Comparative discriminative stimulus effects of 5-methoxy-N,Ndimethyltryptamine and LSD

Rats were trained to discriminate injections of either 5-OMe DMT (1.5 mg/kg) or LSD (0.096 mg/kg) from saline in a two-lever drug discrimination task. After stable discrimination performances were attained (> 85%) in each group, dose-response generalizations between the two groups of animals were examined. The results revealed that the 5-OMe DMT-stimulus response generalized to LSD and that the LSD-stimulus response generalized to 5-OMe DMT. Furthermore, both the 5-OMe DMT-stimulus and the LSD-stimulus could be significantly attenuated by the serotonin antagonist BC-105. However, the pattern of the doserelated antagonism by BC-105 was different between the drug stimuli. It was concluded that while the discriminative stimulus effects of 5-OMe DMT and LSD may be mediated via a common serotonergic system, the receptor interaction of these agents within that pharmacological system may be somewhat different.     

N1-(Benzenesulfonyl)tryptamines as novel 5-HT6 antagonists

N-Benzenesulfonyl-5-methoxy-N,N-dimethyltryptamine (BS/5-OMe DMT; 5) was shown to bind at human 5-HT6 serotonin receptors with high affinity (Ki = 2.3 nM) relative to serotonin (Ki = 78 nM). Structural variation failed to result in significantly enhanced affinity. BS/5-OMe DMT acts as an antagonist of 5-HT-stimulated adenylate cyclase (pA2 = 8.88 nM) and may represent the first member of a novel class of 5-HT6 antagonists.     

Hallucinogens as a discriminative stimuli: generalization of DOM to a 5-methoxy-N, N-dimethyltryptamine stimulus.

Hallucinogens (psychotomimetic agents) are capable of producing various discriminative stimuli for animals. Serotonergic involvement has been implicated as playing a role in the behavioral effects elicited by, for example, mescaline and DOM. Because certain tryptamine analogs possess high serotonin (5-HT) receptor binding affinities, it was of interest to examine one of the more potent agents. Employing a standard operant test chamber, six rats were trained to respond under a variable-interval 15-second schedule of sweetened-milk reinforcement. 5-Methoxy-N, N-dimethyltryptamine (5-OMe DMT), which possesses a 5-HT receptor affinity much higher than that of mescaline, but nearly equivalent to that of DOM, was found to serve as a discriminative stimulus. Furthermore, the 5-OMe DMT stimulus could be attenuated by the 5-HT antagonist BC-105. The 5-OMe DMT stimulus generalized with DOM suggesting that these two hallucinogens produce qualitatively similar interoceptive cues in rats.     

The phenyltetraene lysophospholipid analog PTE-ET-18-OMe as a fluorescent anisotropy probe of liquid ordered membrane domains (lipid rafts) and ceramide-rich membrane domains

The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15′-phenylpentadeca-8′,10′,12′,14′-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine) is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333-5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997-10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.     

X-ray study on homo-oligopeptides t-BOC-(D-Ala)7-OMe and HCl.H(D-Ala)7-OMe.

Observations of extended peptide chains, whose direction is perpendicular to the fiber axis (cross-beta-structures) have hitherto been confined to fibrous proteins and to some synthetic polydisperse polypeptides of rather low molecular weight. This structure has now been found in some monodisperse linear homo-oligopeptides with aliphatic hydrocarbon side chains. X-ray diffraction photographs of t-Boc-(D-Ala)7-OMe and HCl.H(D-Ala)7-OMe show characteristic reflections for this form. In addition, the good orientation of suitably prepared specimens has enabled a fairly complete determination of the unit cell of the heptapeptides to be made. t-Boc-(D-Ala)7-OMe and HCl.H(D-Ala)7-OMe molecules are packed in a monoclinic lattice with a = 4.80, b = 34.5, c = 5.82 A, beta = 65.6 degrees and a = 4.80, b = 29.5, c = 5.82 A, beta = 65.6 degrees, respectively. It has not been possible to establish whether the arrangement of the chains within the sheets is parallel or antiparallel.     

Partial [alphaMe]Aun scan of [l-Leu11-OMe]-trichogin GA IV, a membrane active synthetic precursor of the natural lipopeptaibol.

