In vivo processing of LVV-hemorphin-7 in rat brain and blood utilizing microdialysis combined with electrospray mass spectrometry

In vivo microdialysis in combination with liquid chromatography/electrospray time-of-flight mass spectrometry was used to study the processing of LVV-hemorphin-7, an endogenous decapeptide with opioid activity, in rat brain and blood. A microdialysis probe (flow rate 0.4 microL/min) was used to both introduce LVV-hemorphin-7 into the striatum of the brain (1.0 pmol/microL) or the venous blood (10 pmol/microL) and to collect the metabolic products. LVV-hemorphin-7 was extracellularly metabolized in the striatum to form C-terminal fragments 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, and N-terminal fragments 1-9, 1-8, 1-6. Infusion of the aminopeptidase inhibitor amastatin (1.0 pmol/microL) into the striatum, together with LVV-hemorphin-7, decreased the processing of LVV-hemorphin-7 to form C-terminal fragments 2-10, 3-10, 4-10, but increased the relative levels of fragment 5-10 and N-terminal fragments 1-9, 1-8 and 1-6. The major metabolic product from LVV-hemorphin-7 in the striatum was the C-terminal fragment 5-10, which may be processed by an endopeptidase not sensitive to amastatin. The LVV-hemorphin-7 infusion to the venous blood produced the C-terminal fragments 2-10, 3-10, 4-10, and 5-10, N-terminal fragment 1-9, and internal fragments 4-7 and 4-9. It is concluded that the combination of microdialysis and electrospray mass spectrometry provides a powerful tool for the study of extracellular metabolism and kinetic processes of complex reaction systems in vivo.     

Schiff base derivatives of peptide esters: Relative abundance of N-terminal,C-terminal and ‘internal’ fragments as a function of the blocking group

Electron-impact (EI) mass spectrometry of peptide derivatives is usually interpreted in terms of fragmentation where the charge resides on the N-terminal fragments and to a lesser degree on the less common, charged C-terminal fragments. Substituted and unsubstituted benzylidene, cinnamylidene, α- and β-naphthylidene derivatives of a reference tripeptide, valileala, gave both N- and C-terminal fragments as well as molecular ions. The order of increasing ion current (normalized) in C-terminal fragments was: acetylacetonyl, 4-dimethylaminonaphthylidene, p-dimethyl-aminobenzylidene, 3-pyridylmethylidene, p-diethylaminocinnamylidene, benzylidene, 2-hydroxy-naphthylidene, 4-pyridylmethylidene, p-nitrobenzylidene, p-methoxybenzylidene, p-cyanobenzylidene, cinnamylidene, p-dimethylaminocinnamylidene, β-indolylmethylidene, β-naphthylidene, 2-pyridylmethylidene and α-naphthylidene. The order for this value among the N-terminal fragments is significantly different, however (Day, Falter, Lehman and Hamilton, J. Org. Chem. in press). In addition to N- and C-terminal fragments, many spectra contain internal fragments, arising from loss of fragments from both ends, which provide sequence information. These fragments are found in the mass spectra of Schiff bases formed from various aromatic aldehydes with peptide esters. The interpretation of the latter pattern is facilitate in some cases by deuterium labeling at the α-carbon of the N-terminal amino acid residue of peptides. Such a pattern provides sequence information supplemental to that available involving N- and C-terminal fragmentations. In derivatives of hexaglycine, tetraphenylalanine and tryptophylmethionylaspartyl (β-OEt) phenylalanine amide, for example, substantial sequence information was contained in the internal fragments; in some cases the sequence could be deduced only if the internal fragments were utilized. The 4-dimethylamino-naphthylidene derivatives have proven to be the most useful to date in terms of volatility, tendency to maximize cleavage into N-terminal fragments, intensity of molecular ions and generation of useful mass spectra of certain peptide esters refractory to mass spectrometry in the form of any other derivative investigated.     

SCF Deformation densities and electrostatic potentials of purines and pyrimidines

4-31G wave functions have been computed for five purines and pyrimidines. The calculated deformation densities have been partitioned into atomic fragments, which were integrated to yield atomic multipole moments. The transferability of atomic fragments between related molecules was verified by constructing model maps for uracil and guanine from appropriate fragments of cytosine and adenine. Model electrostatic potentials calculated from the moments of model atoms are similar to the corresponding 4-31G potentials. Comparison of 4-31G and 4-31G** deformation densities of cytosine provides simple rules for estimating the effects of polarization functions on the atomic multipole moments of most atom types occurring in the purines and pyrimidines. These rules were applied to the other molecules and yielded reasonable approximations for their molecular dipole moments. Substituting CH₃ for H has little effect on the deformation density beyond the substitution center.     

