria over debris, specifically post decontamination. Of increasing concern are antibiotic resistant strains that require accurate and rapid post decontamination analysis. Current strategies are fraught with disadvantages and most of them are not selective for viable bacteria. However, bacteria are critically dependent upon iron sequestration, synthesizing and releasing siderophores (SDPs) to tightly bind iron, with the subsequent uptake of iron bound SDPs. This is a highly conserved process that occurs only in intact bacteria. Herein we report a facile method to use bacterial SDPs to selectively and rapidly identify only viable bacteria in complex matrices, and discriminate them from their dead counterparts. Desferrioxamine B (Desf B) tethered to a glass slide is used to specifically capture viable bacteria from a mixture of viable and dead Escherichia coli, as demonstrated by fluorescence microscopy. We report both direct conjugation of Desf B on thin-filmcoated glass slides as well as biotin-streptavidin conjugation strategies, both of which are successful in the said goal. We have analyzed the density of images obtained upon fluorescence staining using edge detection with a Canny edge detector. This novel application of a software analysis tool originally developed for satellite imaging to biological staining allows for accurate quantitation of observed data.

(Bi,Sb)2Te3 + 4 mol%PbTe was quenched in water and on a rotating copper wheel (melt spinning). It was found that PbTe was immiscible in (Bi,Sb)2Te3 when the material is quenched in water and that the thermoelectric figure of merit increases by annealing. Natural nanostructures (nns) were found in melt-spun (Bi,Sb)2Te3, whereas they were hard to detect in (Bi,Sb)2Te3 alloyed with PbTe. There is a correlation between the orientation of the strain field and the nns. Within the grains of melt-spun (Bi,Sb)2Te3 alloyed with PbTe, the chemical composition was homogeneous. An enrichment of Pb was found at the grain boundaries. Quenched (Bi,Sb)2Te3 alloyed with 0.3 wt%PbTe have been spark plasma sintered (SPS). After optimization, the Seebeck coefficients of the melt-spun SPS (MS-SPS) materials were larger than for materials quenched in water and sintered (QW-SPS) materials. In addition, the mobility increases with the carrier concentration in MS-SPS materials, whereas it decreases in QW-SPS materials.

Multicomponent reactions (MCRs) are not only a power- ful tool for drug discovery, they also represent an excellent method- ology for synthesis rationalization. Here we wish to illustrate the potential of MCRs in the production of generic drugs by synthesiz- ing, in racemic form, the antiplatelet agent clopidogrel and the non- steroidal antiandrogen bicalutamide, using Ugi, Petasis and Passe- rini reactions.

M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

We report a general procedure to prepare functional organic thin films for biological assays on oxide surfaces. Silica surfaces were functionalized by self-assembly of an amine-terminated silane film using both vapor- and solution-phase deposition of 3'-aminopropylmethyldiethoxysilane (APMDES). We found that vapor-phase deposition of APMDES under reduced pressure produced the highest quality monolayer films with uniform surface coverage, as determined by atomic force microscopy (AFM), ellipsometry, and contact angle measurements. The amine-terminated films were chemically modified with a mixture of carboxylic acid-terminated poly(ethylene glycol) (PEG) chains of varying functionality. A fraction of the PEG chains (0.1-10 mol %) terminated in biotin, which produced a surface with an affinity toward streptavidin. When used in pseudo-sandwich assays on waveguide platforms for the detection of Bacillus anthracis protective antigen (PA), these functional PEG surfaces significantly reduced nonspecific binding to the waveguide surface while allowing for highly specific binding. Detection of PA was used to validate these films for sensing applications in both buffer and complex media. Ultimately, these results represent a step toward the realization of a robust, reusable, and autonomous biosensor.

We have developed an assay (Endopep-MS) that detects the specific endoproteinase activities of all seven BoNT types by mass spectrometry (MS). Each BoNT type cleaves a unique site on proteins involved in neuronal transmission. Target peptide substrates based on these proteins identify a BoNT type by its enzymatic action on the substrate and the production of two peptide products, which are then detected by matrix-assisted laser desorption/ionization time-of-flight MS or liquid chromatography electrospray ionization MS/MS. We showed the ability to detect all seven toxin types in a multiplexed assay format. The detection limits achieved range from 0.039 to 0.625 mouse LD(50)/mL for toxin types A, B, E, and F in a buffer system. The Endopep-MS assay is the first to differentiate all seven BoNT types, is sensitive, specific, and has the potential to quantify toxin activity.

The synthesis of oligoethylene glycols of defined lengths possessing different end functionalities is described. The utility of these molecules towards the development of a generic membrane anchor is demonstrated.