We synthesized using solution-phase methods three analogs of [l-Leu11-OMe] trichogin GA IV, a membrane active synthetic precursor of the lipopeptaibol antibiotic in which the N-terminal n-octanoyl group and each of the three Aib residues in positions 1, 4 and 8 are replaced by an acetyl group and the lipophilic Calpha,alpha-disubstituted glycine l-(alphaMe)Aun, respectively [partial (alphaMe)Aun scan]. FT-IR absorption and CD analyses unequivocally show that the main three-dimensional structural features of [l-Leu11-OMe] trichogin GA IV are preserved in the analogs. Also, [l-Leu11-OMe] trichogin GA IV and the three Nalpha-acetylated l-(alphaMe)Aun analogs exhibit strictly comparable membrane-modifying properties. Taken together, these results strongly favor the conclusion that a shift of the long hydrocarbon moiety from the Nalpha-blocking group to the side-chain of the 1, 4 or 8 residue does not have any significant effect on the conformational properties or the membrane activity of [l-Leu11-OMe] trichogin GA IV and, by extension, of the natural lipopeptaibol.     

Synthesis of Capped RNA using a DMT Group as a Purification Handle

We report a new method for synthesis of capped RNA or 2′-OMe RNA that uses a N^2?4,4′-dimethoxytrityl (DMT) group as a lipophilic purification handle to allow convenient isolation and purification of the capped RNA. The DMT group is easily removed under mild conditions without degradation of the cap. We have used this approach to prepare capped 10- and 20-mers. This method is compatible with the many condensation reactions that have been reported for preparation of capped RNA or cap analogues.     

Alteration of cross-linking selectivity with the 2′-OMe analogue of 2-amino-6-vinylpurine and evaluation of antisense effects

We previously reported that oligodeoxynucleotides containing 2-amino-6-vinylpurine (2-AVP: 1) exhibit efficient selective cross-linking to cytosine. In this study, the 2′-OMe nucleoside analogue (2) of 2-AVP was designed in order to increase its affinity to RNA and enhance metabolic stability. It has been demonstrated that 2′-OMe oligonucleotides bearing 2 achieve highly selective cross-linking to the thymine base in DNA and show higher antisense effect on luciferase production in cell lysate.     

The phenyltetraene lysophospholipid analog PTE-ET-18-OMe as a fluorescent anisotropy probe of liquid ordered membrane domains (lipid rafts) and ceramide-rich membrane domains

The conjugated phenyltetraene PTE-ET-18-OMe (all-(E)-1-O-(15'-phenylpentadeca-8',10',12',14'-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine) is a recently developed fluorescent lysophospholipid analog of edelfosine, (Quesada et al. (2004) J. Med. Chem. 47, 5333-5335). We investigated the use of this analog as a probe of membrane structure. PTE-ET-18-OMe was found to have several properties that are favorable for fluorescence anisotropy (polarization) experiments in membranes, including low fluorescence in water and moderately strong association with lipid bilayers. PTE-ET-18-OMe has absorbance and fluorescence properties similar to those of diphenylhexatriene (DPH) probes, with about as large a difference between its fluorescence anisotropy in liquid disordered (Ld) and ordered states (gel and Lo) as observed for DPH. Also like DPH, PTE-ET-18-OMe has a moderate affinity for both gel state ordered domains and Lo state ordered domains (rafts). However, unlike fluorescent sterols or DPH (Megha and London (2004) J. Biol. Chem. 279, 9997-10004), PTE-ET-18-OMe is not displaced from ordered domains by ceramide. Also unlike DPH, PTE-ET-18-OMe shows only slow exchange between the inner and outer leaflets of membrane bilayers, and can thus be used to examine anisotropy of an individual leaflet of a lipid bilayer. Since PTE-ET-18-OMe is a zwitterionic molecule, it should not be as influenced by electrostatic interactions as are other probes that do not cross the lipid bilayer but have a net charge. We conclude that PTE-ET-18-OMe has some unique properties that should make it a useful fluorescence probe of membrane structure.     

