Peptide cysteine thiyl radicals abstract hydrogen atoms from surrounding amino acids: the photolysis of a cystine containing model peptide

Peptide cysteine thiyl radicals were generated through UV-photolysis of disulfide precursors, in order to follow intramolecular reactions of those radicals with neighboring amino acids. When reactions were carried out in D(2)O, there was a significant incorporation of deuterium specifically into the C(alpha)-H bonds of glycine residues in positions i+1 and i-1 to the Cys residue, indicating a fast reversible H-atom transfer. This H-atom transfer occurred prior to the formation of final, nonradical products including free thiol, thioaldehyde, and aldehyde. Such fast H-atom transfer is relevant to biologic conditions of oxidative stress and to the stabilization of proteins against oxidation, where the formation of carbon-centered radicals in proteins may lead to fragmentation, intramolecular cross-linking, aggregation and/or epimerization.     

Cyclisation of dienes using phosphorus-centred radicals to form organophosphorus adducts

Reaction of various dienes with phosphites or related phosphorus hydrides, under free radical cyclisation conditions, affords cyclic organophosphorus adducts. This quick, mild and technically clean approach affords 5- and 6-ring carbocycles and heterocycles in good to excellent yield.     

Solid-phase guanidinylation as a diversification strategy of poly-L-proline type II peptide mimic scaffolds

[reaction: see text] Solid-phase guanidinylation of proline-templated amino acids is studied as a diversification strategy of poly-L-proline type II scaffolds.     

Surface derivatization strategy for combinatorial analysis of cell response to mixtures of protein domains

We report a robust strategy for conjugating mixtures of two or more protein domains to nonfouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface-bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. A hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein domains using chloroformate-derived cross-linking. The effectiveness of this synthesis strategy was gauged by using an ELISA to measure competitive, covalent bonding of cell-binding (III(9-10)) and fibronectin-binding (III(1-2)) domains of the cell adhesion protein fibronectin. Cell adhesion, spreading, and fibronectin matrix assembly were examined on surfaces conjugated with single domains, a 1:1 surface mixture of III(1-2) and III(9-10), and a recombinant protein "duplex" containing both domains in one fusion protein. The mixture performed as well as or better than the other surfaces in these assays. Our surface activation strategy is amenable to a wide range of polymer substrates and free amino group-containing protein fragments. As such, this technique may be used to create biologically specific materials through the immobilization of specific protein groups or mixtures thereof on a substrate surface.     

Two ion/ion charge inversion steps to form a doubly protonated peptide from a singly protonated peptide in the gas phase

An approach is described to increase the degree of protonation of a polypeptide ion in the gas phase. Sequential charge inversion reactions involving the reactions of oppositely charged ions are used to yield a net increase in ion charge. The approach is illustrated here with the conversion of singly protonated bradykinin to doubly protonated bradykinin. The first step involves conversion of the singly protonated peptide to the singly deprotonated peptide via reactions with multiply charged anions derived from carboxylate-terminated dendrimers. Some of the singly deprotonated peptide was then converted to doubly protonated peptide via reactions with multiply charged cations derived from amino-terminated dendrimers. The overall approach is illustrative of a general strategy for increasing the absolute charge states of large ions in the gas phase.     

A general strategy to determine a target DNA sequence of a short peptide: application to a d-peptide.

Short peptides could potentially provide a novel element to read-out DNA sequences from the major groove. However, it is difficult to determine sequence-preference of de novo designed monomeric short peptides. Because DNS-binding affinity and specificity of short peptides are usually much lower than those of native DNA-binding proteins, determining the sequence-preference of short peptides by conventional methods utilized to deduce the target sequence of proteins often produces an unclear outcome. We report here a general strategy to defining the sequence-preference of a DNA-binding short peptide by using the heterodimers. A GCN4 basic region peptide tethers a low-affinity DNA-binding peptide adjacent to a GCN4 binding sequence through the cyclodextrin-adamantane association, thereby increasing local concentration of the low-affinity peptide on degenerated DNA sequences. An increase of the local concentration allows one to select a preferential sequence for the low-affinity DNA binding peptide. The method successfully identified specific sequences of short peptides derived from native DNA-binding proteins. The usefulness of this approach has been demonstrated by identifying preferred DNA targets for a peptide composed only of d-amino acids. The method is potentially applicable not only to artificial peptides, but also to other synthethic ligands.     

