Gold(III)‐Induced Oxidation of Amino Acids and Malonic Acid: Reaction Pathways Studied by NMR Spectroscopy with Isotope Labelling

Reactions of Au(III) with biomolecules are of interest in relation to understanding the mechanism of action of therapeutic gold compounds. NMR investigations of ¹³C and ¹⁵N isotopically‐labelled glycine and alanine show that Au(III) induces deamination and subsequent decarboxylation of both amino acids with the same mechanism. For comparison, reactions of Au(III) with sarcosine and the dicarboxylic acid malonic acid were also investigated. The major intermediates and products have been identified.     

Synthesis of azide-fluoro-dehydrocoelenterazine analog as a photoaffinity-labeling probe and photolysis of azide-fluoro-coelenterazine

A photosensitive azide-fluoro-dehydrocoelenterazine analog (Az-F-DCT) was synthesized, starting from 4-fluorophenylacetic acid, as a photoaffinity-labeling probe in order to analyze symplectin active site. To examine the photo-reactivity of Az-F-DCT, azide-fluoro-coelenterazine analog (Az-F-CT) was used as a potent symplectin chromophore model. Photolysis of Az-F-CT in 2,2,2-trifluoroethanol afforded nitrene intermediate to give an insertion product. The structure of this product was confirmed through spectroscopic analyses particularly by using a proton/deuterium (H/D) exchange experiments with ESI-Q-TOF-MS and -MS/MS measurement.     

GC–ICP–MS determination of dimethylselenide in human breath after ingestion of <Superscript>77</Superscript>Se-enriched selenite: monitoring of in-vivo methylation of selenium

The amount of volatile dimethylselenide (DMSe) in breath has been monitored after ingestion of sub-toxic amounts of selenium (300 μg ⁷⁷Se, as selenite) by a healthy male volunteer. The breath samples were collected in Tedlar bags every hour in the first 12 h and then at longer intervals for the next 10 days. The samples were subjected to speciation analysis for volatile selenium compounds by use of cryotrapping–cryofocussing–GC–ICP–MS. Simultaneously, all urine was collected and subjected to total selenium determination by use of ICP–MS. By monitoring m/z 82 and 77, background or dietary selenium and selenium from the administered selenite were simultaneously determined in the urine and in the breath—dietary selenium only was measured by monitoring m/z 82 whereas the amount of spiked ⁷⁷Se (99.1% [enriched spike]) and naturally occurring selenium (7.6% [natural abundance]) were measured by monitoring m/z 77. Quantification of DMSe was performed by using DMSe gas samples prepared in Tedlar bags (linear range 10–300 pg, R ²=0.996, detection limit of Se as DMSe was 10 pg Se, or 0.02 ng L⁻¹, when 0.5 L gas was collected). Dimethylselenide was the only selenium species detected in breath samples before and after the ingestion of ⁷⁷Se-enriched selenite. Additional DM⁷⁷Se was identified as early as 15 min after ingestion of the isotopically-labelled selenite. Although the maximum concentration of ⁷⁷Se in DMSe was recorded 90 min after ingestion, the natural isotope ratio for selenium in DMSe (77/82) was not reached after 20 days. The concentration of DMSe correlated with the total Se concentration in the urine during the experiment (R ²=0.80). Furthermore, the sub-toxic dose of 300 μg selenium led to a significant increase of DMSe and renal excretion of background selenium, confirming that selenium ingested as selenite is homeostatically controlled by excretion. The maximum concentration of DMSe resulting from the spiked selenite was 1.4 ng Se L⁻¹ whereas the dietary background level was less than 0.4 ng Se L⁻¹. Overall excretion as DMSe was calculated to be 11.2% from the ingested selenite within the first 10 days whereas urinary excretion accounts for nearly 18.5%.     

Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique

A new technique for simultaneous recording of multiple fluorophores has been implemented in a confocal scanning laser microscope. Dual excitation wavelengths, intensity-modulated at different frequencies and two lock-in amplifiers tuned to the corresponding frequencies are used. In this way two fluorophores can be independently, albeit simultaneously, excited. This technique, Intensity-modulated Multiple-beam Scanning (IMS) microfluorometry, has the potential to virtually eliminate the cross-talk between fluorophores that often occurs when recording multiple-labelled specimens. Furthermore, it will offer simultaneous information about both the excitation and emission spectra of the fluorophores used. Also, variations in decay time over the image area can be studied independently and simultaneously for two fluorophores.     

Synthesis of 14C-radiolabelled trimethyllead chloride

Two synthetic routes to ¹⁴C-labelled trimethyllead chloride ((CH₃)₃PbCl) from ¹⁴C-methyl iodide (CH₃I) were investigated. Alkylation of (CH₃)₃PbCl with labelled methylmagnesium halide, on a microscale, was less efficient for the synthesis of tetramethyllead ((CH₃)₄Pb) than was an electrochemical reduction of labelled CH₃I at a sacrificial lead cathode. In the Grignard approach, unlabelled decyl bromide served as an initiator for the reaction of ¹⁴C-CH₃l with excess Mg and as a carrier during the subsequent alkylation of (CH₃)₃PbCl. In the electrochemical approach a two-compartment cell, using dimethylformamide as solvent and sodium perchlorate as supporting electrolyte, offered several advantages over a single compartment reactor. The labelled (CH₃)₄Pb from both reactions was isolated by extraction, converted to (CH₃)₃PbCl by controlled oxidation with HCl and purified by thin layer chromatography.     