Synthesis and kinetic studies of new bipyridyl platinum(II) phenoxide complexes by phase transfer catalysis: Crystal structure of [(bipy)Pt(OC6H4-4-OMe)2]

The synthesis of new platinum bipy (bipy = 2,2′-bipyridyl) complexes containing phenoxide ligands is reported, together with kinetic studies of their oxidative addition reactions with MeI to produce phenoxo platinum(IV) complexes. Complexes of the form [(bipy)Pt(OC₆H₄-4-X)₂] (X = OCH₃, CH₃, H, Br, Cl) are prepared by the reaction of the chloro complex [(bipy)PtCl₂] with substituted phenols and KOH in a two phase system of water and chloroform in the presence of benzyl triphenylphosphonium chloride. Platinum(IV) complexes are formed by oxidative addition of MeI to the platinum(II) complexes obtained. The complexes are characterized by elemental analysis, UV-Vis, IR, mass spectrometry and ¹H and ¹³C NMR spectroscopy.The reaction of methyl iodide with [(bipy)Pt(OC₆H₄-4-OMe)₂] to give [(bipy)PtMe(I)(OC₆H₄-4-OMe)₂] follows the rate law rate = k₂[(bipy)Pt(OC₆H₄-4-OMe)₂][MeI]. The values of k₂ increase with increasing polarity of the solvent, suggesting a polar transition state for the reaction.     

Polymorphisms of metal transporter genes DMT1 and ATP7A in Wilson's disease

Wilson's disease (WND) is an inherited disorder of copper metabolism. Divalent metal transporter1 (DMT1) and ATP7A play important roles in metal transport in humans. The frequency of two single nucleotide polymorphisms of the DMT1 gene: DMT1 IVS4 C>A, DMT1 11245 T>C and two of the ATP7A gene: rs1062472 T>C, ATP7A rs 2227291 G>C have been evaluated in a population of 108 Wilson's disease patients and 108 sex- and age-matched healthy volunteers. The DMT1 IVS4 C(+) allele occurred more frequently in WND than in the healthy controls. The allele frequencies of other studied polymorphisms in WND group were in line with frequencies obtained for healthy volunteers. Neither of the polymorphisms had an impact on the age at onset or clinical phenotype of WND.     

Evidence for 5-HT2 involvement in the mechanism of action of hallucinogenic agents

The affinities (Ki values) of twenty two psycho-active agents, including LSD, 5-OMe DMT and a series of phenalkylamine derivatives, for cortical 5-HT1 and 5-HT2 binding sites were compared with two measures of behavioral activity. It was found that a significant correlation (r = 0.938) exists between the 5-HT2 binding affinities of these agents and their ED50 values as determined in tests of stimulus generalization using 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) as the training drug. Furthermore, for fifteen of these agents where human data were available, a significant correlation (r = 0.924) also exists between 5-HT2 binding affinities and their human hallucinogenic potencies. The results of this study suggest that the mechanism of action of these agents involves 5-HT2-related events.     

Synthesis and characterization of ether-derivatized aminophosphines and their application in C-C coupling reactions

Four new bis(phosphino)amine ligands (Ph₂P)₂N-C₆H₃-R, where R = 3,5-OMe (1), 2,5-OMe (2), 2,4-OMe (3) or 3,4-OMe (4), were prepared via aminolysis of the corresponding dimethoxyanilines with 2 equiv. of diphenylphosphine chloride in the presence of triethyl amine. Oxidation of these ligands with aqueous H₂O₂, elemental S₈ or Se powder afforded the corresponding chalcogen oxides 1a-4a, sulfides 1b-4b and selenides 1c-4c in good yields. Reaction of 1-4 with [MCl₂(cod)] (M = Pt, Pd; cod = cycloocta-1,5-diene) in equimolar ratios afforded cis-[MCl₂{(Ph₂P)₂N-C₆H₃-R}] (M = Pt; R = 3,5-OMe 1d, R = 2,5-OMe 2d, R = 2,4-OMe 3d, and R = 3,4-OMe 4d. M = Pd; R = 3,5-OMe 1e, R = 2,5-OMe 2e, R = 2,4-OMe 3e, and R = 3,4-OMe 4e). Similarly, reaction of [Cu(CH₃CN)₄]PF₆ with the 1-4 in 1:2 ratio gave [Cu{(Ph₂P)₂N-C₆H₃-R}₂]PF₆ (R = 3,5-OMe 1f, 2,5-OMe 2f, 2,4-OMe 3f and 3,4-OMe 4f). All new compounds were fully characterized by spectroscopy and elemental analysis and the molecular structures of seven representative compounds were determined by single-crystal X-ray crystallography. In addition, the palladium complexes were investigated as pre-catalysts in C-C coupling reactions.     