2,3-Diphenylquinolizinium bromide as a fluorescent derivatization reagent for amines

2,3-Diphenylquinolizinium bromide (2,3-DPQ) is proposed as a fluorogenic reagent for amino compounds. A spectrofluorimetric method based on its use is described which allows the determination of μg ml^?1 to ng ml^?1 levels of primary and secondary amines, including aromatic and cyclic compounds. The precision of the method was 4–8% (relative standard deviation) (n=10). The influence of several external factors on the derivatization reaction was studied using piperidine as a model compound. The analytical reaction can be effected at room temperature, which avoids the degradation of labile sample amines, e.g., catecholamines, and simplifies the experimental procedure. The presence of an excess of a basic catalyst (triethylamine) was critical for the reaction to develop satisfactorily. Fluorescence due to the reaction product was detected 5 min after the start of the analytical reaction.     

N-Protonated Isomers as Gateways to Peptide Ion Fragmentation

According to the popular “mobile proton model” for peptide ion fragmentation in tandem mass spectrometry, peptide bond cleavage is typically preceded by intramolecular proton transfer from basic sites to an amide nitrogen in the backbone. If the intrinsic barrier to dissociation is the same for all backbone sites, the fragmentation propensity at each amide bond should reflect the stability of the corresponding N-protonated isomer. This hypothesis was tested by using ab initio and force-field computations on several polyalanines and Leu-enkephalin. The results agree acceptably with experimental reports, supporting the hypothesis. It was found that backbone N-protonation is most favorable near the C-terminus. The preference for C-terminal N-protonation, which is stronger for longer polyalanines, may be understood in terms of the well known “helix macrodipole” in the corresponding helical conformations. The opposite stability trend is found for peptides constrained to be linear, which is initially surprising but turns out to be consistent with the reversed direction of the macrodipole in the linear conformation.     

Versatile strategy for oligonucleotide derivatization. Introduction of lanthanide(III) chelates to oligonucleotides

[reaction: see text] Novel nucleosidic phosphoramidite blocks were synthesized by a Mitsunobu reaction between 2'-deoxy-5'-O-(4,4'-dimethoxytrityl)uridine and a primary alcohol containing a conjugate group in its structure (a protected functional group, an organic dye, or a precursor of a lanthanide(III) chelate) followed by phosphitylation. They were used in machine-assisted DNA synthesis in the standard manner. A slightly modified deprotection procedure was used for the preparation of oligonucleotide conjugates tethered to lanthanide(III) chelates. For the latter application one non-nucleosidic block was also synthesized.     

On the question of reversibility in the intramolecular addition of radicals to aldehydes to form cycloalkanols

Experiments with two pairs of epimeric alcohols establish conclusively that the intramolecular addition of a primary radical to an aldehyde to give a cycloalkanoxyl radical is not a reversible process.     

Tandem addition-cyclization mediated by sulfanyl radicals: a versatile strategy for iridoids synthesis

Sulfanyl radicals trigger a tandem addition-cyclization protocol in linalool or citronelene derivatives for the efficient construction of the iridane monoterpene skeleton. Best results in yields and diastereoselectivity were obtained when phenylethylsulfanyl was used as radical initiator. We have proved the utility of this protocol with the enantiospecific synthesis of natural iridane dehydroiridomyrmecin starting from a (−)-linalyl acetate ester derivative in five steps with a 28% overall yield.     

Pyrene as a fluorescent probe for DNA base radicals

The steady-state emission spectra of 5-(1-pyrenyl)-modified pyrimidine and 8-(1-pyrenyl)-modified purine nucleosides in water at different pH values provide important information about the acidity or basicity of photochemically generated DNA base radicals which are key intermediates in DNA-mediated charge transport processes.     