Ox-SLIM: Synthesis of and Site-Specific Labelling with a Highly Hydrophilic Trityl Spin Label

The combination of pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) with site-directed spin labelling is a powerful tool in structural biology. Rational design of trityl-based spin labels has enabled studying biomolecular structures at room temperature and within cells. However, most current trityl spin labels suffer either from aggregation with proteins due to their hydrophobicity, or from bioconjugation groups not suitable for in-cell measurements. Therefore, we introduce here the highly hydrophilic trityl spin label Ox-SLIM. Engineered as a short-linked maleimide, it combines the most recent developments in one single molecule, as it does not aggregate with proteins, exhibits high resistance under in-cell conditions, provides a short linker, and allows for selective and efficient spin labelling via cysteines. Beyond establishing synthetic access to Ox-SLIM, its suitability as a spin label is illustrated and ultimately, highly sensitive PDS measurements are presented down to protein concentrations as low as 45 nm resolving interspin distances of up to 5.5 nm.     

A convergent synthesis of (17R,5Z,8Z,11Z,14Z)-17-hydroxyeicosa-5,8,11,14-tetraenoic acid analogues and their tritiated derivatives

17(R)-OH-AA and 14,15-dehydro-17(R)-OH-AA were synthesized from a common tetraacetylenic precursor and their azide derivatives were obtained in moderate yields via the corresponding p-toluenesulfonates. Since the azido group remained stable during tritiation procedure on Lindlar's catalyst in benzene, both 14,15-dehydro-17(S)-N₃-AA and 14,15-dehydro-17(R)-OH-AA constitute useful intermediates in the synthesis of radio-labelled 17(S)-N₃-AA and 17(R)-OH-AA. In contrast, reduction of azide in methanol afforded 17(S)-NH₂-AA with 95% yield.     

Human Serum Albumin Labelling with a New BODIPY Dye Having a Large Stokes Shift

BODIPY dyes are photostable neutral derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. These are widely used as chemosensors, laser materials, and molecular probes. At the same time, BODIPY dyes have small or moderate Stokes shifts like most other fluorophores. Large Stokes shifts are preferred for fluorophores because of higher sensitivity of such probes and sensors. The new boron containing BODIPY dye was designed and synthesized. We succeeded to perform an annulation of pyrrole ring with coumarin heterocyclic system and achieved a remarkable difference in absorption and emission maximum of obtained fluorophore up to 100 nm. This BODIPY dye was equipped with linker arm and was functionalized with a maleimide residue specifically reactive towards thiol groups of proteins. BODIPY residue equipped with a suitable targeting protein core can be used as a suitable imaging probe and agent for Boron Neutron Capture Therapy (BNCT). As the most abundant protein with a variety of physiological functions, human serum albumin (HSA) has been used extensively for the delivery and improvement of therapeutic molecules. Thiolactone chemistry provides a powerful tool to prepare albumin-based multimodal constructions. The released sulfhydryl groups of the homocysteine functional handle in thiolactone modified HSA were labeled with BODIPY dye to prepare a labeled albumin-BODIPY dye conjugate confirmed by MALDI-TOF-MS, UV-vis, and fluorescent emission spectra. Cytotoxicity of the resulting conjugate was investigated. This study is the basis for a novel BODIPY dye-albumin theranostic for BNCT. The results provide further impetus to develop derivatives of HSA for delivery of boron to cancer cells.     

Elucidation of the Structure of Lignin–Carbohydrate Complexes in Ginkgo CW-DHP by 13C-2H Dual Isotope Tracer

To elucidate the chemical linkages between lignin and carbohydrates in ginkgo cell walls, ¹³C-²H-enriched cell wall-dehydrogenation polymers (CW-DHP) were selectively prepared with cambial tissue from Ginkgo biloba L. by feeding D-glucose-[6-²H₂], coniferin-[α-¹³C], and phenylalanine ammonia-lyase (PAL) inhibitor. The abundant detection of ¹³C and ²H confirmed that D-glucose-[6-²H₂] and coniferin-[α-¹³C] were involved in the normal metabolism of ginkgo cambial cells that had been effectively labelled with dual isotopes. In the ginkgo CW-DHP, ketal and ether linkages were formed between the C-α of lignin side chains and carbohydrates, as revealed by solid state CP/MAS ¹³C-NMR differential spectroscopy. Furthermore, the DMSO/TBAH ionic liquids system was used to fractionate the ball-milled CW-DHP into three lignin-carbohydrate complex (LCC) fractions: glucan–lignin complex (GL), glucomannan–lignin complex (GML), and xylan–lignin complex (XL). The XRD determination indicated that the cellulose type I of the GL was converted into cellulose type II during the separation process. The molecular weight was in the order of Ac-GL > Ac-GML > XL. The ¹³C-NMR and ¹H-NMR differential spectroscopy of ¹³C-²H-enriched GL fraction indicated that lignin was linked with cellulose C-6 by benzyl ether linkages. It was also found that there were benzyl ether linkages between the lignin side chain C-α and glucomannan C-6 in the ¹³C-²H-enriched GML fraction. The formation of ketal linkages between the C-α of lignin and xylan was confirmed in the ¹³C-²H-enriched XL fraction.