Optimization and development of a high-performance liquid chromatography-based one-site immunometric assay with chemiluminescence detection

Various practical and theoretical considerations were examined in the creation and optimization of a high-performance liquid chromatography (HPLC)-based one-site immunometric assay. This method used an HPLC analyte analog column and post-column chemiluminescence detection. The specific analyte chosen as the model for this study was l-thyroxine (also known as T₄). In this technique, a sample containing thyroxine was first combined with an excess of anti-T₄ antibody Fab fragments that had earlier been conjugated with chemiluminescent acridinium ester labels. After incubation, the mixture was injected onto a column that contained immobilized T₄. The amount of thyroxine in the original sample was then determined by measuring the labeled Fab fragments that appeared in the non-retained fraction, or the decrease in excess Fab fragments that were bound to and later eluted from the column. Items considered in creating this assay included the preparation of acridinium ester-labeled Fab fragments, the detection of these fragments with a post-column reactor, and the creation of a suitable immobilized analog column for capturing excess labeled Fab fragments. The final method could measure T₄ in standards at clinically-relevant concentrations and provided a response within 1.5 min of sample injection, following a 20-45 min incubation with the labeled Fab fragments. Possible applications of this method include its use in clinical chemistry and the screening of proteomic or combinatorial libraries.     

Existence of an exceptional reaction pathway for H3(+) formation observed in collision-induced dissociation of methane ions at 1000 eV

Dissociation of CH(4)(+) ions at 1000 eV induced by collision with Ar atoms was investigated by measuring the kinetic energies of the ionized fragments. At small scattering angles, including zero, H(+), H(2)(+), H(3)(+), CH(3)(+), CH(2)(+), CH(+), and C(+) fragments were observed. The attractive part of the potential in the CH(4)(+)-Ar collision system played an important role in the formation of the ionized fragments. Rainbow scattering, leading to a large scattering cross section, was shown to be responsible for the increased formation of H(3)(+). It is proposed that on collision-induced dissociation of CH(4)(+), its three hydrogen atoms, which form a triangle, simultaneously react and move together to form H(3)(+).     

Influence of fluorophor dye labels on the migration behavior of polymerase chain reaction--amplified short tandem repeats during denaturing capillary electrophoresis.

The determination of the length of polymerase chain reaction (PCR)-amplified short tandem repeats (STRs) by denaturing capillary electrophoresis (CE) is a standard procedure for purposes of genotyping. We show that dye-specific mobility anomalies exist for 5'-fluorophor-labelled single-stranded DNA (ssDNA) fragments in CE using the performance-optimized polymer 4 (POP4) buffer sieving matrix, containing the entangled poly(N,N-dimethylacrylamide) polymer, urea, and 2-pyrrolidinone. The dye-specific retardation effects relative to coseparated GeneScan-500 [TAMRA] standard fragments can lead to wrong genotyping, even for allele-specific fragments of pentanucleotide STRs, when comparing the relative calculated sizes of identical fragments, labelled with rhodamine (ROX, TAMRA) or fluorescein dyes (FAM, 6-FAM, HEX, JOE, NED, TET): The size of fluorescein dye-labelled fragments of appr. 100 b in length appears to be smaller by up to 6.5 b. This effect becomes more dramatic with decreasing size: a 6-FAM-labelled 24-mer oligonucleotide appeared to be smaller by 11 b. In contrast, in classical urea/polyacrylamide slab-gel electrophoresis only a small dye-specific retardation of identical fragments is observed. The dye-specific effects are superimposed by weaker size and sequence-dependent anomalies of fragment mobility. Therefore, in denaturing CE the coseparation of a defined allele ladder labelled with the same dye as the unknown sample fragments remains the method of choice for accurate genotyping.     

Screening of mixed poly(ethylene oxide) solutions for microchip separation of double-stranded DNA using an orthogonal design approach

