Labeling of oligonucleotides with DTPA and DOTA on solid phase

Oligonucleotide conjugates labeled with metal chelates of diethylenetriaminepentaacetic acid (DTPA) and tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were synthesized on solid phase using appropriate nucleosidic phosphoramidite building blocks (3, 4) and a modified deprotection-metal chelation protocol. The major differences on the properties of the oligonucleotide conjugates also are discussed.     

Labeling of steroids with tritium

Conditions have been elaborated for the tritium labeling of some steroids to a molar activity of the order of 1 TBk/mol by heterogeneous catalytic isotopic exchange. For steroids without a keto group, isotopic exchange with gaseous tritium is most effective in neutral solvents, whereas for steroids with a keto group an alkaline medium is preferable. At certain pH values it was possible to void hydrogenation of the double bonds in the compounds.     

Effects of anabolic steroids on acute phase responses in intra-abdominal sepsis

The acute phase response is an important adaptive response to sepsis and injury. As anabolic steroids increase protein synthesis we postulated that these agents might also increase hepatic acute phase protein synthesis. Male Wistar rats were pretreated with testosterone or danazol for 48 h prior to caecal ligation and puncture (CLP). Thirty-six h following surgery the animals were killed and blood taken for full blood count, total protein, albumin, alpha, beta and gamma globulin fractions on serum electrophoresis, complement C(3) and transferrin levels. Danazol increased the alpha1, alpha2 and beta1 globulin serum protein fractions in comparison with no surgery and CLP alone groups. These results indicate that danazol increases plasma acute phase proteins, as measured by electrophoresis, in this model of intra-abdominal sepsis.     

Site-specific synthesis of Amadori-modified peptides on solid phase.

Glycation of peptides and proteins is a slow chemical reaction of reducing sugars modifying the amino groups. The first intermediates of this nonenzymatic glycosylation are the Amadori products that can undergo further chemical reactions, finally leading to advanced glycation end products (AGEs). The formation of AGEs was not only linked to aging of tissues and organs in general but also to several diseases such as diabetes mellitus and Alzheimer's disease. Because of the importance of these modifications and their potential use as diagnostic markers, a global postsynthetic approach on solid phase was developed. The peptides were synthesized by Fmoc/(t)Bu-chemistry, with the lysine residue to be modified being protected with the very acid-labile methyltrityl group. Incubation of the peptides with D-glucose in DMF at elevated temperatures resulted in product yields of 35%. Neighboring residues with bulky protecting groups reduced the yields only slightly. The major by-products were the unmodified peptide and an oxidation product. Whereas the unmodified peptide eluted before the glycated peptide, all other by-products eluted later in RP-HPLC, allowing simple purification.     

Preparation of two solid phase immunogens

Dinitrophenyl (DNP) conjugates of Bio-Gel and dinitrophenyl-ornithine conjugates of Bio-Gel and Sepharose were prepared. Their efficacy as immunogens was assessed. It was found that the dinitrophenyl-ornithine gels ellicited generation of large numbers of splenic anti-DNP plaque-forming cells. DNP-Bio-Gel was not immunogenic.     

Peptide N-alkylamides by solid phase synthesis

Three new resins have been developed that allow for the solid phase synthesis of C-terminal peptide N-alkylamides using Boc amino acids, usual side chain protecting groups and hydrogen fluoride cleavage and deprotection. These resins were prepared by reacting the appropriate alkylamine (NH2CH3, NH2CH2CH3, NH2CH2CF3) to Merrifield's 1% divinylbenzene cross-linked chloromethylated polystyrene resin. The application of these resins to the synthesis of C-terminal GnRH N-alkylamides illustrates the versatility of this approach. GnRH analogs were tested for their ability to release LH from cultured rat anterior pituitary cells. [DGlu6, Pro9-NHCH2CH3]-GnRH was synthesized for the first time using the solid phase approach and found to be three times more potent than [DGlu6]-GnRH. Other analogs including [DTrp6, Pro9-NHCH2CH3]-GnRH, [DAla6, Pro9-NHCH2CF3]-GnRH and related peptides were found to be equipotent and to have the same properties (HPLC retention times, amino acid analysis and specific rotation) as the corresponding peptides synthesized using less amenable strategies; yields were equivalent or better than those reported earlier.     

Effects of resin swelling and substitution on solid phase synthesis.

