Affinity of Nicotinoids to a Model Nicotinic Acetylcholine Receptor (nAChR) Binding Pocket in the Human Brain.

The binding affinity of nicotinoids to the binding residues of the α4ß2 variant of the nicotinic acetylcholine receptor (nAChR) was identified as a strong predictor of the nicotinoid's addictive character. Using ab initio calculations for model binding pockets of increasing size composed of 3, 6, and 14 amino acids (3AA, 6AA, and 14AA) that are derived from the crystal structure, the differences in binding affinity of 6 nicotinoids, namely, nicotine (NIC), nornicotine (NOR), anabasine (ANB), anatabine (ANT), myosmine (MYO), and cotinine (COT) were correlated to their previously reported doses required for increases in intracranial self-stimulation (ICSS) thresholds, a metric for their addictive function. By employing the many-body decomposition, the differences in the binding affinities of the various nicotinoids could be attributed mainly to the proton exchange energy between the pyridine and non-pyridine rings of the nicotinoids and the interactions between them and a handful of proximal amino acids, namely Trp156, Trpß57, Tyr100, and Tyr204. Interactions between the guest nicotinoid and the amino acids of the binding pocket were found to be mainly classical in nature, except for those between the nicotinoid and Trp156. The larger pockets were found to model binding structures more accurately and predicted the addictive character of all nicotinoids, while smaller models, which are more computationally feasible, would only predict the addictive character of nicotinoids that are similar to nicotine. The present study identifies the binding affinity of the guest nicotinoid to the host binding pocket as a strong descriptor of the nicotinoid's addiction potential, and as such it can be employed as a fast-screening technique for the potential addiction of nicotine analogs.

Chiral determination of nornicotine, anatabine and anabasine in tobacco by achiral gas chromatography with (1S)-(-)-camphanic chloride derivatization: Application to enantiomeric profiling of cultivars and curing processes.

The alkaloid enantiomers are well-known to have different physiological and pharmacological effects, and to play an important role in enantioselectivity metabolism with enzymes catalysis in tobacco plants. Here, we developed an improved method for simultaneous and high-precision determination of the individual enantiomers of nornicotine, anatabine and anabasine in four tobacco matrices, based on an achiral gas chromatography-nitrogen phosphorus detector (GCNPD) with commonly available Rtx-200 column using (1S)-(-)-camphanic chloride derivatization. The method development consists of the optimization of extraction and derivatization, screening of achiral column, analysis of the fragmentation mechanisms and evaluation of matrix effect (ME). Under the optimized experimental conditions, the current method exhibited excellent detection capability for the alkaloid enantiomers, with coefficients of determination (R2) > 0.9989 and normality test of residuals P > 0.05 in linear regression parameters. The ME can be neglected for the camphanic derivatives. The limit of detection (LOD) and limit of quantitation (LOQ) ranged from 0.087 to 0.24 µg g - 1 and 0.29 to 0.81 µg g - 1, respectively. The recoveries and within-laboratory relative standard deviations (RSDR) were 94.3%~104.2% and 0.51%~3.89%, respectively. The developed method was successfully applied to determine the enantiomeric profiling of cultivars and curing processes. Tobacco cultivars had a significant impact on the nornicotine, anatabine, anabasine concentration and enantiomeric fraction (EF) of (R)-nornicotine, whereas the only significant change induced by the curing processes was an increase in the EF of (R)-anabasine.

Fully automated determination of nicotine and its major metabolites in whole blood by means of a DBS online-SPE LC-HR-MS/MS approach for sports drug testing.

Dried blood spots (DBS) represent a sample matrix collected under minimal-invasive, straightforward and robust conditions. DBS specimens have been shown to provide appropriate test material for different analytical disciplines, e.g., preclinical drug development, therapeutic drug monitoring, forensic toxicology and diagnostic analysis of metabolic disorders in newborns. However, the sample preparation has occasionally been reported as laborious and time consuming. In order to minimize the manual workload and to substantiate the suitability of DBS for high sample-throughput, the automation of sample preparation processes is of paramount interest. In the current study, the development and validation of a fully automated DBS extraction method coupled to online solid-phase extraction using the example of nicotine, its major metabolites nornicotine, cotinine and trans-3'-hydroxycotinine and the tobacco alkaloids anabasine and anatabine is presented, based on the rationale that the use of nicotine-containing products for performance-enhancing purposes has been monitored by the World Anti-Doping Agency (WADA) for several years. Automation-derived DBS sample extracts were directed online to liquid chromatography high resolution/high mass accuracy tandem mass spectrometry, and target analytes were determined with support of four deuterated internal standards. Validation of the method yielded precise (CV <7.5% for intraday and <12.3% for interday measurements) and linear (r(2)>0.998) results. The limit of detection was established at 5 ng mL(-1) for all studied compounds, the extraction recovery ranged from 25 to 44%, and no matrix effects were observed. To exemplify the applicability of the DBS online-SPE LC-MS/MS approach for sports drug testing purposes, the method was applied to authentic DBS samples obtained from smokers, snus users, and e-cigarette users. Statistical evaluation of the obtained results indicated differences in metabolic behavior depending on the route of administration (inhalative versus buccal absorption) in terms of the ratio of nicotine and nornicotine.

