Copper-Catalyzed Covalent Dimerization of Near-Infrared Fluorescent Cyanine Dyes: Synergistic Enhancement of Photoacoustic Signals for Molecular Imaging of Tumors.

Photoacoustic (PA) imaging relies on the absorption of light by chromophores to generate acoustic waves used to delineate tissue structures and physiology. Here, we demonstrate that Cu(II) efficiently catalyzes the dimerization of diverse near-infrared (NIR) cyanine molecules, including a peptide conjugate. NMR spectroscopy revealed a C-C covalent bond along the heptamethine chains, creating stable molecules under conditions such as a wide range of solvents and pH mediums. Dimerization achieved >90% fluorescence quenching, enhanced photostability, and increased PA signals by a factor of about 4 at equimolar concentrations compared to the monomers. In vivo study with a mouse cancer model revealed that dimerization enhanced tumor retention and PA signal, allowing cancer detection at doses where the monomers are less effective. While the dye dimers highlighted peritumoral blood vessels, the PA signal for dimeric tumor-targeting dye-peptide conjugate, LS301, was diffuse throughout the entire tumor mass. A combination of the ease of synthesis, diversity of molecules that are amenable to Cu(II)-catalyzed dimerization, and the high acoustic wave amplification by these stable dimeric small molecules ushers a new strategy to develop clinically translatable PA molecular amplifiers for the emerging field of molecular photoacoustic imaging.

Synthetic Mimics of Native Siderophores Disrupt Iron Trafficking in Acinetobacter baumannii.

Many pathogenic bacteria biosynthesize and excrete small molecule metallophores, known as siderophores, that are used to extract ferric iron from host sources to satisfy nutritional need. Native siderophores are often structurally complex multidentate chelators that selectively form high-affinity octahedral ferric iron complexes with defined chirality recognizable by cognate protein receptors displayed on the bacterial cell surface. Simplified achiral analogues can serve as synthetically tractable siderophore mimics with potential utility as chemical probes and therapeutic agents to better understand and treat bacterial infections, respectively. Here, we demonstrate that synthetic spermidine-derived mixed ligand bis-catecholate monohydroxamate siderophores (compounds 1-3) are versatile structural and biomimetic analogues of two native siderophores, acinetobactin and fimsbactin, produced by Acinetobacter baumannii, a multidrug-resistant Gram-negative human pathogen. The metal-free and ferric iron complexes of the synthetic siderophores are growth-promoting agents of A. baumannii, while the Ga(III)-complexes are potent growth inhibitors of A. baumannii with MIC values <1 µM. The synthetic siderophores compete with native siderophores for uptake in A. baumannii and maintain comparable apparent binding affinities for ferric iron (KFe) and the siderophore-binding protein BauB (Kd). Our findings provide new insight to guide the structural fine-tuning of these compounds as siderophore-based therapeutics targeting pathogenic strains of A. baumannii.

Protonation of Curcumin Triggers Sequential Double Cyclization in the Gas-Phase: An Electrospray Mass Spectrometry and DFT Study.

ESI-protonated natural curcumin (1) undergoes gas-phase cyclization and dissociates via competitive expulsions of 2-methoxy phenol and C4H4O2 (diketene or an isomer). Evidence from mechanistic mass spectrometry and from Density Functional Theory (DFT) reveals that a two-step sequential cyclization occurs for the protonated molecule prior to the unusual loss of the elements of 2-methoxy phenol. Furthermore, the presence of the methoxy group at postion-3 is essential for the second cyclization. The transformation of curcumin upon protonation in the gas phase may be predictive of its solution chemistry and explain how curcumin plays a protective role in biology.

Fimsbactin and Acinetobactin Compete for the Periplasmic Siderophore Binding Protein BauB in Pathogenic Acinetobacter baumannii.