HOMO based two electrons and one-electron oxidation in planar and nonplanar methoxy-substituted nickel tetraphenylporphyrins

A series of methoxy-substituted nickel tetra phenylporphyrins were synthesized by a direct method to correlate their electrochemical properties with nonplanar distortion of the porphyrin rings. From the newly synthesized nickel tetraphenylporphyrin complexes with o,p-dimethoxy [T(2,4-OMe)PPNi], o,m-dimethoxy [T(2,5-OMe)PPNi], m,p-dimethoxy [T(3,4-OMe)PPNi], and o,o′, p-trimethoxy [T(2,4,6-OMe)PPNi] substituted phenyl groups, the planar T(2,4,6-OMe)PPNi structure elucidated by X-ray crystallography shows two well separated one-electron oxidations at +0.85 V and +1.22 V. Among previously reported nickel tetraphenylporphyrins with p-methoxy [T(4-OMe)PPNi] and m,m′,p-trimethoxy [T(3,4,5-OMe)PPNi] substituted phenyl groups, the structurally characterized highly distorted T(3,4,5-OMe)PPNi shows a two-electron oxidation occurring at the potential of +1.05 V. Density functional theory (DFT) calculations performed on the nickel porphine systems show lowering of HOMO-LUMO gap in distorted ruffled or saddled systems with change in the energy level of HOMO and HOMO−1. While the decrease in HOMO-LUMO gap can explain the lowering in the difference between first oxidation potential and first reduction potential, Δ|Ox1 − Red1|, the degeneracy or near-degeneracy of HOMO and HOMO−1 in distorted systems explains the two-electron oxidation process due to pseudo-Jahn-Teller distortion in highly distorted nickel porphyrins.     

Regularly alternating L,D-peptides. II. The double-stranded right-handed antiparallel beta-helix in the structure of t-Boc-(L-Phe-D-Phe)4-OMe

The crystal structure of Boc-(L-Phe-D-Phe)4-OMe has been determined by x-ray diffraction analysis. The peptide crystallizes in the triclinic system, space group P1 with a = 15.290 A, b = 15.163 A, c = 19.789 A, alpha = 102.49 degrees, beta = 96.59 degrees, gamma = 74.22 degrees, and Z = 2. The structure has been solved by coupling of the molecular replacement technique and expansion by tangent formula refinement of the set of known phases. Several cycles of Fourier calculations and least-squares refinement led to the location of 194 atoms of the two independent octapeptide chains and few molecules of cocrystallized solvent (chloroform, water, and methanol). The isotropic refinement converged to R = 0.13 for the 3077 "observed" reflections. The two independent octapeptide molecule form a dimer in the solid state: the two chains are associated by interstrand hydrogen bonds (12 of the type N-H ... O = C) with the formation of a double-stranded antiparallel right-handed -- beta 5.6-helix. These double helices can be represented as a cylinder with a hydrophilic inner core represented by the peptide units and an hydrophobic exterior constituted by the aromatic moieties. The dimensions of the cylinder are equal to those observed for Boc-(L-Val-D-Val)4-OMe. In the solid state the dimers pack with each other in an hexagonal fashion with the formation of layers; between the layers, solvent molecules fill empty spaces.     

Lead and Cadmium Synergistically Enhance the Expression of Divalent Metal Transporter 1 Protein in Central Nervous System of Developing Rats

Divalent metal transporter 1 (DMT1) can transport a large range of ions, including toxic lead (Pb) and cadmium (Cd), across membranes. In this study, a total of 24 rats were divided into four groups for intragastrical perfusion treatment: control, Pb alone, Cd alone, and Pb + Cd. Pb and Cd contents in blood were detected, and the mRNA and protein levels of DMT1 were analyzed in the cerebellum, cortex, and hippocampus. Both Pb and Cd levels were elevated in all groups perfused with Pb and/or Cd, except for Pb level in the Cd-alone group (P < 0.05). The mRNA level of DMT1 did not differ among the four groups (P > 0.05). However, the DMT1 protein expression was significantly increased by 0.9-, 1.0-, and 1.1-fold in cerebellum, cortex, and hippocampus of the Pb + Cd group than in controls, respectively. Pb and Cd exposure can synergistically induce DMT1 protein synthesis and has implications for transportation of toxic ions in the developing rat’s brain. Chengwu Gu and Songjian Chen contributed equally to this work, they are joint first authors.     