Development and characterization of the α3ß4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts.

The α3ß4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3ß4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3ß4 and α3ß4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3ß4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3ß4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3ß4 and α3ß4α5 nAChRs.

Conformational properties of chiral tobacco alkaloids by DFT calculations and vibrational circular dichroism: (-)-S-anabasine.

A thorough DFT and MM study of the conformational landscape, molecular and electronic structures of (-)-S-anabasine is reported aimed to reveal the mechanism controlling its conformational preference. Although the conformational flexibility and diversity of this system is quite extensive, only two structures are populated both in gas-phase and solution (CCl4 and DMSO). NBO-aided electronic structure analyses performed for the eight conformers representing minima in the potential energy surface of (-)-S-anabasine indicate that both steric and electrostatic factors are determinant in the conformational distribution of the sample in gas phase. Nonetheless, hyperconjugative effects are the key force tipping the balance in the conformational equilibrium between the two main rotamers. Increasing the polarity of the medium (using the IEF-PCM formalism) barely affect the conformational energy profile, although a slight increase in the theoretical population of those structures more affected by electrostatic interactions is predicted. The validity of the theoretical models and calculated conformers populations are endorsed by the accurate reproduction of the IR and VCD spectra (recorded in pure liquid and in CCl4 solution) of the sample (that have been firstly recorded and assigned in the present work) which are consistent with the occurrence of a 2:1 conformational ratio.

The effect of intermittent dosing of Nicotiana glauca on teratogenesis in goats.

Sustained inhibition of fetal movement in livestock species, induced by several poisonous plants, can result in numerous skeletal-contracture malformations. Lupines are responsible for a condition in cattle referred to as "crooked calf syndrome" that occurs when pregnant cattle graze teratogenic lupines. Similar malformations are also seen in animals poisoned by Conium maculatum (coniine) and Nicotiana glauca (anabasine). A proposed management strategy to limit these types of birth defects includes utilizing an intermittent grazing schedule to allow short durations of grazing lupine-infested areas interrupted by movement to a lupine-free pasture. The objective of this study was to use a goat model to determine if an intermittent schedule of five continuous days on treatment followed by two days off treatment would be sufficient to decrease, or prevent, the incidence of anabasine-induced malformations. The data from this study suggest that, for N. glauca in goats, the intermittent grazing program of five days exposure with two days of non-exposure is insufficient to prevent significant skeletal malformations from occurring. However, this study did demonstrate an inverse relationship between the amount of serum anabasine in the dam and the extent of fetal movement.

The only African wild tobacco, Nicotiana africana: alkaloid content and the effect of herbivory.

Herbivory in some Nicotiana species is known to induce alkaloid production. This study examined herbivore-induced defenses in the nornicotine-rich African tobacco N. africana, the only Nicotiana species indigenous to Africa. We tested the predictions that: 1) N. africana will have high constitutive levels of leaf, flower and nectar alkaloids; 2) leaf herbivory by the African bollworm Helicoverpa armigera will induce increased alkaloid levels in leaves, flowers and nectar; and 3) increased alkaloid concentrations in herbivore-damaged plants will negatively affect larval growth. We grew N. africana in large pots in a greenhouse and exposed flowering plants to densities of one, three and six fourth-instar larvae of H. armigera, for four days. Leaves, flowers and nectar were analyzed for nicotine, nornicotine and anabasine. The principal leaf alkaloid was nornicotine (mean: 28 µg/g dry mass) followed by anabasine (4.9 µg/g) and nicotine (0.6 µg/g). Nornicotine was found in low quantities in the flowers, but no nicotine or anabasine were recorded. The nectar contained none of the alkaloids measured. Larval growth was reduced when leaves of flowering plants were exposed to six larvae. As predicted by the optimal defense theory, herbivory had a localized effect and caused an increase in nornicotine concentrations in both undamaged top leaves of herbivore damaged plants and herbivore damaged leaves exposed to one and three larvae. The nicotine concentration increased in damaged compared to undamaged middle leaves. The nornicotine concentration was lower in damaged leaves of plants exposed to six compared to three larvae, suggesting that N. africana rather invests in new growth as opposed to protecting older leaves under severe attack. The results indicate that the nornicotine-rich N. africana will be unattractive to herbivores and more so when damaged, but that potential pollinators will be unaffected because the nectar remains alkaloid-free even after herbivory.

