Chlorination as a side reaction in radioiodination with chloramine-T

Chloramine-T is often used as an oxidizing agent for radioiodide in the electrophilic labeling of proteins and phenolic compounds. We have found that chloramine-T is also capable of introducing chlorine under the right conditions. Thus, treatment of fluorescein with no-carrier added¹²⁵I-sodium iodide and chloramine-T resulted in a uv-absorbing, fluorescent product in addition to the (no-carrier-added)¹²⁵I labeled material. The by product was tentatively identified as a chlorinated fluorescein by comparison with a known sample. The potential of macrochlorination should be kept in mind when using chloramine-T for radioiodination.     

125I-Labeled Peptide Mapping and High-Performance Liquid Chromatography I-Peptide Separation of Protein I of Four Strains of Neisseria Gonorrhoeae

Abstract

¹²⁵I-labeled α-chymotryptic peptides of the principal outer membrane proteins (P.Is) of four strains of Neisseria gonorrhoeae were separated and visualized by two-dimensional (2-D) ¹²⁵I-peptide mapping and by high-performance liquid chromatography (HPLC) coupled with a Beckman Biogamma counter. In addition, ¹²⁵I-peptides were recovered from the HPLC separation and re-separated by the 2-D ¹²⁵I-peptide mapping system. The results indicated that the 2-D ¹²⁵I-peptide mapping procedure was best suited for comparative analyses of α-chymotryptic digests whereas the HPLC system, which is able to detect many more peptides than the 2-D system, is ideally suited for preparative separation of the ¹²⁵I-peptides. ¹²⁵I-peptides separated by HPLC could be recovered, rerun on the 2-D system, and the location of each peptide ascertained. The coupling of these two procedures allows for the isolation of specific ¹²⁵I-labeled peptides for further immunological and structural analyses of these outer membrane proteins.     

Purification of Radioiodinated Peptides with PRP-1 Polystyrene Cartridges and HPLC: Application to Atrial Natriuretic Factor and Vasopressin

Abstract

A simple and rapid cleanup procedure is described for the purification of iodinated peptides using PRP-1 polystyrene cartridges following the radioiodination process. The method is validated using different volumes and solvent systems and compared to the standard Sep-Pak C₁₈ procedure. In this study, the method is used to prepare ¹²⁵I-labeled atrial natriuretic factor and arginine-vasopressin which are further purified by reverse phase HPLC giving maximally obtainable specific activity required for the radioimmunoassays of these peptides.     

Optimization of Iodination of [125I]-N-Succinimidyl-Para-Iodobenzoate Using Chloramine-T for Labeling of Proteins

Generally, indirect radioiodination of proteins using N-succinimidyl-iodobenzoates preserve the biological function of the protein due the mild labeling conditions. However, a drawback is that the two-step procedure often gives a low overall labeling yield. For this reason the production of [¹²⁵I] N-succinimidyl-para-iodobenzoate ([¹²⁵I]SPIB), using Chloramine-T as an oxidant, was optimized regarding substrate, oxidant, reaction time, and volume of the reaction mixture and found to be 86±6%. Produced [¹²⁵I]SPIB was successfully used for the labeling of monoclonal antibodies.     

Facile Preparation of (S)-N-[(1-Ethyl-2-pyrrolidinyl)methyl]-2,3-dimethoxy-5-(tributyltin)benzamide from Isoremoxipride: The Precursor of [125I]- and [123I]Epidepride

Abstract

[¹²⁵I]Epidepride, (S)-(?)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-[¹²⁵I]iodo-2,3-dimethoxybenzamide ([¹²⁵I]NCQ 219), is a new, extremly potent radioligand, useful in the study of the distribution of the dopamine D-2 receptors in the brain. Its synthesis requires radioiodination of the corresponding 5-(tributyltin) derivative. The aryltin precursor (TDP 526) can be conveniently prepared in high yield from isoremoxipride (FLB 457) by tetrakis(triphenylphosphine)palladium(0)-catalyzed stannylation using bis(tri-n-butyltin) in triethylamine. An improved method for the preparation of isoremoxipride from o-vanillin was developed.     

Preparation of 2-125I-Testosterone and Its Use as Ligand for Radioimmunoassay in Serum

Abstract

A radioiodination technique is described which consists of heating Testosterone (T) with Na¹²⁵I and hydrogen peroxide in acetic acid in sealed vial. The binding capacity of labelled substances was measured after thin layer chromatography of the reaction mixture. One compound was selected for radioimmunoassay on the basis of its binding capacity for anti-T serum. Standard curves utilizing tritiated and radioiodinated T, under identical conditions, were superimposible. Serum hormone concentrations determined by the two tracers were comparable. Accuracy, precision and sensitivity were satisfactory. Mass spectroscopy and nuclear magnetic resonance of the unlabelled iodinated T indicated that iodine is in C-2.     