Structural Determinants of the 5′-Methylthioinosine Specificity of Plasmodium Purine Nucleoside Phosphorylase

Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5′-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5′-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5′-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5′-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket.     

Hydration of short DNA, RNA and 2'-OMe oligonucleotides determined by osmotic stressing

Studies on hydration are important for better understanding of structure and function of nucleic acids. We compared the hydration of self-complementary DNA, RNA and 2′-O-methyl (2′-OMe) oligonucleotides GCGAAUUCGC, (UA)₆ and (CG)₃ using the osmotic stressing method. The number of water molecules released upon melting of oligonucleotide duplexes, Δn_W, was calculated from the dependence of melting temperature on water activity and the enthalpy, both measured with UV thermal melting experiments. The water activity was changed by addition of ethylene glycol, glycerol and acetamide as small organic co-solutes. The Δn_W was 3–4 for RNA duplexes and 2–3 for DNA and 2′-OMe duplexes. Thus, the RNA duplexes were hydrated more than the DNA and the 2′-OMe oligonucleotide duplexes by approximately one to two water molecules depending on the sequence. Consistent with previous studies, GC base pairs were hydrated more than AU pairs in RNA, whereas in DNA and 2′-OMe oligonucleotides the difference in hydration between these two base pairs was relatively small. Our data suggest that the better hydration of RNA contributes to the increased enthalpic stability of RNA duplexes compared with DNA duplexes.     

Solution-state structure of a fully alternately 2��-F/2��-OMe modified 42-nt dimeric siRNA construct

A high-resolution solution structure of a stable 42-nt RNA dimeric construct has been derived based on a high number of NMR observables including nuclear overhauser effects (NOEs), J-coupling constants and residual dipolar couplings (RDCs), which were all obtained with isotopically unlabeled molecules. Two 21-nt siRNA that efficiently hybridize consist of ribose units that were alternately substituted by 2′-fluoro or 2′-methoxy groups. Structure calculations utilized a set of H-F RDC values for all 21 2′-fluoro modified nucleotides under conditions of weak alignment achieved by Pf1 phages. A completely 2′-F/2′-OMe modified dimeric RNA construct adopts an antiparallel double-helical structure consisting of 19 Watson–Crick base pairs with additional 3′ UU overhangs and a 5′ phosphate group on the antisense strand. NMR data suggest that the stability of individual base pairs is not uniform throughout the construct. While most of the double helical segment exhibits well dispersed imino resonances, the last three base pairs either display uncharacteristic chemical shifts of imino protons or absence of imino resonances even at lower temperatures. Accessibility of imino protons to solvent exchange suggests a difference in stability of duplex ends, which might be of importance for incorporation of the guide siRNA strand into a RISC.     

Degradation of Dimethyl Isophthalate by Viarovorax paradoxus Strain T4 Isolated from Deep-Ocean Sediment of the South China Sea

Viarovorax paradoxusT4 strain was isolated from deep-ocean sediment and demonstrated to be able to degrade dimethyl isophthalate (DMI). When DMI was utilized as the sole source of carbon and energy, it was transformed by hydrolysis initially, forming monomethyl isophthalate (MMI) and isophthalate acid (IA) as degradation intermediates. DMI and MMI were completely transformed to MMI and IA in about 100 h, respectively. Degradation of IA was completed in about 55 h. Analysis of total organic carbon in the culture medium confirmed that more than 80% of the substrate carbon was mineralized. Bacterial esterase induced by a range of substrates could be assessed using p-nitrophenyl acetate as the common substrate using crude enzyme preparation. The decreasing trend of K _m values derived from the Michaelis–Menten equation was dimethyl phthalate (DMP) > monomethyl phthalate (MMP) > dimethyl terephthalate (DMT) > Liver esterase > DMI > MMI > monomethyl terephthalate (MMT), indicating that higher K _m values were obtained by di-esters than mono-ester and the esters induced by terephthalate esters showed the highest activity. This investigation suggests that biochemical pathways for phthalate esters share many common characteristics and the esterases induced by different substrates are highly specific.