Environmental and pathogenic microbes produce siderophores as small iron-binding molecules to scavenge iron from natural environments. It is common for microbes to produce multiple siderophores to gain a competitive edge in mixed microbial environments. Strains of human pathogenic Acinetobacter baumannii produce up to three siderophores: acinetobactin, baumannoferrin, and fimsbactin. Production of acinetobactin and baumannoferrin is highly conserved among clinical isolates while fimsbactin production appears to be less common. Fimsbactin is structurally related to acinetobactin through the presence of catecholate and phenolate oxazoline metal-binding motifs, and both are derived from nonribosomal peptide assembly lines with similar catalytic domain orientations and identities. Here we report on the chemical, biochemical, and microbiological investigation of fimsbactin and acinetobactin alone and in combination. We show that fimsbactin forms a 1:1 complex with iron(III) that is thermodynamically more stable than the 2:1 acinetobactin ferric complex. Alone, both acinetobactin and fimsbactin stimulate A. baumannii growth, but in combination the two siderophores appear to compete and collectively inhibit bacterial growth. We show that fimsbactin directly competes with acinetobactin for binding the periplasmic siderophore-binding protein BauB suggesting a possible biochemical mechanism for the phenomenon where the buildup of apo-siderophores in the periplasm leads to iron starvation. We propose an updated model for siderophore utilization and competition in A. baumannii that frames the molecular, biochemical, and cellular interplay of multiple iron acquisition systems in a multidrug resistant Gram-negative human pathogen.

Crystal Structure of the Siderophore Binding Protein BauB Bound to an Unusual 2:1 Complex Between Acinetobactin and Ferric Iron.

The critical role that iron plays in many biochemical processes has led to an elaborate battle between bacterial pathogens and their hosts to acquire and withhold this critical nutrient. Exploitation of iron nutritional immunity is being increasingly appreciated as a potential antivirulence therapeutic strategy, especially against problematic multidrug resistant Gram-negative pathogens such as Acinetobacter baumannii. To facilitate iron uptake and promote growth, A. baumannii produces a nonribosomally synthesized peptide siderophore called acinetobactin. Acinetobactin is unusual in that it is first biosynthesized in an oxazoline form called preacinetobactin that spontaneously isomerizes to the final isoxazolidinone acinetobactin. Interestingly, both isomers can bind iron and both support growth of A. baumannii. To address how the two isomers chelate their ferric cargo and how the complexes are used by A. baumannii, structural studies were carried out with the ferric acinetobactin complex and its periplasmic siderophore binding protein BauB. Herein, we present the crystal structure of BauB bound to a bis-tridentate (Fe3+L2) siderophore complex. Additionally, we present binding studies that show multiple variants of acinetobactin bind BauB with no apparent change in affinity. These results are consistent with the structural model that depicts few direct polar interactions between BauB and the acinetobactin backbone. This structural and functional characterization of acinetobactin and its requisite binding protein BauB provides insight that could be exploited to target this critical iron acquisition system and provide a novel approach to treat infections caused by this important multidrug resistant pathogen.

Enterobacteria secrete an inhibitor of Pseudomonas virulence during clinical bacteriuria.

Escherichia coli and other Enterobacteriaceae are among the most common pathogens of the human urinary tract. Among the genetic gains of function associated with urinary E. coli isolates is the Yersinia high pathogenicity island (HPI), which directs the biosynthesis of yersiniabactin (Ybt), a virulence-associated metallophore. Using a metabolomics approach, we found that E. coli and other Enterobacteriaceae expressing the Yersinia HPI also secrete escherichelin, a second metallophore whose chemical structure matches a known synthetic inhibitor of the virulence-associated pyochelin siderophore system in Pseudomonas aeruginosa. We detected escherichelin during clinical E. coli urinary tract infection (UTI) and experimental human colonization with a commensal, potentially probiotic E. coli bacteriuria strain. Escherichelin production by colonizing enterobacteria may help human hosts resist opportunistic infections by Pseudomonas and other pyochelin-expressing bacteria. This siderophore-based mechanism of microbial antagonism may be one of many elements contributing to the protective effects of the human microbiome. Future UTI-preventive probiotic strains may benefit by retaining the escherichelin biosynthetic capacity of the Yersinia HPI while eliminating the Ybt biosynthetic capacity.