A simple method for the determination of the specific activity of125I-tracer used in radioimmunoassay

Knowledge of the specific activity of the¹²⁵I-tracer is essential for optimization and for calculation of RIA parameters. The specific activity of the¹²⁵I-thyroxin used in thyroxin radioimmunoassay /RIA/ has been determined by a simple method involving combination of RIA and displacement analysis. It has been compared with the value obtained by the conventional method based on radioiodination data. Our studies indicate that even for a non-protein hormone like thyroxin the specific activity of¹²⁵I-thyroxin derived from iodination data is not reliable. The specific activities obtained by displacement analysis were consistent with the experimental findings.     

Potential of radioiodinated anti cancer compounds of natural origin for cancer therapy

Plumbagin and Quercetin are naturally occurring compounds which exhibit anti-cancerous activity. To evaluate the effect of radioiodination on cytotoxicity, both Plumbagin and Quercetin were radioiodinated with ¹²⁵I ¹²⁵I-Plumbagin and ¹²⁵I-Quercetin could be prepared in moderate yields and good radiochemical purity and were charactenzed using reverse phase HPLC. In Swiss mice bearing fibrosarcoma, ¹²⁵I-Plumbagin showed a tumor uptake of ∼2.5%ID/g at 3 h p.i. and ∼0.5% ID/g at 24 h p.i. on i.v. injection. When injected intratum orally, greater tumor uptake and retention was observed (∼20%ID/g at 3 h p.i. and ∼14%ID/g at 24 h p.i. respectively).     

Selenomethionine Extraction from Selenized Yeast: an LC-MS Study of the Acid Hydrolysis of a Synthetic Selenopeptide

A synthetically prepared seleno-peptide (AHPDVLTVXLQMLDDGR) was used as a model system for the acid hydrolysis of selenized yeast proteins. The seleno-peptide is a tryptic peptide of a heat shock protein 104 from Saccharomyces cerevisiae, was subjected to acid hydrolysis using methanesulfonic acid over a time period of 8 hours. Aliquots of the solution were sub-sampled at predetermined time intervals and the peptide fragments characterized by reversed phase LC MSⁿ. Similarly, the appearance of amino acid residues in the solution was monitored. It was found that after about 8 hours the synthetic peptide completely hydrolyzed. The use of a selenopeptide as a model for hydrolysis of selenized yeast hydrolysis was validated by comparing the decomposition time profile of the synthetic peptide with that of a selenized yeast sample. The rate of hydrolysis was identical in both systems, suggesting that the employed acid hydrolysis yields to the complete decomposition of the Se containing proteins in yeast and consequently to the liberation of selenomethionine.     

Labeling of atenolol with radioactive iodine-125 using <Emphasis Type="Italic">N</Emphasis>-bromosuccinimide and hydrogen peroxide as oxidizing agents

An adopted method for the preparation of high radiochemical purity ¹²⁵I-atenolol was investigated. Direct radioiodination of atenolol was carried out using N-bromosuccinamide or hydrogen peroxide as an oxidizing agent. The reaction proceeds well within 30 min at room temperature (25 ± 1 °C) and afforded a radiochemical yield up to 97% as pure as ¹²⁵I-atenolol. Different chromatographic techniques (electrophoresis, TLC and HPLC) were used to determine the radiochemical yield and purity of the labeled product. Biodistribution studies were carried out in normal Albino Swiss mice and the results indicate that ¹²⁵I-atenolol can be used safely as myocardial imaging agent.     

Preparation of radioiodinated khellin for the urinary tract imaging

Khellin, 4,9-dimethoxy-7-methyl-5H-furo[3,2-g]chromen-5-one, the most biologically active furochromone present in Ammi visnaga fruits indigenous to Egypt. A procedure for radioiodination of khellin with ¹²⁵I is carried out via an electrophilic substitution reaction. The reaction parameters studied were khellin amount, pH of the reaction mixture, reaction time, temperature, different oxidizing agents and different organic media to optimize the conditions for the labeling of khellin and to obtain a high radiochemical yield of the ¹²⁵I-khellin (¹²⁵I-Khel). Using 3.7 MBq of Na¹²⁵I, 100 μg (0.96 mM) of khellin as substrate, 100 μg (1 mM) of chloramine-T as oxidizing agent in ethanol at 60 °C for 10 min, a maximum radiochemical yield of ¹²⁵I-Khel (78 %) was obtained. The specific activity of ¹²⁵I-Khel was 3 MBq/0.5 mM. The biological distribution in normal mice indicates that radioiodinated khellin is a novel agent for urinary tract infection imaging.     

