Lysergic N,N-diethylamide (LSD) is perhaps one of the most intriguing psychoactive substances known and numerous analogs have been explored to a varying extent in previous decades. In 2013, N6-allyl-6-norlysergic acid diethylamide (AL-LAD) and (2’S,4’S)-lysergic acid 2,4-dimethylazetidide (LSZ) have appeared on the ‘research chemicals’ / new psychoactive substances (NPS) market in both powdered and blotter form. This study reports the analytical characterization of powdered AL-LAD and LSZ tartrate samples and their semi-quantitative determination on blotter paper. Included in this study was the use of nuclear magnetic resonance spectroscopy, gas chromatography mass spectrometry (MS), low and high-resolution electrospray MS(/MS), high performance liquid chromatography diode array detection and GC solid-state infrared analysis. One feature shared by serotonergic psychedelics, such as LSD, is the ability to mediate behavioral responses via activation of 5-HT2A receptors. Both AL-LAD and LSZ displayed LSD-like responses in male C57BL/6J mice when employing the head-twitch response (HTR) assay. AL-LAD and LSZ produced nearly identical inverted-U-shaped dose-dependent effects, with the maximal responses occurring at 200 µg/kg. Analysis of the dose-responses by nonlinear regression confirmed that LSZ (ED50 = 114.2 nmol/kg) was equipotent to LSD (ED50 = 132.8 nmol/kg) in mice, whereas AL-LAD was slightly less potent (ED50 = 174.9 nmol/kg). The extent to which a comparison in potency can be translated directly to humans requires further investigation. Availability of both chemical and pharmacological data obtained from NPS as they appear on the market provides important data to research communities that are interested in various aspects related substance use and forensic identification.

To better understand why aminoacyl-tRNAs (aa-tRNAs) have evolved to bind bacterial elongation factor Tu (EF-Tu) with uniform affinities, mutant tRNAs with differing affinities for EF-Tu were assayed for decoding on Escherichia coli ribosomes. At saturating EF-Tu concentrations, weaker-binding aa-tRNAs decode their cognate codons similarly to wild-type tRNAs. However, tighter-binding aa-tRNAs show reduced rates of peptide bond formation due to slow release from EF-Tu•GDP. Thus, the affinities of aa-tRNAs for EF-Tu are constrained to be uniform by their need to bind tightly enough to form the ternary complex but weakly enough to release from EF-Tu during decoding. Consistent with available crystal structures, the identity of the esterified amino acid and three base pairs in the T stem of tRNA combine to define the affinity of each aa-tRNA for EF-Tu, both off and on the ribosome.T he ternary complex of bacterial elongation factor Tu (EF-Tu), GTP and aminoacyl-tRNA (aa-tRNA) binds to the ribosome and participates in a multistep decoding pathway in which GTP is hydrolyzed, EF-Tu•GDP is released, and the aa-tRNA enters the ribosomal A site (1-6). Although all elongator aa-tRNAs bind EF-Tu•GTP with similar affinities (7-9), studies with misacylated tRNAs reveal that the protein shows substantial specificity for both the esterified amino acid and the tRNA body (10-12). The nearly uniform EF-Tu binding affinity observed for tRNAs acylated with their correct (cognate) amino acid occurs because the sequence of each tRNA has evolved to compensate for the variable thermodynamic contribution of the esterified amino acid. Thus, weak-binding esterified amino acids such as glycine and alanine have corresponding tRNAs that bind the protein tightly, while tight-binding amino acids such as tyrosine or glutamine have corresponding tRNAs that bind poorly. The crystal structure of Thermus aquaticus EF-Tu•GTP bound to Saccharomyces cerevisiae Phe-tRNA Phe (13) reveals that the protein primarily forms extensive interactions with the helical phosphodiester backbone of the acceptor and T stems of tRNA Phe . Recent protein (14) and tRNA (15, 16) mutagenesis experiments indicate that much of the specificity is the result of interactions made between three amino acids of EF-Tu and three adjacent base pairs in the T stem (16). Additional mutagenesis experiments indicate that the thermodynamic contribution of each of the three base pairs is independent of the others, making it possible to adjust the affinity of aa-tRNAs to EF-Tu in a predictable manner.Although a detailed structural and thermodynamic understanding of how EF-Tu achieves uniform binding with different aa-tRNAs is beginning to emerge, the underlying selective pressures that lead to uniform binding are less clear. While aa-tRNAs must bind EF-Tu tightly enough to participate in translation, the high intracellular concentration of EF-Tu (17) ensures that they do not significantly compete with one another for the protein.It therefore seems unlikely that a minimum threshold binding af...

Measuring the binding affinities of 42 single base pair mutants in the acceptor and T-stems of S. cerevisiae tRNAPhe to T. thermophilus EF-Tu revealed that most of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into E. coli tRNALeuCAG resulted in similar changes in the binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNAPhe resulted in chimeras with EF-Tu binding affinities similar to the donor tRNA. Finally, analysis of double and triple base pair mutants of tRNAPhe shows that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the T-stem primarily account for tRNA binding specificity to EF-Tu.

The 5-HT2A receptor is thought to be the primary target for psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) and other serotonergic hallucinogens (psychedelic drugs). Although a large amount of experimental work has been conducted to characterize the pharmacology of psilocybin and its dephosphorylated metabolite psilocin (4-hydroxy-N,N-dimethyltryptamine), there has been little systematic investigation of the structure–activity relationships (SAR) of 4-substituted tryptamine derivatives. In addition, structural analogs of psilocybin containing a 4-acetoxy group, such as 4-acetoxy-N,N-dimethyltryptamine (4-AcO-DMT), have appeared as new designer drugs, but almost nothing is known about their pharmacological effects. To address the gap of information, studies were conducted with 17 tryptamines containing a variety of symmetrical and asymmetrical N,N-dialkyl substituents and either a 4-hydroxy or 4-acetoxy group. Calcium mobilization assays were conducted to assess functional activity at human and mouse 5-HT2 subtypes. Head-twitch response (HTR) studies were conducted in C57BL/6J mice to assess 5-HT2A activation in vivo. All of the compounds acted as full or partial agonists at 5-HT2 subtypes, displaying similar potencies at 5-HT2A and 5-HT2B receptors, but some tryptamines with bulkier N-alkyl groups had lower potency at 5-HT2C receptors and higher 5-HT2B receptor efficacy. In addition, O-acetylation reduced the in vitro 5-HT2A potency of 4-hydroxy-N,N-dialkyltryptamines by about 10- to 20-fold but did not alter agonist efficacy. All of the compounds induce head twitches in mice, consistent with an LSD-like behavioral profile. In contrast to the functional data, acetylation of the 4-hydroxy group had little effect on HTR potency, suggesting that O-acetylated tryptamines may be deacetylated in vivo, acting as prodrugs. In summary, the tryptamine derivatives have psilocybin-like pharmacological properties, supporting their classification as psychedelic drugs.

Background:The cognate-esterified amino acid is critical for optimal delivery of aminoacyl-tRNAs to the ribosome by EF-Tu. Results: Mutation of His-66 in EF-Tu alters the specific binding of many, but not all aminoacyl-tRNAs, but does not affect decoding. Conclusion: His-66 is critical for the specificity of EF-Tu for the esterified amino acid. Significance: Selective mutation of His-66 could improve the incorporation efficiency of unnatural amino acids into proteins.