Synthesis of radiolabelled compounds

Compounds labelled with radioisotopes such as¹⁴C,³H,³²P,³⁵S and¹²⁵I, are essential tools for research and especially in the life sciences. Syntheses can be directed to isotopic labelling or to non-isotopic labelling. In isotopic labelling a compound is labelled with an isotope of an element already present in the compound whereas non-isotopic labelling is achieved with an isotope is foreign to the material or compound being labelled. This paper reviews the properties of the more important and commonly used radioisotopes in research and methods currently employed for the synthesis of radiolabelled compounds. The relative ease of measurement of radioactivity in large numbers of samples, when compared for example with the measurement of mass, and the great sensitivity with which very small quantities of radioactive compounds can be accurately measured, has resulted in the widespread need and use of radiolebelled compounds. Methods for the practicable labelling of compounds for use as radiotracers all into three main categories, namely, chemical systhesis, biochemical methods and isotope exchange reactions. Examples are chosen to illustrate these methods for the labelling of amino acids, peptides, proteins, carbohydrates, lipids, neurochemicals, nucleic acids, steroids and miscellaneous compounds of interest in biological research. Most methods of labelling employed lead to specific labelling, that is, to modecules in which the position(s) of the radioactive atom(s) is known with certainty. However, isotope exchange reactions which are especially useful for labelling molecules with tritium are often non-specific. The routine use of tritium nuclear magnetic resonance spectroscopy has resolved any uncertainties in the specificity of labelling with tritium. Examples are given illustrating the effectiveness of this valuable analytical technique. A review of the synthesis of radiolabelled compounds would not be complete without reference to the special properties of the labelled compounds themselves which affect their useful shelf-life, self-decomposition, purification and analysis; factors which all need to be clearly understood in order to use radiolabelled compounds with confidence in scientific research.     

99mTc-labelled human serum albumin: Chemical and electrochemical labelling methods

The labelling of human serum albumin /HSA/ with^99mTc has been investigated using a chemical method /stannous citrate/ and electrolytically generated tin/II/ ions. A comparative study of various chemical parameters and current intensities has been carried out in order to find the optimal conditions for labelling. The labelling yield was over 95%, for the chemical and electrolytical methods.     

Evaluation of the 99mtechnetium labelling effect on Pseudomonas aeruginosa surface properties

Gamma emitter isotopes present some advantages over beta emitters as radioisotopic microbial labels. The labelling of bacteria with ^99mtechnetium (^99mTc) has recently been described. However, it was not ascertained weether the labelling process modifies microbial physicochemical surface properties important in the interaction between bacteria and eukaryotic cells. In the present study, we evaluated the effect of the labelling process on Pseudomonas aeruginosa surface charge, hydrophobicity, adherence to human buccal epithelial cells and phagocytosis by human leukocytes. No significant differences in electrophoretic mobility or cationized ferritin distribution was observed on the cell surface of labelled and unlabelled bacteria. ^99mTc labelling did not modify the hydrophobicity adhesiveness or phagocytosis of P. aeruginosa. It is concluded that bacterial labelling with ^99mTc may be a useful method for the numeration of bacteria and the analysis of their functional properties.     

A trifluoromethylphenyl diazirine-based SecinH3 photoaffinity probe

The synthesis of a trifluoromethylphenyl diazirine photoaffinity probe of the cytohesin inhibitor SecinH3 is described. The probe exhibits improved labelling efficiency over a benzophenone-based probe and thus is more suitable for photoaffinity labelling in complex biological samples.     

Tyrosine Conjugation Methods for Protein Labelling

Over the last two decades, the development of chemical biology and the need for more defined protein conjugates have fostered active research on new bioconjugation techniques. In particular, a wide range of biorthogonal labelling strategies have been reported to functionalise the phenol side chain of tyrosines (Tyr). Tyr occur at medium frequency and are partially buried at the protein surface, offering interesting opportunities for site-selective labelling of the most reactive residues. Tyr-targeting has proved effective for designing a wide range of important biomolecules including antibody–drug conjugates, fluorescent or radioactive protein probes, glycovaccines, protein aggregates, and PEG conjugates. Innovative methods have also been reported for site-specific labelling with ligand-directed anchors and for the specific affinity capture of proteins. This review will present and discuss these promising alternatives to the conventional labelling of the nucleophilic lysine and cysteine residues.     