Peptide-Level Interactions between Proteins and Small-Molecule Drug Candidates by Two Hydrogen-Deuterium Exchange MS-Based Methods: The Example of Apolipoprotein E3.

We describe a platform utilizing two methods based on hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 µM for the three different regions, giving an overall binding affinity of 7.4 µM. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides site-specific binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, ΔC1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein-ligand interactions.

Protonated N-Alkyl-2-nitroanilines Undergo Intramolecular Oxidation of the Alkyl Chain upon Collisional Activation.

The collisional activation of protonated N-propyl-2-nitroaniline obtained by electrospray ionization shows two major competitive dissociation pathways: the elimination of the elements of propionic acid, [M + H - C3H6O2]+ to give an m/z 107 ion, and of the elements of ethanol, [M + H - C2H6O]+ to give an m/z 135 ion. The mechanistic study reported here addresses these unusual fragmentations to reveal that both occur via a common intermediate formed by the transfer of an oxygen atom from the nitro group to the first carbon atom of the propyl group, allowing elimination of propionic acid and (H2O + ethene), respectively. The corresponding loss of CH4O does not occur when the propyl group is replaced by an ethyl group, but elimination of the elements of propanol does occur when propyl is replaced by a butyl group. Further, the product ions of m/z 107 and 135 are also formed when the propyl chain is replaced with a hexyl group.

Metal selectivity by the virulence-associated yersiniabactin metallophore system.

Uropathogenic Escherichia coli secrete siderophores during human infections. Although siderophores are classically defined by their ability to bind iron(III) ions, the virulence-associated siderophore yersiniabactin was recently found to bind divalent copper ions during urinary tract infections. Here we use a mass spectrometric approach to determine the extent of non-iron(III) metal interactions by yersiniabactin and its TonB-dependent outer membrane importer FyuA. In addition to copper, iron and gallium ions, yersiniabactin was also observed to form stable nickel, cobalt, and chromium ion complexes. In E. coli, copper(II) and all other non-iron(III) yersiniabactin complexes were imported by FyuA in a TonB-dependent manner. Among metal-yersiniabactin complexes, copper(II) yersiniabactin is predicted to be structurally distinctive and was the only complex not to competitively inhibit iron(III) yersiniabactin import. These results are consistent with yersiniabactin as part of a metallophore system able to prioritize iron(III) complex uptake in high copper environments.

Dosimetry determines the initial OH radical concentration in fast photochemical oxidation of proteins (FPOP).

Fast photochemical oxidation of proteins (FPOP) employs laser photolysis of hydrogen peroxide to give OH radicals that label amino acid side-chains of proteins on the microsecond time scale. A method for quantitation of hydroxyl radicals after laser photolysis is of importance to FPOP because it establishes a means to adjust the yield of •OH, offers the opportunity of tunable modifications, and provides a basis for kinetic measurements. The initial concentration of OH radicals has yet to be measured experimentally. We report here an approach using isotope dilution gas chromatography/mass spectrometry (GC/MS) to determine quantitatively the initial •OH concentration (we found ~0.95 mM from 15 mM H2O2) from laser photolysis and to investigate the quenching efficiencies for various •OH scavengers.

The Magic-Size Nanocluster (CdSe)34 as a Low-Temperature Nucleant for Cadmium Selenide Nanocrystals; Room-Temperature Growth of Crystalline Quantum Platelets.

Reaction of Cd(OAc)2·2H2O and selenourea in primary-amine/secondary-amine cosolvent mixtures affords crystalline CdSe quantum platelets at room temperature. Their crystallinity is established by X-ray diffraction analysis (XRD), high-resolution transmission electron microscopy (TEM), and their sharp extinction and photoluminescence spectra. Reaction monitoring establishes the magic-size nanocluster (CdSe)34 to be a key intermediate in the growth process, which converts to CdSe quantum platelets by first-order kinetics with no induction period. The results are interpreted to indicate that the critical crystal-nucleus size for CdSe under these conditions is in the range of (CdSe)34 to (CdSe)68. The nanocluster is obtained in isolated form as [(CdSe)34(n-octylamine)16(di-n-pentylamine)2], which is proposed to function as crystal nuclei that may be stored in a bottle.

