[2-3H]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-llb-H-benzo[a]-quinolizine), a derivative of the neuroleptic tetrabenazine, binds to the membrane ofpurified bovine chromaffin granules. The chromaffin granules of the adrenal medulla and the ghosts derived from their membranes accumulate catecholamines by a two-step ATP-dependent process (1-4): (i) inward translocation of protons by an electrogenic ATP-dependent H' pump (5-10), and (ii) a monoamine carrier driven by the H' electrochemical gradient (9,(11)(12)(13)(14)(15) and specifically blocked by reserpine or tetrabenazine (16). Whereas the H' pump has been functionally and structurally related to the mitochondrial ATPase complex (17,18), the catecholamine carrier has not yet been characterized. It is assumed to be a protein because the transport is temperature sensitive, shows some stereoselectivity (19), and is affected by the histidine-specific reagent diethylpyrocarbonate (20). The carrier has been solubilized and reconstituted in lipid vesicles (21,22), but this technique has not allowed purification. The carrier activity is difficult to assay because vesicles have to be energized to accumulate catecholamines, thus introducing possible artifacts.An alternate approach to the study of the carrier is the use ofbinding techniques. We have recently shown that, of several inhibitors (tetrabenazine, reserpine, haloperidol, and chlorpromazine), tetrabenazine has the most specific interaction with the carrier (23). In the present communication, we describe the binding of 2-[2-3H]hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3, 4,6,7-hexahydro-llb-H-benzo[a]quinolizine (dihydrotetrabenazine, [3H]TBZOH), a derivative of tetrabenazine to bovine chromaffin granule membrane. A preliminary account of some of these experiments has been published (24).[3H]Dihydrotetrabenazine MATERIALS AND METHODS TBZOH was obtained by reduction of tetrabenazine (Fluka) by NaBH4 in methanol (25).[3H]TBZOH (12 Ci/mmol; 1 Ci = 3.7 x 1010 Bq) was prepared as described (24). Its purity was periodically checked by TLC and, when necessary, the product was repurified (24). Stock solutions (80 ,AM) were made in 100 mM HCl and diluted in water.Bovine chromaffin granule membranes were prepared by osmotic lysis of granules isolated by centrifugation on a 1.6 M sucrose layer (26,27). Membranes were frozen in liquid nitrogen and were stored at -80°C. They were rapidly thawed at 370C, centrifuged at 100,000 x g for 15 min, and resuspended -in 20 mM Hepes KOH buffer containing 0.3 M sucrose.For [3H]TBZOH binding studies, membranes (5-15 ,ug of protein per ml) were incubated at 25°C with various concentrations of [3H]TBZOH in 0.3 M sucrose/20 mM KOH Hepes, pH 7.5. Bound ligand was measured by filtration on Millipore HAWP filters or by centrifugation at full speed in a Beckman Airfuge with cellulose propionate tubes. For filtration, 0.2-to 2.0-ml aliquots of the incubation mixture were diluted in 4 ml of ice-cold 0.3 M sucrose/10 mM KOH Hepes, pH 7.5, containing 125 ...
An adiabatic conformational analysis of serotonin (5-hydroxytryptamine, 5-HT) using quantum chemistry led to six stable conformers that can be either +gauche (Gp), -gauche (Gm), and anti (At) depending upon the value taken by ethylamine side chain and 5-hydroxyl group dihedral angles φ1, φ2, and φ4, respectively. Further vibrational frequency analysis of the GmGp, GmGm, and GmAt conformers with the 5-hydroxyl group in the anti position revealed an additional red-shifted N-H stretch mode band in GmGp and GmGm that is absent in GmAt. This band corresponds to the 5-HT side-chain N-H bond involved in an intramolecular nonbonded interaction with the 5-hydroxy indole ring. The influence of this nonbonded interaction on the electronic distribution was assessed by analysis of the spin-spin coupling constants of GmGp and GmGm that show a marked increase for C2-C3 and C8-C9 bonds in GmGm and GmGp, respectively, with a decrease of their double bond character and an increase of their length. The Atoms in Molecules (AIM), Natural Bond Orbital (NBO), and fluorescence and CD spectra (TDDFT method) analyses confirmed the existence in GmGp and GmGm of a through-space charge-transfer between the HOMO and the HOMO-1 π-orbital of the indole ring and the LUMO σ* N-H antibonding orbital of the ammonium group. The strength of the cation-π interaction was determined by calculating binding energies of the NH4(+)/5-hydroxyindole complexes extracted from stable conformers. The energy decomposition analysis indicated that cationic-π interactions in the GmGp and GmGm conformers are governed by the electrostatic term with significant contributions from polarization and charge transfer. The lower stability of the GmGm over the GmGp comes from a higher exchange repulsion and a weaker polarization contributions. Our results provide insight into the nature of intramolecular forces that influence the conformational properties of 5-HT.