核酸探针技术

本文对核酸探针技术进行了较全面的综述，介绍了核探针的制备及非放射性标记方法，并对核酸的Ｓｏｕｔｈｅｒｎ转印杂交，Ｎｏｒｔｈｅｒｎ转印杂交，ＩｎＳｉｔｕ转印杂交及斑点杂交法的原理及应用进行了简明的评述。     

Visualisation of biopolymer mixtures using confocal scanning laser microscopy (CSLM) and covalent labelling techniques

Confocal scanning laser microscopy (CSLM) has been used to study the behaviour of mixtures of proteins, gelatine, whey proteins and β-lactoglobulin, and polysaccharides, dextran, gellan gum, carrageenan, gum Arabic, and starch. CSLM proved to be a suitable technique to visualise the microstructure of these (phase separated) mixtures in two and three-dimensional images. Contrast through fluorescence is obtained either by covalent labelling (polysaccharides and proteins) or non-covalent labelling (proteins and starch). Double and triple labelling allows the visualisation of individual components in a complex mixture of biopolymers.     

Efficient solid phase strategy for preparation of modified xanthene dyes for biolabelling

An efficient solid phase strategy for the versatile functionalisation of xanthene dyes for conjugation and labelling of biomolecules is presented. The low cost, high purity and excellent spectral properties of the obtained materials provide an attractive alternative for the labelling of a wide range of molecules.     

Optimal labelling of proteins with acridinium ester

The effects of reaction conditions on the labelling of model proteins (human IgG and BSA) with an acridinium hydroxsuccinimidyl ester are studied. The effect of changes in reaction temperature were minimal. An increase in reaction time increased the amount of labelling, eventually reaching saturation. A higher label/protein ratio produced a proportionally higher label incorporation. The optimal pH of the labelling buffer was found to be pH 8.5. The acridinium-labelled proteins can be detected and quantified in the 4 × 10^?18?3 × 10^?14 mol range.     

Design and synthesis of a highly efficient labelling reagent for incorporation of tetrafluorinated aromatic azide into proteins

Chemical labelling can significantly extend the structures and functions of proteins for advanced applications. Herein, a highly efficient bench-stable reagent diazo-azide was designed and synthesized for the incorporation of tetrafluorinated aromatic azides into proteins via the diazonium coupling. The diazo-azide-labelled proteins could be further functionalized via the nonhydrolysis Staudinger reaction to achieve fluorescence labelling, PEGylation and biotinylation. The whole protein labelling processes were catalysis-free and could be finished within several hours under the mild conditions. To this end, we have prepared thickened viral nanoparticles with controllable diameters.     

13C-isotope labelling for the facilitated NMR analysis of a complex dynamic chemical system

(13)C-isotope labelling is presented as a novel tool for the study of complex chemical systems. (13)C-isotope labelling permits the quantification of all 26 members of a dynamic library from a single (13)C NMR spectrum without the need for advanced instrumentation or sophisticated experimental protocols.     

Detection and quantitative estimation of metalloporphyrins in vivo

Spectrofluorometry, radioisotope (RI) labelling and high performance liquid chromatography (HPLC) can be used to estimate photosensitizer concentrations. The biodistribution of porphyrins and metalloporphyrins containing the bifunctional chelating agent diethylenetriamine pentaacetic acid (DTPA) was examined in tumour-bearing mice by nitrogen-pulsed laser spectrofluorometry (PLS) and RI labelling. The biodistribution of metalloporphyrin amino acid derivatives containing alkoxyl groups was also examined by PLS and HPLC analysis using an acetone powder extraction method. Spectrofluorometry is useful for estimating the biodistribution of porphyrins in tumour, lung, kidney and serum, but not in liver. However, spectrofluorometry cannot be used to evaluate the concentration of certain metalloporphyrins such as manganese complexes. The concentrations of porphyrins in liver measured by PLS and two other methods showed remarkable differences. RI labelling and HPLC analysis are obviously tedious methods. Therefore it seems practical to screen for a number of compounds using the spectrofluorometric method (PLS). Subsequently, the porphyrins which give good results with PLS should be measured using RI labelling and HPLC.     

(13)C/(12)C isotope labelling to study leaf carbon respiration and allocation in twigs of field-grown beech trees

In situ (13)C/(12)C isotopic labelling was conducted in field-grown beech (Fagus sylvatica) twigs to study carbon respiration and allocation. This was achieved with a portable gas-exchange open system coupled to an external chamber. This method allowed us to subject leafy twigs to CO(2) with a constant carbon isotope composition (delta(13)C of -51.2 per thousand) in an open system in the field. The labelling was done during the whole light period at two different dates (in June 2002 and October 2003). The delta(13)C values of respiratory metabolites and CO(2) that is subsequently respired during the night were measured. It was found that night-respired CO(2) is not completely labelled (only ca. 58% and 27% of new carbon is found in respired CO(2) immediately after the labelling in June 2002 and October 2003, respectively) and the labelling level progressively disappeared during the next day. It is concluded that the carbon respired by beech leaves after illumination was supplied by a mixture of carbon sources in which current carbohydrates were not the only contributors. In addition, as has been found in herbaceous plants, isotopic data before labelling showed that carbon isotope discrimination favoring the (13)C isotope occurred during the night respiration of beech leaves.     