A new route is presented to prepare analogs of nucleosides homologated at the 3′- and 5′-positions. This route, applicable to both the D- and L-enantiomeric forms, is suitable for the preparation of monomeric bis-homonucleosides needed for the synthesis of oligonucleotide analogs. It begins with the known monobenzyl ether 3 of pent-2-yne-1,5-diol, which is reduced to alkenol 4. Sharpless asymmetric epoxidation of 4, followed by opening of the epoxide 5 with allylmagnesium bromide, gives a mixture of diols 6 and 7. Protection of the primary alcohol as a silyl ether followed by treatment with OsO4, NaIO4, and mild acid in MeOH, followed by reduction, yields (2R,3R) {{[(tert-butyl)diphenylsilyl]oxy}methyl}tetrahydro-2-(2-hydroxyethyl)-5-methoxyfuran (=methyl 3-{{[(tert-butyl)diphenylsilyl]oxy}methyl}-2,3,5-trideoxy-α/β-D-erythro-hexafuranoside; 10) (Scheme 1). Protected nucleobases are added to this skeleton with the aid of trimethylsilyl triflate (Scheme 2). The o-toluoyl (2-MeC6H4CO) and p-anisoyl (4-MeOC6H4CO) groups were used to protect the exocyclic amino group of cytosine. The bis-homonucleoside analogs 11 and 14a are then converted to monothiol derivatives suitable for coupling (Schemes 3 and 4) to oligonucleotide analogs with bridging S-atoms. This synthesis replaces a much longer synthesis for analogous nucleoside analogs that begins with diacetoneglucose (=1,2 : 5,6-di-O-isopropylideneglucose), with the stereogenic centers in the final products derived from the Sharpless asymmetric epoxidation. The new route is useful for large-scale synthesis of these building blocks for the synthesis of oligonucleotide analogs.

A convergent, solution-phase synthesis was developed for the bis(methylene) sulfone-bridged oligodeoxynucleotide analogs (SNA) 5′-d(HOCH2-Tso2Tso2Tso2Cso2Tso2Tso2Tso2T-CH2SO)-3′ (35b) and 5′-d(HOCH2-Tso2Tso2Tso2Tso2Tso2Tso2Tso2T-CH2SO)-3′ (34c) (SO2 corresponds to CH2SO2CH2 instead of OP(O)(O−)(O). In these, the phosphodiester linkages are replaced by non-ionic bis(methylene) sulfone linkers. The general strategy involved convergent coupling of 3′,5′-bishomo-β-D-deoxyribonucleotide analogs functionalized at the 6′-end (CH2C(5′)) as bromides or mesylates and at the CH2C(3′) position as thiols, with the resulting thioether being oxidized to the corresponding sulfone. A single charge was introduced at the terminal CH2C(3′) position of the octamers to increase their solubility in water. During the synthesis, it became apparent that the key intermediates generated secondary structures through either folding or aggregation in a variety of solvents. This generated unusual reactivity and was unique for very similar structures. For example, although the dimeric thiol d(BzOCH2-Tso2C-CH2SH) (14b) was a well-behaved synthetic intermediate, the tetrameric thiol d(TrOCH2-Tso2Tso2Tso2toC-CH2SH) derived from the corresponding thioacetate was rapidly converted to a disulfide by very small amounts of oxidant (2829, Scheme 6), while the analogous tetrameric thiol d(BzOCH2-Tso2TsTso2T-CH2SH) (26), differing only by a single heterocycle, was oxidized much more slowly (Bz=PhCO, Tr=Ph3C, to=2-MeC6H4CO (at N4 of dc)). The sequence-dependent reactivity, well known in many classes of natural products (including polypeptides), is not prominent in natural oligonucleotides. These results are discussed in light of the proposal that the repeating negative charge in nucleic acids is key to their ability to serve as genetic molecules, in particular, their capability to support Darwinian evolution. The ability of 5′-d(HOCH2-Tso2Tso2Tso2Cso2Tso2Tso2Tso2T-CH2SO)-3′ (35b) to bind as a third strand to duplex DNA was also examined. No triple-helix-forming propensity was detected in this molecule.

We have developed a large-scale “one-pot” procedure for the conversion of commercially available [13C]- or [2H3,13C]methanol to [13C]- or [2H3,13C]methyl phenyl sulfide. [13C]methyl phenyl sulfide is a potentially versatile, chemically stable, and nonvolatile labeling precursor. In addition, we report an efficient method for the oxidation of [13C]methyl phenyl sulfide to [13C]methyl phenyl sulfone. Finally, we have used [13C]methyl phenyl sulfide to produce 13C-labeled methyl iodide, containing exactly one or two deuterons.