Standard addition method applied to the urinary quantification of nicotine in the presence of cotinine and anabasine using surface enhanced Raman spectroscopy and multivariate curve resolution.

In this work, urinary nicotine was determined in the presence of the metabolite cotinine and the alkaloid anabasine using surface enhanced Raman spectroscopy and colloidal gold as substrate. Spectra were decomposed using the multivariate curve resolution-alternating least squares method, and pure contributions were recovered. The standard addition method was applied by spiking urine samples with known amounts of the analyte and relative responses from curve resolution were employed to build the analytical curves. The use of multivariate curve resolution in conjunction with standard addition method showed to be an effective strategy that minimized the need for reagent and time-consuming procedures. The determination of the alkaloid nicotine was successfully accomplished at concentrations 0.10, 0.20 and 0.30 µg mL(-1) and total error values less than 10% were obtained.

Direct functionalization processes: a journey from palladium to copper to iron to nickel to metal-free coupling reactions.

The possibility of finding novel disconnections for the efficient synthesis of organic molecules has driven the interest in developing technologies to directly functionalize C-H bonds. The ubiquity of these bonds makes such transformations attractive, while also posing several challenges. The first, and perhaps most important, is the selective functionalization of one C-H bond over another. Another key problem is inducing reactivity at sites that have been historically unreactive and difficult to access without prior inefficient prefunctionalization. Although remarkable advances have been made over the past decade toward solving these and other problems, several difficult tasks remain as researchers attempt to bring C-H functionalization reactions into common use. The functionalization of sp(3) centers continues to be challenging relative to their sp and sp(2) counterparts. Directing groups are often needed to increase the effective concentration of the catalyst at the targeted reaction site, forming thermodynamically stable coordination complexes. As such, the development of removable or convertible directing groups is desirable. Finally, the replacement of expensive rare earth reagents with less expensive and more sustainable catalysts or abandoning the use of catalysts entirely is essential for future practicality. This Account describes our efforts toward solving some of these quandaries. We began our work in this area with the direct arylation of N-iminopyridinium ylides as a universal means to derivatize the germane six-membered heterocycle. We found that the Lewis basic benzoyl group of the pyridinium ylide could direct a palladium catalyst toward insertion at the 2-position of the pyridinium ring, forming a thermodynamically stable six-membered metallocycle. Subsequently we discovered the arylation of the benzylic site of 2-picolonium ylides. The same N-benzoyl group could direct a number of inexpensive copper salts to the 2-position of the pyridinium ylide, which led to the first description of a direct copper-catalyzed alkenylation onto an electron-deficient arene. This particular directing group offers two advantages: (1) it can be easily appended and removed to reveal the desired pyridine target, and (2) it can be incorporated in a cascade process in the preparation of pharmacologically relevant 2-pyrazolo[1,5-a]pyridines. This work has solved some of the challenges in the direct arylation of nonheterocyclic arenes, including reversing the reactivity often observed with such transformations. Readily convertible directing groups were applied to facilitate the transformation. We also demonstrated that iron can promote intermolecular arylations effectively and that the omission of any metal still permits intramolecular arylation reactions. Lastly, we recently discovered a nickel-catalyzed intramolecular arylation of sp(3) C-H bonds. Our mechanistic investigations of these processes have elucidated radical pathways, opening new avenues in future direct C-H functionalization reactions.

Potential state-selective hydrogen bond formation can modulate activation and desensitization of the α7 nicotinic acetylcholine receptor.

A series of arylidene anabaseines were synthesized to probe the functional impact of hydrogen bonding on human α7 nicotinic acetylcholine receptor (nAChR) activation and desensitization. The aryl groups were either hydrogen bond acceptors (furans), donors (pyrroles), or neither (thiophenes). These compounds were tested against a series of point mutants of the ligand-binding domain residue Gln-57, a residue hypothesized to be proximate to the aryl group of the bound agonist and a putative hydrogen bonding partner. Q57K, Q57D, Q57E, and Q57L were chosen to remove the dual hydrogen bonding donor/acceptor ability of Gln-57 and replace it with hydrogen bond donating, hydrogen bond accepting, or nonhydrogen bonding ability. Activation of the receptor was compromised with hydrogen bonding mismatches, for example, pairing a pyrrole with Q57K or Q57L, or a furan anabaseine with Q57D or Q57E. Ligand co-applications with the positive allosteric modulator PNU-120596 produced significantly enhanced currents whose degree of enhancement was greater for 2-furans or -pyrroles than for their 3-substituted isomers, whereas the nonhydrogen bonding thiophenes failed to show this correlation. Interestingly, the PNU-120596 agonist co-application data revealed that for wild-type α7 nAChR, the 3-furan desensitized state was relatively stabilized compared with that of 2-furan, a reversal of the relationship observed with respect to the barrier for entry into the desensitized state. These data highlight the importance of hydrogen bonding on the receptor-ligand state, and suggest that it may be possible to fine-tune features of agonists that mediate state selection in the nAChR.