The serotonergic system plays a critical role in a wide variety of physiological and behavioral processes. Dysregulation of the tightly controlled extracellular concentration of serotonin (5-hydroxytryptamine, 5-HT) appears to be at the origin of a host of metabolic and psychiatric disorders. Since the plasma membrane 5-HT transporter (SERT) is the major protagonist in regulating extracellular 5-HT concentration, SERT is the target of most drugs interacting with the serotonergic system. Unfortunately, some of the drugs towards SERT (e.g. amphetamine derivatives) interfere with cell homeostasis leading to cell toxicity. Developing new SERT ligands devoid of any side-effect represents a major priority in the treatment of 5-HT-associated pathologies. Here, we report structure-activity relationships (SAR) and three-dimensional QSAR (3D-QSAR) studies of a library of 121 compounds including 5-HT analogs, harmanes, benzothiazoles, indanones, amphetamine derivatives and substrate-type 5-HT releasers, with the goal of identifying the structural determinants crucial for SERT uptake. In the absence of data about the bioactive form of 5-HT, conformational analysis of 5-HT was performed using quantum chemistry calculations. This led to three 5-HT stable conformers with anti, -gauche and +gauche side-chain conformation. These conformers, used as templates for superimposition with all the library compounds, enabled the design of a reliable 6-points pharmacophore representative of SERT uptake activity. Molecular dynamics (MD) simulations performed with compounds that are efficiently, moderately, poorly or not transported by SERT allowed to assess the validity of our pharmacophore. Altogether, our data provide for the first time a reliable pharmacophore of SERT uptake activity, which may help to the design of new drugs targeting SERT.
The proton transfer equilibrium reactions involving 3-penten-Z-one, 3-methyl-3-buten-Z-one, crotonic acid and methacrylic acid were carried out in an ion Cyclotron resonance (ICR) spectrometer. The semiempirical method MNDO, used to estimate the heats of formation for 14 protonated [C,&,O]' and [Cd-170z]' ions and the energetic aspect of the fragmentations of metastable [CJ3uO]" and [Cd3uOz]+' ions, leads to the conclusion that the ions corresponding to protonation at the carbonyl oxygen are the most stable. Thus the experimentally determined heats of formation of protonated olefinic carbonyl compounds can be attributed to the following structure: [CH3COHCHCHCH31+ (AH, = 490 kJ mol-'1, [CH3COHC(CH3)CH21' (AH, = 502 kJ mol-'1, [HOCOHCHCHCH,I+ (AH, = 330 M mol-') and [HOCOHC(CH3)CH21+
Dissociative ionization of 1,Zepoxy n-alkanes gives rise to abundant [C,H,O]+ ions of structure [CH30CHCHCH21+.Tbis conclusion is drawn from metastable ion analysis and from collisional activation spectra. This fragmentation involves the C-C ring opening and a 1,4-H migration leading to the corresponding enol ether [CH,OCHCHCH,R]+' precursor of [CHJOCHCHCH,]+ fragment. The same isomerization scheme applies to 1,2-epoxy methyl substituted alkanes and 2,3-epoxy n-alkanes. 3 4 7 8 11 12 0 CH, OCH, 13 14 17
The unimolecular dissociations of C, epoxides ions monoor disubstituted at C, give exclusive loss of CH, and exclusive formation of methoxyvinyl carbenium ion, both in the source and in the 2nd field-free region. In the case of the 1,2-disubstituted ion in the 2nd field-free region the loss of ethene is the only pathway, while a competition occurs for the trisubstituted ion leading to [C3H60]"' and [C,H,O]+' ions, the structure of which are demonstrated. The first step of the different mechanisms is the cleavage of the heterocyclic C-C bond.
Mass spectra of the four isomeric ethylbutyl ketones, C2H5CO‐n‐C4H9 (1), C2H5CO‐iso‐C4H9 (2), C2H5CO‐sec‐C4H9 (3) and C2H5 CO‐t‐C4H9 (4), are reported. The structure of the fragment ions produced from precursors of both high and low internal energy were identified by collision experiments in a six‐sector mass spectrometer. Metastable molecular ions 1+˙ and 2+˙ behave identically in expelling CH3˙, C3H6 and C2H5˙ to produce the same fragment ions: [C2H5C(OH)CHCHCH3]+, [C2H5C(OH)CH2]+˙ and a mixture of [CHOHCHCHC2H5]+ and [CHOHCHC(CH3)2]+, respectively. The key isomerization step between 1+˙ and 2+˙ is a 1,2‐enol–olefin shift. Metastable molecular ions 3+˙ and 4+˙ give essentially ethene and ethyl losses, respectively; the fragment ions are [C2H5C(OH)CHCH3]+˙ and [t‐C4H9CO]+. Keto–enol isomerization also occurs to a significant extent for ions 1+˙ and 2+˙ and 3+˙. In contrast, 1,2‐ or 1,3‐ethylhydroxycarbene migrations are only marginal processes.
Experimental gas‐phase acidities of 2‐oxopropanaloximes, XCH2COCHNOH (X H, CH3S, CH3SO, CH3SO2; compounds 1–4), were determined by Fourier transform ion cyclotron resonance (FT‐ICR) spectrometry. The values are δ Gacid° = 1401, 1381, 1360 and 1351 kJ mol−1 for 1, 2, 3, and 4, respectively. Molecular orbital calculations using the semi‐empirical AM1 method provided information on the geometry and relative energy of neutrals species 1–4 and their conjugate bases, together with charge distributions and entropies of deprotonation. It is demonstrated that the proton abstraction occurs preferentially at the oxime function; the formation of an enolate as a conjugate base is unfavourable by 70–140 kJ mol−1. The large variation of the gas‐phase acidities for 1–4 is explained in terms of the field/inductive empirical substituent constant σF. The variation of solution acidities appears to be comparatively strongly attenuated. This attenuation is attributed mainly to charge delocalization in the anion, which was confirmed by charge density calculations.
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