Most of the currently used reflective coatings for EUV and X-ray mirrors are periodic nanometer multilayers. Depending on the number of periods and the absorption in the multilayer stack a certain band width of the incoming radiation can be reflected. In order to increase the integral reflectance or to accept larger ranges of incidence angles, non-periodic multilayers are needed. With the transition from periodic to non-periodic multilayers new challenges arise for the deposition process. Since the reflectance spectra are sensitive to every single layer thickness a precise coating control and an exact knowledge of the interface reactions are required. Furthermore substrate roughness influences the reflectance spectra. With an advanced coating process using additional ion bombardment during thin film growth the integrated reflectance of broadband mirrors can be conserved even for an initial substrate roughness of about 0.7 nm rms.

Spark Plasma Sintering (SPS) is used for the fabrication of wafers of n- and p-type thermoelectric V2VI3 materials. The SPS process did not change the overall chemical composition. X-ray diffraction analysis and the electron backscattered selected area diffraction prove the preferential orientation after the SPS procedure expecting anisotropic thermoelectric prop- erties. The mechanical properties of the SPS material are enormously enhanced, so that the fabrication of thin wafers with only 100 µm thickness suitable for the development of Peltier devices with high cooling power density will be possible. (© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The hexane extract from the leaves of Dasymaschalon sootepenseCraib (Annonaceae) showed strong cytotoxic activity against the L1210 tumor cell line. Activity-directed fractionation of the extract by column chromatography on silica gel and high-pressure liquid chromatography led to the isolation of the acetogenins 1 – 4 as the main active principles. The structures of the two novel structures named sootepensin A (1) and sootepensin B (2) were elucidated by spectroscopic analysis (UV, EI- and ESI-MS, 1D- and 2D-1H- and 13C-NMR). The absolute configurations were established by 2D-NMR experiments utilizing Mosher esters. Two recently described compounds, tonkinin C (3) and tonkinesin C (4), were also isolated and are new to the genus Dasymaschalon. All four acetogenins were found to be highly cytotoxic against the L1210 tumor cell line.

A research program has applied the tools of synthetic organic chemistry to systematically modify the structure of DNA and RNA oligonucleotides to learn more about the chemical principles underlying their ability to store and transmit genetic information. Oligonucleotides (as opposed to nucleosides) have long been overlooked by synthetic organic chemists as targets for structural modification. Synthetic chemistry has now yielded oligonucleotides with 12 replicatable letters, modified backbones, and new insight into why Nature chose the oligonucleotide structures that she did.

Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.

Lipochitooligosaccharides (LCOs) secreted by rhizobia play a key role in the induction of nodules on the roots of legumes by activating signaling events that, at very low concentration, initiate cell division at distinct sites in the root (1, 2). Possibly these signals affect regulation of the plant cell cycle (3), and evidence is accumulating that LCOs play a general role as plant growth regulators (4–6). Previously we have developed methods to synthesize LCOs and demonstrated that these signaling molecules, which trigger nodulation in legumes, also promote cell division in tobacco protoplasts grown in the absence of auxin and cytokinin (7). Notably, glycolipid signals carrying fatty acids with double bonds in the trans configuration were efficient at femtomolar concentration to alleviate the requirement for micromolar concentrations of auxin and cytokinin to sustain growth of cultured tobacco protoplasts. LCOs increased the levels of AXI1 transcripts in protoplasts grown in the absence of auxin. The AXI1 gene is auxin- and cytokinin-responsive and its deregulated expression uncouples protoplasts from the normal effects of auxin on cell division (8). In this study, we have investigated the minimum structural requirements for LCOs to induce cell division of tobacco protoplasts. We have used the AXI1 promoter in transient expression assays to show that multiple signaling pathways for AXI1 expression exist in tobacco cells.

Lipoarabinomannan (LAM) is a critical virulence factor in the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. LAM is secreted in urine and serum from infected patients and is being studied as a potential diagnostic indicator for the disease. Herein, we present a novel ultra-sensitive and specific detection strategy for monomeric LAM based on its amphiphilic nature and consequent interaction with supported lipid bilayers. Our strategy involves the capture of LAM on waveguides functionalized with membrane mimetic architectures, followed by detection with a fluorescently labeled polyclonal antibody. This approach offers ultra-sensitive detection of lipoarabinomannan (10 fM, within 15 min) and may be extended to other amphiphilic markers. We also show that chemical deacylation of LAM completely abrogates its association with the supported lipid bilayers. The loss of signal using the waveguide assay for deacylated LAM, as well as atomic force microscopy (AFM) images that show no change in height upon addition of deacylated LAM support this hypothesis. Mass spectrometry of chemically deacylated LAM indicates the presence of LAM-specific carbohydrate chains, which maintain antigenicity in immunoassays. Further, we have developed the first three-dimensional structural model of mannose-capped LAM that provides insights into the orientation of LAM on supported lipid bilayers.