We describe a quick and systematic optimization of molecular weights (MWs) and concentrations of a series of mixed poly(ethylene oxide) (PEO) matrices for separating specific double-stranded DNA fragments on polymethylmethacrylate-based microchips by using an orthogonal design (ORD) approach. The mixed matrices are composed of PEOs in four MW ranges (M(w) 8 x 10(6), 1 x 10(6), 4 x 10(5), and 1 x 10(5)) with varying concentration ratios. In the mixed solutions, PEO with an intermediate MW of 4 x 10(5) is found to be a dominant factor for separating small DNA fragment pairs (e.g., 82 and 88 bp), while PEO with a high MW of 8 x 10(6) plays an important role in separating intermediate and large fragments (e.g., 271 and 281 bp, 506 and 517 bp, 7 and 10 kbp). High-concentration PEO mixtures give better resolution for short fragments, while dilute PEO mixtures show better resolution for long fragments. The optimized matrices are suitable for high-resolution separation of multiplex polymerase chain reaction-amplified products and restriction digest fragments ranging in size from 20 bp to 40 kbp within 4 min at a constant field strength of 177 V/cm. The experimental results indicate the robustness and speediness of the ORD to screen the contribution of PEO MWs and to tune optimally the PEO concentration ratio of different MWs with reference to the performance of specific DNA fragments separated.     

Charge distribution between formally identical fragments: The retro Diels-Alder cleavage

The electron impact induced retro Diels-Alder cleavage of 4-vinylcyclohexene, 1,4-dimethyl-4-vinylcyclo-hexene, limonene, dicyclopentadiene and dicyclohexadiene has been investigated using deuterium labelled analogues. With limonene, dicylopentadiene and dicyclohexadiene the charged diene fragments are produced with the same probability from both subunits of the molecular ion. With 4-vinylcyclohexene and 1,4-dimethyl-4-vinylcyclohexene, diene fragments containing the former vinyl group are charged preferentially. This small asymmetry of charge distribution was found to be independent of the ionizing energy. A new, qualitative rationalization is suggested to account for the unsymmetrical charge distribution in 4-vinylcyclohexene and 1,4-dimethyl-4-vinylcyclohexene. Metastable spectra and hydrogen scrambling are discussed.     

Dissociative excitation of SiH4, SiD4, Si2H6 and GeH4 by 0–100 eV electron impact

We have measured emission cross sections of various electronically excited fragments produced by electron-impact dissociation of SiH₄, SiD₄, Si₂H₆ and GeH₄. At low impact energy (10–20 eV), the measured appearance potentials are correlated to specific dissociation processes. Below 22 eV superexcited states of SiH₄ play a dominant role in the formation of neutral excited fragments. In agreement with the results obtained on alkanes, the cross sections for fragment emission from Si₂H₆ are lower than those for SiH₄. On the other hand, the comparison of cross sections at 100 eV for fragment emission, dissociation and ionization on going from CH₄ to SiH₄ and GeH₄ shows an increase of the probability for production of neutral ground-state fragments at the cost of excited or ionic fragments. Both effects can be explained by a growing probability for internal conversion among the decay channels of superexcited states with increasing number of atoms or electrons in the parent molecule. For each molecule, the H Balmer-emission cross sections at 100 eV are proportional to n^−b, where n 3 is the principal quantum number of the upper state of H and 3 < b < 5 is a parameter characteristic of the parent molecule. Finally, a detailed analysis of the isotopic effect between SiH₄ and SiD₄ on both fragment emission and ionization cross sections from 0 to 100 eV gives strong evidence of the competition between dissociation and autoionization in the decay of superexcited states.     

Activation energy of single-stranded DNA moving through cross-linked polyacrylamide gels at 300 V/cm effect of temperature on sequencing rate in high-electric-field capillary gel electrophoresis

In DNA sequencing, single-stranded DNA fragments are separated by gel electrophoresis. This separation is based on a sieving mechanism where DNA fragments are retarded as they pass through pores in the gel. In this paper, we present the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T LongRanger gels at an electric field of 300 V/cm. The temperature dependence is compared with the predictions of the biased reptation model. The model predicts that the fragment length for the onset of biased reptation with stretching increases with the square of temperature; the data show that the onset of biased reptation with stretching decreases with temperature. Biased reptation fails to model accurately the temperature dependence of mobility. We analyzed the data and extracted the activation energy for passage of sequencing fragments through the gel. For fragments containing less than ca. 200 bases, the activation energy increases linearly with the number of bases at a rate of 25 J/mol per base; for longer fragments, the activation energy increases at a rate of 6.5 J/mol per base. This transition in the activation energy presumably reflects a change in conformation of the DNA fragments; small fragments exist in a random coil configuration and larger fragments migrate in an elongated configuration.     