The role of methoxy group in the Nazarov cyclization of 1,5-bis-(2-methoxyphenyl)-1,4-pentadien-3-one in the gas phase and condensed phase.

ESI-protonated 1,5-bis-(2-methoxyphenyl)-1,4-pentadien-3-one (1) undergoes a gas-phase Nazarov cyclization and dissociates via expulsions of ketene and anisole. The dissociations of the [M + D](+) ions are accompanied by limited HD scrambling that supports the proposed cyclization. Solution cyclization of 1 was effected to yield the cyclic ketone, 2,3-bis-(2-methoxyphenyl)-cyclopent-2-ene-1-one, (2) on a time scale that is significantly shorter than the time for cyclization of dibenzalacetone. The dissociation characteristics of the ESI-generated [M + H](+) ion of the synthetic cyclic ketone closely resemble those of 1, suggesting that gas-phase and solution cyclization products are the same. Additional mechanistic studies by density functional theory (DFT) methods of the gas-phase reaction reveals that the initial cyclization is followed by two sequential 1,2-aryl migrations that account for the observed structure of the cyclic product in the gas phase and solution. Furthermore, the DFT calculations show that the methoxy group serves as a catalyst for the proton migrations necessary for both cyclization and fragmentation after aryl migration. An isomer formed by moving the 2-methoxy to the 4-position requires relatively higher collision energy for the elimination of anisole, as is consistent with DFT calculations. Replacement of the 2-methoxy group with an OH shows that the cyclization followed by aryl migration and elimination of phenol occurs from the [M + H](+) ion at low energy similar to that for 1.

Cupric yersiniabactin is a virulence-associated superoxide dismutase mimic.

Many Gram-negative bacteria interact with extracellular metal ions by expressing one or more siderophore types. Among these, the virulence-associated siderophore yersiniabactin (Ybt) is an avid copper chelator, forming stable cupric (Cu(II)-Ybt) complexes that are detectable in infected patients. Here we show that Ybt-expressing E. coli are protected from intracellular killing within copper-replete phagocytic cells. This survival advantage is highly dependent upon the phagocyte respiratory burst, during which superoxide is generated by the NADPH oxidase complex. Chemical fractionation links this phenotype to a previously unappreciated superoxide dismutase (SOD)-like activity of Cu(II)-Ybt. Unlike previously described synthetic copper-salicylate (Cu(II)-SA) SOD mimics, the salicylate-based natural product Cu(II)-Ybt retains catalytic activity at physiologically plausible protein concentrations. These results reveal a new virulence-associated adaptation based upon spontaneous assembly of a non-protein catalyst.

New protein footprinting: fast photochemical iodination combined with top-down and bottom-up mass spectrometry.

We report a new approach for the fast photochemical oxidation of proteins (FPOP) whereby iodine species are used as the modifying reagent. We generate the radicals by photolysis of iodobenzoic acid at 248 nm; the putative iodine radical then rapidly modifies the target protein. This iodine-radical labeling is sensitive, tunable, and site-specific, modifying only histidine and tyrosine residues in contrast to OH radicals that modify 14 amino-acid side chains. We iodinated myoglobin (Mb) and apomyoglobin (aMb) in their native states and analyzed the outcome by both top-down and bottom-up proteomic strategies. Top-down sequencing selects a certain level (addition of one I, two I's) of modification and determines the major components produced in the modification reaction, whereas bottom-up reveals details for each modification site. Tyr146 is found to be modified for aMb but less so for Mb. His82, His93, and His97 are at least 10 times more modified for aMb than for Mb, in agreement with NMR studies. For carbonic anhydrase and its apo form, there are no significant differences of the modification extents, indicating their similarity in conformation and providing a control for this approach. For lispro insulin, insulin-EDTA, and insulin complexed with zinc, iodination yields are sensitive to differences in insulin oligomerization state. The iodine radical labeling is a promising addition to protein footprinting methods, offering higher specificity and lower reactivity than ∙OH and SO(4)(-∙), two other radicals already employed in FPOP.