Site-selective and covalent labelling of the cysteine-containing peptide glutathione with a ferrocenyl group

Site-specific labelling of the cysteine-containing peptide glutathione with a ferrocene group was achieved by reaction with ferrocenylmethanol in aqueous acidic medium. The resulting peptide was shown to be a potent competitive inhibitor of the biologically important enzyme glutathione-(S)-transferase. This approach may prove general for the labelling of proteins with ferrocene.     

New rapid methods of labelling by radioiodine

New methods for labelling of organic molecules by iodine radionuclides have been developed on the basis of a detailed study of processes occurring during radioiodination. Kits were preparations of iodinated radiophyarmaceuticals (e. g. hippurate, Bengal Rose, sulfobromophythalein). Further iodogene-impregnated filters were developed for labelling of non-iodinated compounds, especially proteins. Their use and preparation is quite simple and easy.     

用^3HNMR波谱法分析微波激发催化交换制备的^3H标记颠茄类生物碱

用~3H NMR波谱法测定了微波激发催化交换制备的四种~3H标记颠茄类生物碱示踪剂的氚标记位置及其相对百分含量。并在用~1H NMK归属原料纯度及标记物分子各基因化学位移时采用了水峰抑制技术(简称WEFT),既能同时消除溶剂氘代甲醇及水峰的影响;又能提高灵敏度(近10倍),从而计算出所得标记产物的化学纯度值与放化纯度值。为快速、准确寻找制备标记生物碱的最佳实验条件和控制标记物质量提供了唯一的物理无损分析方法。     

The effect of cobalt/II/ and copper/II/ ions on the preparation of sodium123I-o-iodohippurate

The role of Cu/II/ and Co/II/ ions in the preparation of sodium¹²³I-o-iodohippurate /Hippuran/ was investigated. It was found that Cu/II/ ions insure the highest radioactive labelling yield /97.61±0.92%/; while Co/II/ ions decrease the labelling yield. The role of Cu/II/ ions does not, however, appear to be catalytic in nature since in the absence of any metallic ion, the labelling yield was /95.00±1.01%/.Presented at the 9th Symposium on the Biological Aspects of Saudi Arabia held on 24–27 March 1986 in Riyadh, Saudi Arabia.     

Formamidine sulfinic acid as reducing agent in technetium-99m rhenium sulfide labelling

Labelling kinetic studies, radiochemical characterization and particle size evaluation of technetium-99m rhenium sulfide colloid using formamidine sulfinic acid as reducing agent are described. Comparison with the same colloid which makes use of Sn-sodium pyrophosphate complex as reducing agent has shown higher labelling yields, simplification of labelling procedure and a longer shelf life when formamidine sulfinic acid is used.     

The use of fluorescent probes in immunochemistry

The limitations and advantages of particular dyes for labelling proteins and other biological materials are discussed. Methods available for conjugating dyes to proteins are outlined. Following a discussion of double labelling methods the use of photoactivatable fluorochromes and time resolved fluorescence methodologies are outlined. The reasons for the photoinstability of some fluorochromes are discussed and methods for overcoming the problem are described.     

A Comparison of Cysteine-Conjugated Nitroxide Spin Labels for Pulse Dipolar EPR Spectroscopy

The structure-function and materials paradigms drive research on the understanding of structures and structural heterogeneity of molecules and solids from materials science to structural biology. Functional insights into complex architectures are often gained from a suite of complementary physicochemical methods. In the context of biomacromolecular structures, the use of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) has become increasingly popular. The main interest in PDS is providing long-range nanometre distance distributions that allow for identifying macromolecular topologies, validating structural models and conformational transitions as well as docking of quaternary complexes. Most commonly, cysteines are introduced into protein structures by site-directed mutagenesis and modified site-specifically to a spin-labelled side-chain such as a stable nitroxide radical. In this contribution, we investigate labelling by four different commercial labelling agents that react through different sulfur-specific reactions. Further, the distance distributions obtained are between spin-bearing moieties and need to be related to the protein structure via modelling approaches. Here, we compare two different approaches to modelling these distributions for all four side-chains. The results indicate that there are significant differences in the optimum labelling procedure. All four spin-labels show differences in the ease of labelling and purification. Further challenges arise from the different tether lengths and rotamers of spin-labelled side-chains; both influence the modelling and translation into structures. Our comparison indicates that the spin-label with the shortest tether in the spin-labelled side-group, (bis-(2,2,5,5-Tetramethyl-3-imidazoline-1-oxyl-4-yl) disulfide, may be underappreciated and could increase the resolution of structural studies by PDS if labelling conditions are optimised accordingly.