Mitragynine concentrations in two fatalities.

Two cases of fatalities are reported of which the recreational use of Mitragyna speciosa ("kratom") could be confirmed. One of these cases presents with one of the highest postmortem mitragynine concentrations published to date. Our results show that even extremely high mitragynine blood concentrations following the consumption of kratom do not necessarily have to be the direct cause of death in such fatalities as a result of an acute overdose. The two cases are compared with regard to the differences in mitragynine concentrations detected and the role of mitragynine in the death of the subjects. Irrespective of the big differences in mitragynine concentrations in the postmortem blood samples, mitragynine was not the primary cause of death in either of the two cases reported here. Additionally, by rough estimation, a significant difference in ratio of mitragynine to its diastereomers in the blood and urine samples between the two cases could be seen.

Detection of methaemoglobinaemia and its application in 'poppers' abuse: maintaining the right balance between reduction and autooxidation during storage.

In our study, we analysed the effect of a variety of storage conditions on the methaemoglobin (MetHb) content of blood samples obtained from altogether 110 deceased subjects with diverse causes of death, including three 'poppers'-related fatalities. The obtained results were compared to data from blood samples of six living, healthy subjects. Results obtained from the spectrophotometric measurement of blood MetHb content suggest that storage at room temperature (RT) and storage at -20 °C result in either highly fluctuating values, as was the case for the RT samples, or values much higher than the initial MetHb concentrations when stored at -20 °C. Blood samples at 4 °C showed more stable MetHb levels, which, however, increased with up to 4 % of the initial value after only 3 weeks of storage. These factors pose a problem in forensic toxicology, especially in nitrite abuse cases, where the involvement of such substance abuse is often unknown at the time of blood sampling and thus often requires longer storage times. Nevertheless, even after the storage of blood samples over several months at 4 and -20 °C, 'poppers' cases still show a significantly higher MetHb concentration as compared to non-'poppers' samples that were stored for the same time period under identical conditions.

Synthesis and Biological Evaluation of Pentacycloundecylamines and Triquinylamines as Voltage-Gated Calcium Channel Blockers.

Preclinical studies for neurodegenerative diseases have shown a multi-targeted approach to be successful in the treatment of these complex disorders with several pathoetiological pathways. Polycyclic compounds, such as NGP1-01 (7a), have demonstrated the ability to target multiple mechanisms of the complex etiology and are referred to as multifunctional compounds. These compounds have served as scaffolds with the ability to attenuate Ca(2+) overload and excitotoxicity through several pathways. In this study, our focus was on mitigating Ca(2+) overload through the L-type calcium channels (LTCC). Here, we report the synthesis and biological evaluation of several novel polycyclic compounds. We determined the IC50 values for both the pentacycloundecylamines and the triquinylamines by means of a high-throughput fluorescence calcium flux assay utilizing Fura-2/AM. The potential of these compounds to offer protection against hydrogen peroxide-induced cell death was also evaluated. Overall, 8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6) .0(3,10) .0(5,9) ]undecane (NGP1-01, 7a) had the most favorable pharmacological profile with an IC50 value of 86 µM for LTCC inhibition and significant reduction of hydrogen peroxide-induced cell death. In general, the triquinylamines were more active as LTCC blockers than the oxa-pentacycloundecylamines. The aza-pentacycloundecylamines were potent LTCC inhibitors, with 8-hydroxy-N-phenylethyl-8,11-azapentacyclo[5.4.0.0(2,6) .0(3,10) .0(5,9) ]undecane (8b) also able to offer significant protection in the cell viability assays.

Intermolecular 'cross-torque': the N4-cytosine propargyl residue is rotated to the 'CH'-edge as a result of Watson-Crick interaction.

Propargyl groups are attractive functional groups for labeling purposes, as they allow CuAAC-mediated bioconjugation. Their size minimally exceeds that of a methyl group, the latter being frequent in natural nucleotide modifications. To understand under which circumstances propargyl-containing oligodeoxynucleotides preserve base pairing, we focused on the exocyclic amine of cytidine. Residues attached to the exocyclic N4 may orient away from or toward the Watson-Crick face, ensuing dramatic alteration of base pairing properties. ROESY-NMR experiments suggest a uniform orientation toward the Watson-Crick face of N(4)-propargyl residues in derivatives of both deoxycytidine and 5-methyl-deoxycytidine. In oligodeoxynucleotides, however, UV-melting indicated that N(4)-propargyl-deoxycytidine undergoes standard base pairing. This implies a rotation of the propargyl moiety toward the 'CH'-edge as a result of base pairing on the Watson-Crick face. In oligonucleotides containing the corresponding 5-methyl-deoxycytidine derivative, dramatically reduced melting temperatures indicate impaired Watson-Crick base pairing. This was attributed to a steric clash of the propargyl moiety with the 5-methyl group, which prevents back rotation to the 'CH'-edge, consequently preventing Watson-Crick geometry. Our results emphasize the tendency of an opposing nucleic acid strand to mechanically rotate single N(4)-substituents to make way for Watson-Crick base pairing, providing no steric hindrance is present on the 'CH'-edge.

A modified guanosine phosphoramidite for click functionalization of RNA on the sugar edge.

A propargyl containing guanosine phosphoramidite was synthesized and incorporated into siRNA, enabling click-ligation with an azido fluorophore onto the nucleobase sugar edge. Duplex stability was not affected by labeling at this new site, which allowed deconvolution of the effects of label, structure and attachment site on RNAi activity.