New strategy to identify radicals in a time evolving EPR data set by multivariate curve resolution-alternating least squares

Electron paramagnetic resonance (EPR) spectroscopy is a powerful technique that is able to characterize radicals formed in kinetic reactions. However, spectral characterization of individual chemical species is often limited or even unmanageable due to the severe kinetic and spectral overlap among species in kinetic processes. Therefore, we applied, for the first time, multivariate curve resolution-alternating least squares (MCR-ALS) method to EPR time evolving data sets to model and characterize the different constituents in a kinetic reaction. Here we demonstrate the advantage of multivariate analysis in the investigation of radicals formed along the kinetic process of hydroxycoumarin in alkaline medium. Multiset analysis of several EPR-monitored kinetic experiments performed in different conditions revealed the individual paramagnetic centres as well as their kinetic profiles. The results obtained by MCR-ALS method demonstrate its prominent potential in analysis of EPR time evolved spectra.     

Hydrophobic nitroxide radicals as probes to investigate the hydrophobic interaction

The EPR spectrum of 2,2,6,6-tetramethylpiperidine-N-oxide (TEMPO) has been systematically investigated in several solvents and in aqueous solutions of glycerol,t-BuOH, Bu₄NBr,n-HexNH₃Br,n-OctNH₃Br, NaBPh₄, and Ph₄AsCl. Most of the results have been obtained at 25°C, though the temperature dependence of the linewidths has been examined in water and several organic solvents. Data on the spectrum of ditertiarybutylnitroxide in aqueous solutions of glycerol and Bu₄NBr are also presented. The spectrum was simulated to determine W_H, the linewidth after allowance for the unresolved proton hyperfine interaction, in each manifold of the triplet due to the nitrogen hyperfine interaction. The linewidth W_H is analyzed in terms of the reorientational correlation time Äθ and the angular velocity correlation time ÄJ. In most solutions Äθ is determined only by the bulk solution viscosity, except in situations where significant clustering of hydrophobic solute molecules occurs. In pure nonaqueous solvents the temperature dependence results are consistent with ÄJ being determined by the bulk viscosity, while in water or aqueous solutions, a very different behavior is found which is interpreted as a manifestation of clathrate-like hydration of the hydrophobic radical. This interpretation is incorporated in a two-state model developed to account for the W_H data of TEMPO in aqueous solutions of various hydrophobic solutes. The equilibrium parameters derived from the model, for the association of TEMPO with several hydrophobic solutes, support the concept of hydrophobic interactions as pictured from thermodynamic excess functions.     

Photolithographic synthesis of cyclic peptide arrays using a differential deprotection strategy

We report here a strategy for the photolithographic synthesis of diverse, spatially addressable arrays of cyclic peptides which employs a differential deprotection strategy for the combinatorial addition of side chains to a pre-fabricated cyclic core.     

N-methyl-N-alkylsilyltrifluoroacetamide-I2 as a new derivatization reagent for anabolic steroid control.

Analytical methods for the control of growth promoters have to be specific and sensitive. At low concentration levels, it is difficult to identify some molecules unambiguously even with the improved performance of analytical methods. GC-MS analysis of 17 beta-trenbolone and its major metabolite, 17 alpha-trenbolone, is a good example. A new derivatization agent has been developed which is based on silylation of the 3- and 17-oxygenated functions and nucleophilic substitution in the 4-position. The structure of the derivatized products was demonstrated using a simple model, cyclohex-2-en-1-one, by NMR and MS spectrometry. In contrast to data found in the literature, this derivative permitted specific mass spectra for trenbolone, sensitive signals for high mass ions and reproducible gas chromatograms to be obtained. The addition of an N(CH3)COCF3 radical to the steroid nucles allowed highly specific detection in GC-high resolution MS even following extraction from complex matrices; sensitive responses were also observed in the negative chemical ionization mode. Moreover, there are significant differences in the electron ionization mass spectra of the two stereoisomers, 17 alpha- and 17 beta-trenbolone. These preliminary results and those obtained for androsta-1,4-dien-3-one and pregna-4,6-dien-3-one indicate useful advances for the determination of steroids and potential applications for metabolism studies on such compounds.