Thermal decomposition of generation-4 polyamidoamine dendrimer films: decomposition catalyzed by dendrimer-encapsulated Pt particles

The thermal decomposition of hydroxyl-terminated generation-4 polyamidoamine dendrimer (G4OH) films deposited on Au surfaces has been compared with decomposition of the same dendrimer encapsulating an approximately 40-atom Pt particle (Pt-G4OH). Infrared absorption reflection spectroscopy studies showed that, when the films were heated in air to various temperatures up to 275 degrees C, the disappearance of the amide vibrational modes occurred at lower temperature for the Pt-G4OH film. Dendrimer decomposition was also investigated by thermogravimetric analysis (TGA) in both air and argon atmospheres. For the G4OH dendrimer, complete decomposition was achieved in air at 500 degrees C, while decomposition of the Pt-G4OH dendrimer was completed at 400 degrees C, leaving only platinum metal behind. In a nonoxidizing argon atmosphere, a greater fraction of the G4OH decomposed below 300 degrees C, but all of the dendrimer fragments were not removed until heating above 550 degrees C. In contrast, Pt-G4OH decomposition in argon was similar to that in air, except that decomposition occurred at temperatures approximately 15 degrees C higher. Thermal decomposition of the dendrimer films on Au surfaces was also studied by temperature programmed desorption (TPD) and X-ray photoelectron spectroscopy (XPS) under ultrahigh vacuum conditions. Heating the G4OH films to 250 degrees C during the TPD experiment induced the desorption of large dendrimer fragments at 55, 72, 84, 97, 127, 146, and 261 amu. For the Pt-G4OH films, mass fragments above 98 amu were not observed at any temperature, but much greater intensities for H(2) desorption were detected compared to that of the G4OH film. XPS studies of the G4OH films demonstrated that significant bond breaking in the dendrimer did not occur until temperatures above 250 degrees C and heating to 450 degrees C caused dissociation of C=O, C-O, and C-N bonds. For the Pt-G4OH dendrimer films, carbon-oxygen and carbon-nitrogen bond scission was observed at room temperature, and further decomposition to atomic species occurred after heating to 450 degrees C. All of these results are consistent with the fact that the Pt particles inside the G4OH dendrimer catalyze thermal decomposition, allowing dendrimer decomposition to occur at lower temperatures. However, the Pt particles also catalyze bond scission within the dendrimer fragments so that decomposition of the dendrimer to gaseous hydrogen is the dominant reaction pathway compared to desorption of the larger dendrimer fragments observed in the absence of Pt particles.     

Catalytic isomerization of terminal olefins in liquid-crystalline polyesters at hydrosilylation with 1-(1′-arylethoxy)-1,1,3,3-tetramethyldisiloxanes

Liquid-crystalline compounds based on terephthaloyl-bis(4-oxybenzoates) with terminal double carbon-carbon bonds in aliphatic fragments were prepared. The introduction of polar and easily polarizable groups into the aliphatic fragments was shown to reduce the mesomorphic capacity. Hydrosilylation of the polyesters catalyzed by rhodium and platinum complexes was studied. The isomerization of the olefin fragment was demonstrated to be the main process.     

A single-strand conformation polymorphism method for the large-scale analysis of mutations/polymorphisms using capillary array electrophoresis

We present a high-throughput single-strand conformation polymorphism (SSCP) method, performed on a commercially available capillary array DNA sequencer. We tested various sieving matrices and electrophoretic conditions, using 51 DNA fragments which included 45 fragments carrying only one single nucleotide polymorphism (SNP), 4 fragments having two SNPs and 2 fragments with insertion or deletion. Resolution of alleles was improved by increasing concentrations of both sieving matrices and buffers, and all examined polymorphisms of DNA fragments were detected, most of them (45 fragments) as clearly split allele peaks in heterozygotes. Allele frequencies of SNPs can be estimated accurately by determining the relative amounts of alleles in pooled DNA. In this method, the turn-around time for the analysis of 96 samples is less than 3 h. These results demonstrate that capillary array-based SSCP is an efficient and accurate technique for the large-scale quantitative analysis of mutations/polymorphisms.     

Cation- and anion-assisted binding of triethylenediamine by zinc bisporphyrinates

The complexation of zinc calix[4]arene or calix[4]pyrrole bisporphyrinates with alkali metal cations, halide anions, and triethylenediamine was studied by ¹H NMR spectroscopy. It was established that the binding of molecules and/or charged particles by various fragments of calix[4]arene and calix[4]pyrrole porphyrins are interrelated processes. This makes it possible to use one process (for example, complexation of the calix[4]arene fragment of the macrocycle with alkali metal cations or complexation of the calix[4]pyrrole fragment with halide ions) as a tool for controlling another process (complexation of the porphyrin fragments of the macrocycle with neutral molecules).     