Establishing a quality control protocol for the radiochemical evaluation of radioiodinated biomolecules with application in nuclear medicine

Quality Control (QC) of radiopharmaceuticals is important, providing products of high standards. The current study refers to an enzymic radioiodination method applied to the labeling of antibodies used in nuclear medicine and to the respective QC procedures. Readily available materials such as¹²⁵I, polyclonal immunoglobulin G (IgG) and a collection of simple chromatographic techniques helped establish a QC protocol suitable for the radiochemical evaluation of radioiodinated biomolecules. A high pre-purification radiolabeling yield (90%) for total protein was detected by paper electrophoresis (PE) and size exclusion chromatography (SEC), but only the inclusion of SDS-PAGE confirmed the existence of “self-iodination” of lactoperoxidase (LPO) and revealed the true radiolabeling yield for¹²⁵I-IgG (79%).     

Radioiodination of N-[3-(4-morphilino)propyl]-N-methyl-2-hydroxy-5-iodo-3-methyl-benzylamine (ERC9) via isotopic exchange reaction

Summary A simple method for labeling ERC9 with radioactive iodine has been carried out via nucleophilic substitution radioiodination. The factors affecting the radiochemical yield of the labeling of ERC9, such as pH of the medium, substrate concentration and temperature were investigated. Chromatographic analysis technique was used to determine the radiochemical yield and purity. Kinetics and thermodynamic parameters indicated a second order reaction. Activation energy for the isotopic exchange reaction between ERC9 and ¹²⁵I^- was calculated to be 24.4 kJ/mol.     

Peptide Profiles of Surface-Labeled Proteins Shed or Released by Enzyme Digestion from Staphylococcus Aureus Determined by Reverse Phase High Performance Liquid Chromatography

Abstract

The surface proteins of Staphylococcus aureus were radio-labeled and subsequently characterized by autoradiography and reverse phase-high performance liquid chromatography. Approximately 25% of the surface-labeled proteins were shed from non-growing cells; the shed proteins contributed to the labeled-protein pool obtained by protease digestion of whole cells. The elution profiles of ¹²⁵I-labeled peptides allowed direct comparisons of shed surface proteins (untreated or protease digested) with surface proteins cleaved from the cells by protease treatment.     

Site-Specific Radioiodination of HER2-Targeting Affibody Molecules using 4-Iodophenethylmaleimide Decreases Renal Uptake of Radioactivity

Affibody molecules are small scaffold-based affinity proteins with promising properties as probes for radionuclide-based molecular imaging. However, a high reabsorption of radiolabeled Affibody molecules in kidneys is an issue. We have shown that the use of ¹²⁵I-3-iodo-((4-hydroxyphenyl)ethyl)maleimide (IHPEM) for site-specific labeling of cysteine-containing Affibody molecules provides high tumor uptake but low radioactivity retention in kidneys. We hypothesized that the use of 4-iodophenethylmaleimide (IPEM) would further reduce renal retention of radioactivity because of higher lipophilicity of radiometabolites. An anti-human epidermal growth factor receptor type 2 (HER2) Affibody molecule (Z_HER2:2395) was labeled using ¹²⁵I-IPEM with an overall yield of 45±3 %. ¹²⁵I-IPEM-Z_HER2:2395 bound specifically to HER2-expressing human ovarian carcinoma cells (SKOV-3 cell line). In NMRI mice, the renal uptake of ¹²⁵I-IPEM-Z_HER2:2395 (24±2 and 5.7±0.3 % IA g⁻¹at 1 and 4 h after injection, respectively) was significantly lower than uptake of ¹²⁵I-IHPEM-Z_HER2:2395 (50±8 and 12±2 % IA g⁻¹at 1 and 4 h after injection, respectively). In conclusion, the use of a more lipophilic linker for the radioiodination of Affibody molecules reduces renal radioactivity.     

Radioiodination, purification and bioevaluation of Piroxicam in comparison with Meloxicam for imaging of inflammation

The present study is performed to compare the electrophilic substitution radioiodination reaction of two non-steroidal anti-inflammatory drugs namely, Piroxicam (Pirox) and Meloxicam (Melox) with ¹²⁵I where both chloramine-T (CAT) and iodogen were used as oxidizing agents. The factors affecting the percent of radiochemical yields such as drug concentration, pH of the reaction mixtures, different oxidizing agents, reaction time, temperature and different organic media were studied to optimize the conditions for labeling of Pirox and Melox and to obtain high radiochemical yields. The maximum radiochemical yield of ¹²⁵I-Piroxicam (¹²⁵I-Pirox) was 94% using 3.7 MBq of Na¹²⁵I, 0.4 mM of Pirox as substrate, 3.6 mM of chloramine-T (CAT) as oxidizing agent in acetone at neutral pH = 7 and at 60 °C within 20 min where the maximum radiochemical yield of ¹²⁵I-Melox was 92% using 0.7 mM of Melox as substrate, 0.62 mM of iodogen as oxidizing agent in acetone at neutral pH = 7 and at 25 °C within 30 min. The radiochemical yields were determined by TLC and high-pressure liquid chromatography (HPLC). Tracers showed good localization in inflamed muscle either septic or sterile. The collected data indicates that Pirox and Melox can be used as antiinflammatory imaging agents at 24 and 2 h post injection, respectively.     

