Intermolecular crosslinking of fatty acyl chains in phospholipids: Use of photoactivable carbene precursors

Phospholipids containing photolysable carhene precursors (beta-trifluoro-a-diazopropionoxy and m-diazirinophenoxy groups) in w-positions of sn-2 fatty acyl chains were prepared. Photolysis of their vesicles produced crosslinked products in 40-60 % yields. Crosslinking was mostly intermolecular and occurred bv carbene insertion into the C-H bonds of a second fatty acyl chain. Crosslinking products were characterized by (i) their gel permeation behavior, (ii) analysis of produets formed by base-catalyzed transesterification. (iii) degradation with phosphoiipases A2 and C, (iv) gas chromatography/mass spectrometry, and-(v) use of mixtures of phospholipids carrying thf' carhene precursors and a phospholipid containing radioactively labeled fatty acyl groups. Nitrenes generated from the aliphatic or aromatic azido groups in phospholipids were unsatisfactory for forming crosslinks by insertion in C-H bond

Sites of intermolecular crosslinking of fatty acyl chains in phospholipids carrying a photoactivable carbene precursor

Sonicated vesicles of l-fatty acyl-2-w-(2-diazo-3.3,3-trifluoropropionoxy) fatty acyl sn-glycero-3-phosphorylcholines were shown recently to form intermolecular crosslinks by insertion of the photogenerated carbene into a C-H bond of a neighboring hydrocarbon chain. We now report that photolysis of multilamellar dispersions gives a second series of products in which carbene insertion is accompanied by elimination of a molecule of hydrogen fluoride. The sites of crosslinking in the latter compounds have been studied by mass spectrometry using phospholipids with varying chain lengths of the fatty acyl groups carrying the carbene precursor. The patterns observed show that the point of maximum crosslinking is consistent with the recent conclusion that in phospholipids the sn-2 fatty acyl chain trails the sn-1 chain by 2-4 atoms.

Highlights in Analytical Chemistry in Switzerland: OMA & OPA - A Software Tool for Mass Spectrometric Sequencing of Nucleic Acids

Oligonucleotides comprising unnatural building blocks, which interfere with the translation machinery, have gained increased attention for the treatment of gene-related diseases (e.g. antisense, RNAi). Due to structural modifications, synthetic oligonucleotides exhibit increased biostability and bioavailability upon administration. Consequently, classical enzyme-based sequencing methods are not applicable to their sequence elucidation and verification. Tandem mass spectrometry is the method of choice for performing such tasks, since gas-phase dissociation is not restricted to natural nucleic acids. However, tandem mass spectrometric analysis can generate product ion spectra of tremendous complexity, as the number of possible fragments grows rapidly with increasing sequence length. The fact that structural modifications affect the dissociation pathways greatly increases the variety of analytically valuable fragment ions. The gas-phase dissociation of oligonucleotides is characterized by the cleavage of one of the four bonds along the phosphodiester chain, by the accompanying loss of nucleases, and by the generation of internal fragments due to secondary backbone cleavage. For example, an 18-mer oligonucleotide yields a total number of 272’920 theoretical fragment ions. In contrast to the processing of peptide product ion spectra, which nowadays is highly automated, there is a lack of tools assisting the interpretation of oligonucleotide data. The existing web-based and stand-alone software applications are primarily designed for the sequence analysis of natural nucleic acids, but do not account for chemical modifications and adducts. Consequently, we developed a software to support the interpretation of mass spectrometric data of natural and modified nucleic acids and their adducts with chemotherapeutic agents.

Deficiency of ECHS1 causes mitochondrial encephalopathy with cardiac involvement.