Resins sold by several companies were examined for swelling, uniformity of beads, and substitution in the case of resins sold with the first amino acid attached. Effects of resin swelling, uniformity, and substitution on the solid phase synthesis of long, structured and/or branched peptides were evaluated.     

In vivo sampling with solid phase microextraction

This review discusses the most recent developments and future challenges in the application of solid phase microextraction (SPME) for sampling of live biological samples. The emphasis is placed on applications of fiber SPME for analysis of volatile emissions and drugs in biological fluids. The method development section highlights the main parameters that need to be considered in the case of in vivo experiments: extraction techniques, selection of extraction phases, calibration procedures, determination of free concentrations, and automation.     

Matrix solid phase dispersion (MSPD)

A review of the many uses of matrix solid phase dispersion (MSPD) in the extraction and analysis of a variety of compounds from a range of samples is provided. Matrix solid phase dispersion (MSPD) has found particular application as a somewhat generic analytical process for the preparation, extraction and fractionation of solid, semi-solid and/or highly viscous biological samples. Its simplicity and flexibility contribute to it being chosen over more classical methods for these purposes. MSPD is based on several simple principles of chemistry and physics, involving forces applied to the sample by mechanical blending to produce complete sample disruption and the interactions of the sample matrix with a solid support bonded-phase (SPE) or the surface chemistry of other solid support materials. These principles are discussed as are the factors to be considered in conducting a MSPD extraction.     

Matrix solid phase dispersion (MSPD)

A review of the many uses of matrix solid phase dispersion (MSPD) in the extraction and analysis of a variety of compounds from a range of samples is provided. Matrix solid phase dispersion (MSPD) has found particular application as a somewhat generic analytical process for the preparation, extraction and fractionation of solid, semi-solid and/or highly viscous biological samples. Its simplicity and flexibility contribute to it being chosen over more classical methods for these purposes. MSPD is based on several simple principles of chemistry and physics, involving forces applied to the sample by mechanical blending to produce complete sample disruption and the interactions of the sample matrix with a solid support bonded-phase (SPE) or the surface chemistry of other solid support materials. These principles are discussed as are the factors to be considered in conducting a MSPD extraction.     

In vivo sampling with solid phase microextraction

This review discusses the most recent developments and future challenges in the application of solid phase microextraction (SPME) for sampling of live biological samples. The emphasis is placed on applications of fiber SPME for analysis of volatile emissions and drugs in biological fluids. The method development section highlights the main parameters that need to be considered in the case of in vivo experiments: extraction techniques, selection of extraction phases, calibration procedures, determination of free concentrations, and automation.     

Combinatorial solid phase peptide synthesis and bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.     

A solid phase method of determining DNA sequence

A solid-phase method for DNA sequencing has been developed which involves immobilization of the terminally labeled DNA fragment on the DEAE-paper followed by chemical modification and cleavage at G, A + G, C + T, and C sites. As compared to the Maxam and Gilbert method, the new technique is more rapid and less laborious, being of the same efficiency.     

Labeling on the surface

A strategy for labeling cell-surface proteins in living cells with small-molecule fluorophores allowed scientists to study receptor trafficking by single-cell FRET imaging in real time.     

Evaluation of exudates by solid phase microextraction-gas chromatography

Head-space solid phase microextraction combined with gas chromatography (SPME-GC) was used for the determination of bacterial volatile fatty acid (VFA) patterns. The method was validated with cultures of reference bacterial strains. It was confirmed that VFA production depends on the composition of the cultivation medium, which limits accurate characterisation of particular bacterial species. A set of 195 clinical exudates of various origin and consistence was analysed using SPME-GC and compared with 73 samples extracted using tert-butyl methyl ether. Approximate agreement of VFA profiles with cultivation findings was found in most cases. However, 20.5% of clinical exudates with distinct VFA profiles appeared to be false-negative by cultivation. Using SPME-GC of exudates, the frequency of false-negative cultivations was higher than that of solvent extraction of exudates or blood cultures found previously. The described method is suitable for preliminary detection of bacteria, particularly non-sporulating anaerobes, in clinical samples. It can reveal false-negative findings due to cultivation. Analysis can be performed in 30 min without the need for cultivation.     

A facile solid phase synthesis of tetramic acid

The condensation of N-acylated amino acids with polymer-supported cyclic malonic acid ester was carried out in the presence of DCC/DMAP. The cyclisation of the intermediate resin, by heating in an organic solvent, gave the N-protected tetramic acid derivatives in good yields and high purity.