Potential of radioiodinated anticancer compounds of traditional Chinese medicine for cancer therapy

23-Hydroxybetulinic acid (23-HBA) is the efficient antitumor compound extracted from the roots of a Chinese Medicinal Herb, Pulsatilla chinensis (Bge) Regel. To evaluate the effect of radioiodination on cytotoxicity, 23-HBA was radioiodinated with ¹²⁵I. ¹²⁵I-23-HBA could be prepared in high yields and good radiochemical purity and was characterized using reverse phase HPLC. In ICR mice bearing Liver Cancer HepA tumor, ¹²⁵I-23-HBA showed a tumor uptake of 2.1% ID/g at 2 h p.i. and 0.15% ID/g at 48 h p.i on i.v. injection. When injected intratumorally, greater tumor uptake and retention was observed (20% ID/g at 2 h p.i. and 4.6% ID/g at 48 h p.i. respectively).     

Radioimmunoassay of Human Follicle Stimulating Hormone /HFSH/

A method for determining Human FSH in blood/serum by RIA is described. The radioiodination of FSH with¹²⁵I is carried out under carefully controlled conditions such as, amount of initial activity of¹²⁵I take for iodination, the reaction volume, and the reaction time, etc. The tracer FSH obtained thus is with minimum damage and optimum specific activity which is ideally suited for RIA. The shelf-life of the tracer is enhanced by the addition of benzyl alchol. The tracer can be conveniently stored at+4–6°C upto 10 weeks, avoiding the repeated freezing and thawing process. Antiserum to FSH is raised in rabbits by repeated injections via intramuscular route. The method utilizes polyethylene glycol /PEG/ as the separation system. Using this method, a number of control samples of men and women of reproductive age group are screened. This sensitive assay has a good validity and has an inter-assay variation less than 15%.     

Synthesis of insulin fragments with disulfide bridges between intact chains A20-B19

The synthesis of six insulin fragments is described, in which various sequences of the two chains are linked by the disulfide bridge between A²⁰ and B¹⁹. The fragments in question are: A^20–21–B^19–21, A^20–21–B^18–21, A^20–21–B^17–21, A^19–21–B^19–21, A^16–21–B^18–21 and A^20–21–B^12–21. In order to build up the simpler fragments the disulfide bridge was established by oxidation with iodine of two S-trityl cysteine peptides in which the carboxyl and amino groups were protected by the t-butyl and t-butyloxycarbonyl residue. From the mixture obtained the unsymmetrical cystine peptide was separated in all cases from the two symmetrical ones by counter-current distribution. In the synthesis of the more complex fragments advantageous use was made of smaller unsymmetrical fragments prepared as above but having one amino group protected by the N-trityl residue. After selective elimination of this group it was possible to lengthen the peptide chain at this position. The free peptides were obtained by removal of the protecting groups with strong acids, in particular concentrated hydrochloric acid. While in this deprotecting step the disulfide bond was stable, conditions are discussed under which disproportionation was observed. None of the six synthetic insulin fragments showed activity in stimulating rat adipose tissue to convert ¹⁴C-labelled glucose to CO₂ in vitro.     

Collision-induced reporter fragmentations for identification of covalently modified peptides

Collision-induced reporter fragmentations of the currently most important covalent peptide modifications as detected by tandem mass spectrometry are summarized. These fragmentations comprise the formation of reporter ions, which are preferentially immonium ions, immonium ion-derived fragments or side chain fragments. In addition, the reporter neutral loss reactions for covalently modified amino acid residues are summarized. For each individual covalent modification which can be recognized by a reporter fragmentation, the accurate mass shift and the gross formula shift of the modified amino acid residue are given. The same set of data is provided for the reporter fragmentations. Finally, an extensive accurate mass and gross formula list is presented as supplementary material, describing mostly regular and modified y₁ and dipeptide a and b ions, which are helpful for identification of the peptide ends of covalently modified peptides. Figure When modified peptides are fragmented by collision-induced dissociation in a tandem mass spectrometer, the modification is either lost as part of a charged fragment, so that a reporter ion for the modification is generated or it is lost as part of a neutral fragment, so that a modification-specific reporter neutral loss is observed in the fragment ion spectrum. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chien-Wen Hung and Andreas Schlosser contributed equally to this work.