OBJECTIVE Short-chain enoyl-CoA hydratase (ECHS1) is a multifunctional mitochondrial matrix enzyme that is involved in the oxidation of fatty acids and essential amino acids such as valine. Here, we describe the broad phenotypic spectrum and pathobiochemistry of individuals with autosomal-recessive ECHS1 deficiency. METHODS Using exome sequencing, we identified ten unrelated individuals carrying compound heterozygous or homozygous mutations in ECHS1. Functional investigations in patient-derived fibroblast cell lines included immunoblotting, enzyme activity measurement, and a palmitate loading assay. RESULTS Patients showed a heterogeneous phenotype with disease onset in the first year of life and course ranging from neonatal death to survival into adulthood. The most prominent clinical features were encephalopathy (10/10), deafness (9/9), epilepsy (6/9), optic atrophy (6/10), and cardiomyopathy (4/10). Serum lactate was elevated and brain magnetic resonance imaging showed white matter changes or a Leigh-like pattern resembling disorders of mitochondrial energy metabolism. Analysis of patients' fibroblast cell lines (6/10) provided further evidence for the pathogenicity of the respective mutations by showing reduced ECHS1 protein levels and reduced 2-enoyl-CoA hydratase activity. While serum acylcarnitine profiles were largely normal, in vitro palmitate loading of patient fibroblasts revealed increased butyrylcarnitine, unmasking the functional defect in mitochondrial β-oxidation of short-chain fatty acids. Urinary excretion of 2-methyl-2,3-dihydroxybutyrate - a potential derivative of acryloyl-CoA in the valine catabolic pathway - was significantly increased, indicating impaired valine oxidation. INTERPRETATION In conclusion, we define the phenotypic spectrum of a new syndrome caused by ECHS1 deficiency. We speculate that both the β-oxidation defect and the block in l-valine metabolism, with accumulation of toxic methacrylyl-CoA and acryloyl-CoA, contribute to the disorder that may be amenable to metabolic treatment approaches.

Distributed Atomic Polarizabilities of Amino Acids and their Hydrogen-Bonded Aggregates

With the purpose of rational design of optical materials, distributed atomic polarizabilities of amino acid molecules and their hydrogen-bonded aggregates are calculated in order to identify the most efficient functional groups, able to buildup larger electric susceptibilities in crystals. Moreover, we carefully analyze how the atomic polarizabilities depend on the one-electron basis set or the many-electron Hamiltonian, including both wave function and density functional theory methods. This is useful for selecting the level of theory that best combines high accuracy and low computational costs, very important in particular when using the cluster method to estimate susceptibilities of molecular-based materials.

Determination of fatty acid ethyl esters in dried blood spots by LC-MS/MS as markers for ethanol intake: application in a drinking study.

The forensic utility of fatty acid ethyl esters (FAEEs) in dried blood spots (DBS) as short-term confirmatory markers for ethanol intake was examined. An LC-MS/MS method for the determination of FAEEs in DBS was developed and validated to investigate FAEE formation and elimination in a drinking study, whereby eight subjects ingested 0.66-0.84 g/kg alcohol to reach blood alcohol concentrations (BAC) of 0.8 g/kg. Blood was taken every 1.5-2 h, BAC was determined, and dried blood spots were prepared, with 50 μL of blood, for the determination of FAEEs. Lower limits of quantitation (LLOQ) were between 15 and 37 ng/mL for the four major FAEEs. Validation data are presented in detail. In the drinking study, ethyl palmitate and ethyl oleate proved to be the two most suitable markers for FAEE determination. Maximum FAEE concentrations were reached in samples taken 2 or 4 h after the start of drinking. The following mean peak concentrations (c̅ max) were reached: ethyl myristate 14 ± 4 ng/mL, ethyl palmitate 144 ± 35 ng/mL, ethyl oleate 125 ± 55 ng/mL, ethyl stearate 71 ± 21 ng/mL, total FAEEs 344 ± 91 ng/mL. Detectability of FAEEs was found to be on the same time scale as BAC. In liquid blood samples containing ethanol, FAEE concentrations increase post-sampling. This study shows that the use of DBS fixation prevents additional FAEE formation in blood samples containing ethanol. Positive FAEE results obtained by DBS analysis can be used as evidence for the presence of ethanol in the original blood sample. Graphical Abstract Time courses for fatty acid ethyl ester (FAEE) concentrations in DBS and ethanol concentrations for subject 1 over a period of 7 h. Ethanol ingestion occured during the first hour of the time course.