Quantification of N-acetyl-l-aspartate in dried blood spots: A simple and fast LC-MS/MS neonatal screening method for the diagnosis of Canavan disease.

BACKGROUND: Canavan disease is a devastating neurometabolic disorder caused by accumulation of N acetylaspartate in brain and body fluids due to genetic defects in the aspartoacylase gene (ASPA). New gene therapies are on the horizon but will require early presymptomatic diagnosis to be fully effective. METHODS: We therefore developed a fast and highly sensitive liquid chromatography mass spectrometry (LC-MS/MS)-based method for quantification of N-acetylaspartate in dried blood spots and established reference ranges for neonates and older controls. With this test, we investigated 45 samples of 25 Canavan patients including 8 with a neonatal sample. RESULTS: Measuring N-acetylaspartate concentration in dried blood with this novel test, all Canavan patients (with variable severity) were well separated from the control group (median; range: 5.7; 1.6-13.6 µmol/L [n = 45] vs 0.44; 0.24-0.99 µmol/L [n = 59] (p < 0.05)). There was also no overlap when comparing neonatal samples of Canavan patients (7.3; 5.1-9.9 µmol/L [n = 8]) and neonatal controls (0.93; 0.4-1.8 µmol/L [n = 784]) (p < 0.05). CONCLUSIONS: We have developed a new LC-MS/MS-based screening test for early postnatal diagnosis of Canavan disease that should be further evaluated in a population-based study once a promising treatment becomes available. The method meets the general requirements of newborn screening and should be appropriate for multiplexing with other screening approaches that combine chromatographic and mass spectrometry techniques.

State of the Art in the Preparation and Properties of Molecular Monomeric s-Heptazines: Syntheses, Characteristics, and Functional Applications.

This review gives an account on the fast expanding field of monomeric (or molecular) heptazines, at the exclusion of their various polymeric forms, often referred to as carbon nitrides. While examples of monomeric heptazines were extremely limited until the beginning of this century, the field has started expanding quickly since then, as has the number of reports on polymeric materials, though previous reviews did not separate these fields. We provide here a detailed report on the synthetic procedures for molecular heptazines. We also extensively report on the different achievements realized from these new molecules, in the fields of physical chemistry, spectroscopy, materials preparation, (photo)catalysis, and devices. After a comprehensive summary and discussion on heptazines syntheses and characteristics, we show that starting from well-defined molecules allows a versatility of approaches and a wide tunability of the expected properties. It comes out that the field of monomeric heptazines is now emerging and possibly heading toward maturity, while diverging from the one of polymeric carbon nitrides. It is likely that this area of research will quickly surge to the forefront of the search for active organic molecules, with special attention to the domains of catalysis and organic-based functional materials and devices.

Synthesis of Thiocyameluric Acid C6 N7 S3 H3 , Its Reaction to Alkali Metal Thiocyamelurates and Organic Tris(dithio)cyamelurates.

Thiocyameluric acid C6 N7 S3 H3 , the tri-thio analogue of cyameluric acid, is a key compound for the synthesis of new s-heptazine (tri-s-triazine) derivatives. Here, two different routes for the synthesis of thiocyameluric acid and its reaction to tris(aryldithio)- and tris(alkyldithio)cyamelurates C6 N7 (SSR)3 are reported as well as transformation to alkali metal thiocyamelurates M3 [C6 N7 S3 ], M=Na, K. These compounds were characterised by FTIR, Raman, solution 13 C and 1 H NMR spectroscopies, thermal gravimetric analysis (TGA) and elemental analysis. The three (de)protonation steps of thiocyameluric acid were investigated by acid-base titration followed via UV/Vis absorption spectroscopy. While it was not possible to determine the three pKa values, it could be postulated that the acid strength probably increases in the following order: cyanuric acid (C3 N3 O3 H3 ) < thiocyanuric acid (C3 N3 S3 H3 ) < cyameluric acid (C6 N7 O3 H3 ) < thiocyameluric acid (C6 N7 S3 H3 ). Single crystals of Na3 [C6 N7 S3 ]⋅10 H2 O and K3 [C6 N7 S3 ]⋅6 H2 O were obtained and the structures analyzed by single crystal X-ray diffraction. Additionally, quantum chemical calculations were performed to get insights into the electronic structure of thiocyameluric acid and to clarify the thiol-thione tautomerism. Based on a comparison of calculated and measured vibrational spectra it can be concluded that thiocyameluric acid and the di- and mono-protonated anions exist in the thione form.

Sulfides and Disulfides of s-Triazine: Potential Thermal Thiyl Radical Generators.

A series of aliphatic and aromatic thioethers and dithioethers of s-triazine were synthesised to study their thermal properties, in particular the thermally induced thiyl radical generation ability. Four symmetric s-triazine sulfides of the type (RS)3 C3 N3 , namely 2,4,6-tris(phenylthio)- (1), 2,4,6-tris(para-tolylthio)- (3), 2,4,6-tris(ethylthio)- (5) and 2,4,6-tris(tert-butylthio)-1,3,5-triazine (7), as well as four symmetric s-triazine disulfides of the type (RSS)3 C3 N3 , namely 2,4,6-tris(phenyldithio)- (2), 2,4,6-tris(para-tolyldithio)- (4), 2,4,6-tris(ethyldithio)- (6) and 2,4,6-tris(tert-butyldithio)-1,3,5-triazine (8) were synthesised. All compounds were comprehensively characterised by 1 H and 13 C NMR, infrared and Raman spectroscopy as well as elemental analyses. Single-crystal X-ray diffraction analyses of 1, 2 and 5 are discussed. The thermal behaviour was studied by thermogravimetric analyses coupled with mass spectrometry (TGA-MS) and quantum chemical calculations. Limiting oxygen index (LOI) flammability tests showed that the disulfides are the most promising radical generators, and are most likely suitable flame retardants for selected polymers.

Arylthio- and Arylseleno-Substituted s-Heptazines.

In contrast to numerous thio- and selenocyanuric acid derivatives, sulfur- or selenium-containing s-heptazines have not been reported so far. Thio- and selenocyameluric acid esters were obtained by substitution reactions of s-heptazine chloride C6 N7 Cl3 with aromatic thiols and selenophenol; the resultant white or yellow solids were stable in air. They were comprehensively characterized by elemental analysis, 1 H, 13 C NMR, and IR spectroscopy. The thiocyameluric acid phenyl ester (1, C6 N7 (S(C6 H5 ))3 ) and the corresponding selenium compound (7) formed co-crystals with mesitylene, which were analyzed by single-crystal X-ray diffraction. Both structures showed unusually large channels of ≈12 Šdiameter. The thermal stability was measured by TGA (thermogravimetric analysis), and the flame retardancy of compound 1 was tested in PP by carrying out limiting oxygen index (LOI) measurements, which gave promising results. Quantum chemical calculations of the title compounds were performed to explain the observed properties and structural characteristics.