Deprotonated N-(2,4-Dinitrophenyl)amino Acids Undergo Cyclization in Solution and the Gas Phase.

The collisionally activated mass spectral fragmentations of N-(2,4-dinitrophenyl)alanine and phenylalanine [M - H](-) may be gas-phase analogs of the base-catalyzed cyclization of N-(2,4-dinitrophenyl)amino acids in aqueous dioxane. This latter reaction is one source of the 2-substituted 5-nitro-1H-benzimidazole-3-oxides, which are antibacterial agents. The fragmentation of both compounds, established by tandem mass spectrometric experiments and supported by molecular modeling using DFT methods, indicate that the [M - H](-) ions dissociate via sequential eliminations of CO(2) and H(2)O to produce deprotonated benzimidazole-N-oxide derivatives. The gas-phase cyclization reactions are analogous to the base-catalyzed cyclization in solution, except that in the latter case, the reactant must be a dianion for the reaction to occur on a reasonable time scale. The cyclization of N-(2-nitrophenyl)phenylalanine, which has one less nitro group, requires a stronger base for the cyclization than the compound with a second nitro group at the 4-position. Following losses of CO(2) and H(2)O are expulsions of both neutral molecules and free radicals, the latter being examples of violations of the even-electron ion rule.

Structural determinant of chemical reactivity and potential health effects of quinones from natural products.

Although many phenols and catechols found as polyphenol natural products are antioxidants and have putative disease-preventive properties, others have deleterious health effects. One possible route to toxicity is the bioactivation of the phenolic function to quinones that are electrophilic, redox-agents capable of modifying DNA and proteins. The structure-property relationships of biologically important quinones and their precursors may help understand the balance between their health benefits and risks. We describe a mass-spectrometry-based study of four quinones produced by oxidizing flavanones and flavones. Those with a C2-C3 double bond on ring C of the flavonoid stabilize by delocalization of an incipient positive charge from protonation and render the protonated quinone particularly susceptible to nucleophilic attack. We hypothesize that the absence of this double bond is one specific structural determinant that is responsible for the ability of quinones to modify biological macromolecules. Those quinones containing a C2-C3 single bond have relatively higher aqueous stability and longer half-lives than those with a double bond at the same position; the latter have short half-lives at or below ∼1 s. Quinones with a C2-C3 double bond show little ability to depurinate DNA because they are rapidly hydrated to unreactive species. Molecular-orbital calculations support that quinone hydration by a highly structure-dependent mechanism accounts for their chemical properties. The evidence taken together support a hypothesis that those flavonoids and related natural products that undergo oxidation to quinones and are then rapidly hydrated are unlikely to damage important biological macromolecules.

Gas-phase nazarov cyclization of protonated 2-methoxy and 2-hydroxychalcone: an example of intramolecular proton-transport catalysis.

Upon CA, ESI generated [M + H](+) ions of chalcone (benzalacetophenone) and 3-phenyl-indanone both undergo losses of H(2)O, CO, and the elements of benzene. CA of the [M + H](+) ions of 2-methoxy and 2-hydroxychalcone, however, prompts instead a dominant loss of ketene. In addition, CA of the [M + H](+) ions of 2-methoxy-beta-methylchalcone produces an analogous loss of methylketene instead. Furthermore, the [M + D](+) ion of 2-methoxychalcone upon CA eliminates only unlabeled ketene, and the resultant product, the [M + D - ketene](+) ion, yields only the benzyl-d(1) cation upon CA. We propose that the 2-methoxy and 2-hydroxy (ortho) substituents facilitate a Nazarov cyclization to the corresponding protonated 3-aryl-indanones by mediating a critical proton transfer. The resultant protonated indanones then undergo a second proton transport catalysis facilitated by the same ortho substituents producing intermediates that eliminate ketene to yield 2-methoxy- or 2-hydroxyphenyl-phenyl-methylcarbocations, respectively. The basicity of the ortho substituent is important; for example, replacement of the ortho function with a chloro substituent does not provide an efficient catalyst for the proton transports. The Nazarov cyclization must compete with an alternate cyclization, driven by the protonated carbonyl group of the chalcone that results in losses of H(2)O and CO. The assisted proton transfer mediated by the ortho substituent shifts the competition in favor of the Nazarov cyclization. The proposed mechanisms for cyclization and fragmentation are supported by high-mass resolving power data, tandem mass spectra, deuterium labeling, and molecular orbital calculations.