We are exploring the feasibility of using recently developed flu orogen-activating proteins (FAPs) as reporters for single-molecule imaging. FAPs are single-chain antibodies choosen to specifically bind small chromophoric molecules termed f1uorogens. Upon binding to its cognate FAP the fluorescence quantum yield of the fluorogen can increase substantially giving rise to a fluorescent complex. Based on the seminal work of Szent-Gyorgyi et al. (Nature Biotechnology, Volume 26, Number 2, pp 235-240, 2008) we have chosen to study two fluorogen-activating single-chain antibodies, HL 1.0.1-TOI and H6-MG bound to their cognate fluorogens, thiazole orange and malachite green derivatives, respectively. Here we use fluorescence correlation spectroscopy study the photophysics of these fluorescent complexes.

A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 μL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure. © 2010 American Chemical Society.

We report the modular synthesis of robust, biotinylated biantennary sialylglycoconjugates and their ability to differentiate between two type A influenza strains. This is the first demonstration of glycoconjugate-based discriminatory capture and detection of two strains of intact influenza virus, in the presence of the innate enzymatic activity of viral neuraminidases. We also demonstrate a “carboassay” using glycoconjugates as capture and reporter elements, which therefore, does not require antibodies. The capture of intact influenza viruses is of potential benefit for clinical diagnostics.

A novel one-pot two-step multi component reaction of acrylic aldehydes, bromoanilines, acids and isocyanides yielding polysubstituted indoles is described. The reaction is based on the combination of an Ugi four-component reaction followed by an intramolecular Heck-reaction. The simultaneous use of formic acid and cinnamaldehydes affords in situ generation of 1H-indoles. Convertible isocyanides can also be used with success in this Ugi/Heck strategy and enable synthesis of 1H-indole-2-carboxylic acid building blocks.

We report here a procedure for the functioalization of SiO2-coated, SiONx waveguides for biological assays. Surface functionalization occurs by self-assembly of an amine-terminated silane monolayer on the waveguide, followed by partial chemical modification with functionalized polyethylene glycol (PEG) groups. Functionalized surfaces were characterized by atomic force microscopy and contact angle measurements. When combined with a BSA blocking step, these functional PEG surfaces significantly reduced non-specific binding and allowed for specific binding to occur. An antibody sandwich assay for detection of Bacillus anthracis protective antigen was used to validate these surfaces for sensing applications.

A new rapid, mass spectrometry-based method to detect and differentiate botulinal neurotoxins is described., Botulinum neurotoxins (BoNTs) are proteases that cleave specific cellular proteins essential for neurotransmitter release. Seven BoNT serotypes (A–G) exist; 4 usually cause human botulism (A, B, E, and F). We developed a rapid, mass spectrometry–based method (Endopep-MS) to detect and differentiate active BoNTs A, B, E, and F. This method uses the highly specific protease activity of the toxins with target peptides specific for each toxin serotype. The product peptides derived from the endopeptidase activities of BoNTs are detected by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. In buffer, this method can detect toxin equivalents of as little as 0.01 mouse lethal dose (MLD)50 and concentrations as low as 0.62 MLD50/mL. A high-performance liquid chromatography–tandem mass spectrometry method for quantifying active toxin, where the amount of toxin can be correlated to the amount of product peptides, is also described.

Ion-exchange chromatography is a well-established method for the analysis and purification of phosphodiester-linked oligonucleotides. If elution is carried out under alkaline conditions, the secondary structure of G- and C-rich oligomers is disrupted. Furthermore, elution times become more sensitive to the G and T content of the oligomer, because G and T are deprotonated at pH 10. In recent work on peptide-nucleic acids (PNAs) we noted that mixtures of PNA oligomers G(sub 4), G(Sub 6), G(sub 8), and G(sub 10) are readily separated by elution at pH 12 on an RPC-5 column. Here we show that this separation method is more generally applicable.

The common nodulation genes of rhizobial bacteria code for enzymes involved in the synthesis of lipo-chitooligosaccharide signal molecules. These molecules not only trigger the development of nodules on the roots of leguminous plants, but similar signals may affect the growth and development of non-leguminous plants.

The stability of the PNA (peptide nucleic acid) thymine monomer ¿N-[2-(thymin-1-ylacetyl)]-N-(2-aminoaminoethyl)glycine¿ and those of various PNA oligomers (5-8-mers) have been measured at room temperature (20 degrees C) as a function of pH. The thymine monomer undergoes N-acyl transfer rearrangement with a half-life of 34 days at pH 11 as analyzed by 1H NMR; and two reactions, the N-acyl transfer and a sequential degradation, are found by HPLC analysis to occur at measurable rates for the oligomers at pH 9 or above. Dependent on the amino-terminal sequence, half-lives of 350 h to 163 days were found at pH 9. At pH 12 the half-lives ranged from 1.5 h to 21 days. The results are discussed in terms of PNA as a gene therapeutic drug as well as a possible prebiotic genetic material.