Automated site-directed drug design: The generation of a basic set of fragments to be used for automated structure assembly

Summary If a method is to be developed to assemble putative ligands structures in site-directed drug design, from molecular graphs generated in the site, then basic building blocks are needed. Structure assembly is a combinatoric process that needs to be optimised if it is to be tractable. What has to be determined is whether small molecular fragments can have transferable properties from one molecule to another. In this paper we determine all possible combinations of 3-, 4- and 5-atom aliphatic fragments from a small set of atoms H, C, N, O, F or Cl. The frequency of occurrence of these candidate fragments is searched for in the Cambridge Structural Database. A similar analysis is performed on charged fragments. A more restricted search is carried out for P and S and aromatic structures. A basic set of fragments can be derived that have a significant frequency in known crystal structures. The transferability of fragment properties is discussed in subsequent papers.     

Exchange coupling through diamagnetic [Fe(CO)4]2- bridging ligands in a xenophilic cluster

The electronic structure of so-called 'xenophilic' clusters, which contain both organometallic fragments and Werner-type paramagnetic transition metal centres, presents a challenge to simple theories of bonding. Density functional theory shows clearly that the cluster Mn(2)(thf)(4)(Fe(CO)(4))(2) is best described as an exchange-coupled Mn(II)(2) dimer, the closed-shell organometallic [Fe(CO)(4)](2-) fragments acting simply as bridging ligands. The high-spin configuration of the Mn(II) ions leads to single occupation of the Mn-Fe σ* orbitals and therefore substantially weaker metal-metal bonding than in conventional low-valent organometallic clusters. The transition metal fragments are effective mediators of superexchange (J(calc) = -44 cm(-1)), leading to the measured effective magnetic moment of ~5 μ(B) at 300 K, considerably lower than the limiting value of 8.37 μ(B) for two uncoupled S = 5/2 Mn(II) centres.     

Synthesis and Properties of Cobalt(II) Tetrakis-[4,5-di(4′-carboxy)phenylene(oxy/sulfanyl/amino)]phthalocyanines

Russian Journal of General Chemistry - Phthalonitriles with fragments of 4-hydroxy, 4-mercapto and 4-aminobenzoic acids were synthesized by nucleophilic substitution in...     

A Formylglycine-Peptide for the Site-Directed Identification of Phosphotyrosine-Mimetic Fragments**

Discovery of protein-binding fragments for precisely defined binding sites is an unmet challenge to date. Herein, formylglycine is investigated as a molecular probe for the sensitive detection of fragments binding to a spatially defined protein site . Formylglycine peptide 3 was derived from a phosphotyrosine-containing peptide substrate of protein tyrosine phosphatase PTP1B by replacing the phosphorylated amino acid with the reactive electrophile. Fragment ligation with formylglycine occurred in situ in aqueous physiological buffer. Structures and kinetics were validated by NMR spectroscopy. Screening and hit validation revealed fluorinated and non-fluorinated hit fragments being able to replace the native phosphotyrosine residue. The formylglycine probe identified low-affinity fragments with high spatial resolution as substantiated by molecular modelling. The best fragment hit, 4-amino-phenyl-acetic acid, was converted into a cellularly active, nanomolar inhibitor of the protein tyrosine phosphatase SHP2.     

Electron-impact mass spectra of natural phenylethanoids

The electron-impact mass spectra of a number of phenylpropanoids and iridoid glycosides containing a phenylethanoid fragment in their structure have been studied. This has revealed features of the formation of fragments of 4-hydroxyphenylethyl and 3,4-dihydroxyphenylethyl alcohols and also of fragments of phenylethylamine and indolylethylamine derivatives present in the structures of the compounds investigated.     

Calix[4]arene methylenebisphosphonic acids as calf intestine alkaline phosphatase inhibitors

Calix[4]arenes bearing one or two methylenebisphosphonic acid fragments were prepared via addition of diethylphosphite to the parent calix[4]arene aldehydes. The resulting compounds displayed stronger inhibition of calf intestine alkaline phosphatase than simple methylenebisphosphonic or 4-hydroxyphenyl methylenebisphosphonic acids. The action of these phosphorylated calix[4]arenes is concordant with partial mixed-type inhibition. The inhibition constants Ki and Ki' for the calix[4]arene bis(methylenebisphosphonic) acid in Tris-HCl buffer at pH 9 are 0.38 microM and 2.8 microM respectively. The replacement of the phosphoric acid moieties on the macrocycle with diethylphosphonates results in a sharp decrease of its inhibitory action. Preorganizing phosphonic acid fragments using a calixarene platform therefore provides a promising approach for the design of efficient alkaline phosphatase inhibitors.