2-Nitrophenyl Aryl Sulfides Undergo Both Intramolecular and Electrospray-Induced Intermolecular Oxidation of Sulfur: An Experimental and Theoretical Case Study.

Aromatic sulfides bearing a nitro group undergo sulfur oxidation upon electrospray ionization in the positive-ion mode. For example, 2-nitrophenyl phenyl sulfide, its para nitro isomer, and its chloro and methyl substituted analogs pick up an oxygen atom to afford [M + H + O](+) and [M + Na + O](+) ions upon ESI. Elemental-composition determination and tandem mass spectrometry confirm the reactions. Another oxidation of the sulfur, by the ortho nitro group of the [M + H](+) ions, occurs as intramolecular oxygen-transfer processes, evidenced by characteristic losses of SO, SO(2) and SO(2)H(*), the latter yielding the carbazole radical cation, and the generation of the aryl-SO(+) product ion. The intramolecular oxidation via oxygen transfer from the nitro group to the sulfur was corroborated by molecular modeling. The results substantiate both inter- and intramolecular oxidation and provide more evidence that care must be taken when analyzing not only methionine-containing peptides but also small sulfides.

Radical Cation/Radical Reactions: A Fourier Transform Ion Cyclotron Resonance Study of Allyl Radical Reacting with Aromatic Radical Cations.

A method for the study of reactions of open-shell neutrals (radicals) and radical cations is described. Pyrolysis (25-1500 degrees C) of thermally labile compounds, such as, 1,5-hexadiene via a Chen nozzle yields a seeded beam of reactive species in helium. The pyrolysis products are then analyzed by electron ionization (EI) or reacted with stored ions. Electron ionization of the pyrolysis products of 1,5-hexadiene shows that both the allyl radical and allene are generated. Reactions of benzene radical cations and the pyrolysis products of 1,5-hexadiene result in carbon-carbon bond formation. Those reactions of allyl radical with the benzene radical cation yield the C(7)H(7) (+) ion of m/z 91, permitting an unusual entry into arenium ions. The reaction of allene with benzene radical cation in contrast yields C(9)H(10) (+). and C(9)H(9) (+).

Protonated nitro group as a gas-phase electrophile: experimental and theoretical study of the cyclization of o-nitrodiphenyl ethers, amines, and sulfides.

A novel gas-phase electrophilic cyclization, initiated by the protonation of a nitro group, occurs for 2-nitrophenyl phenyl ether and for the analogous sulfide and amine, leading to heterocyclic intermediates in each case. Subsequently, the cyclic intermediates dissociate via two pathways: (1) unusual step-wise eliminations of two OH radicals to afford heterocyclic cations, [phenoxazine - H](+), [phenothiazine - H](+), and [phenazine + H](+), and (2) expulsion of H(2)O, to yield a heterocyclic ketone, followed by loss of CO. The proposed structures of the gas-phase product ions and reaction mechanisms are supported by chemical substitution, deuterium labeling, accurate mass measurements at high mass resolving power, product-ion mass spectra obtained by tandem mass spectrometry, mass spectra of reference compounds, and molecular orbital calculations. Using a mass spectrometer as a reaction vessel, we demonstrate that, upon protonation, a nitro group becomes an electrophile and participates in cyclization reactions in the gas phase.