Application of catalytic dynamic resolution of N-Boc-2-lithiopiperidine to the asymmetric synthesis of 2-aryl and 2-vinyl piperidines.

The highly enantioselective synthesis of 2-aryl- and 2-vinyl-piperidines has been accomplished through a catalytic dynamic resolution (CDR) of N-Boc-2-lithiopiperidine. The method has been applied to the synthesis of both enantiomers of the tobacco alkaloid anabasine.

The conformational landscape of nicotinoids: solving the conformational disparity of anabasine.

The conformational landscape of the alkaloid anabasine (neonicotine) has been investigated by using rotational spectroscopy and ab initio calculations. The results allow a detailed comparison of the structural properties of the prototype piperidinic and pyrrolidinic nicotinoids (anabasine vs. nicotine). Anabasine adopts two most stable conformations in isolation conditions, for which we determined accurate rotational and nuclear quadrupole coupling parameters. The preferred conformations are characterized by an equatorial pyridine moiety and additional N-H equatorial stereochemistry at the piperidine ring (eq-eq; eq=equatorial). The two rings of anabasine are close to a bisecting arrangement, with the observed conformations differing by an approximately 180 degrees rotation of the pyridine subunit, denoted either syn or anti. The preference of anabasine for the eq-eq-syn conformation has been established by relative intensity measurements (syn/anti approximately 5(2)). The conformational preferences of free anabasine are directed by a weak N...H-C hydrogen bond interaction between the nitrogen lone pair at piperidine and the closest C-H bond in pyridine, with N...H distances ranging from 2.686 (syn) to 2.667 A (anti). Supporting ab initio calculations by using MP2 and the recent M05-2X density functional are provided, evaluating the predictive performance of both methods.

Activation and desensitization of nicotinic alpha7-type acetylcholine receptors by benzylidene anabaseines and nicotine.

Nicotinic receptor activation is inextricably linked to desensitization. This duality affects our ability to develop useful therapeutics targeting nicotinic acetylcholine receptor (nAChR). Nicotine and some alpha7-selective experimental partial agonists produce a transient activation of alpha7 receptors followed by a period of prolonged residual inhibition or desensitization (RID). The object of the present study was to determine whether RID was primarily due to prolonged desensitization or due to channel block. To make this determination, we used agents that varied significantly in their production of RID and two alpha7-selective positive allosteric modulators (PAMs): 5-hydroxyindole (5HI), a type 1 PAM that does not prevent desensitization; and 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596), a type 2 PAM that reactivates desensitized receptors. The RID-producing compounds nicotine and 3-(2,4-dimethoxybenzylidene)anabaseine (diMeOBA) could obscure the potentiating effects of 5HI. However, through the use of nicotine, diMeOBA, and the RID-negative compound 3-(2,4-dihydroxybenzylidene)anabaseine (diOHBA) in combination with PNU-120596, we confirmed that diMeOBA produces short-lived channel block of alpha7 but that RID is because of the induction of a desensitized state that is stable in the absence of PNU-120596 and activated in the presence of PNU-120596. In contrast, diOHBA produced channel block but only readily reversible desensitization, whereas nicotine produced desensitization that could be converted into activation by PNU-120596 but no demonstrable channel block. Steady-state currents through receptors that would otherwise be desensitized could also be produced by the application of PNU-120596 in the presence of a physiologically relevant concentration of choline (60 microM), which may be significant for the therapeutic development of type 2 PAMs.

Sequential preconcentration by coupling of field amplified sample injection with pseudo isotachophoresis-acid stacking for analysis of alkaloids in capillary electrophoresis.

A novel on-column sequential preconcentration method based on the combination of field-amplified sample injection induced by acetonitrile and pseudo isotachophoresis (ITP)-acid stacking is developed for simply but efficiently concentrating alkaloid cations in a high-salt sample matrix in capillary electrophoresis. Acetonitrile (70%) added to a sample solution with a high-salt sample matrix not only induces field-amplified sample stacking by decreasing conductivity but also acts as a termination reagent in the succeeding pseudo ITP. After sample injection had been completed, a plug of H(+) was injected electrokinetically and a neutralization reaction between H(+) and tartrate from the buffer solution produced a low conductivity zone, in which the injected analyte cations were further concentrated. With the sequential preconcentration method, a 3 orders of magnitude detection sensitivity (1,400-fold) increase could be observed compared with the conventional electrokinetic injection method, without compromising separation efficiency and peak shape, and detection limits of 0.1 ng/mL for myosmine and 0.3 ng/mL for anabasine with the conditions selected were achieved. The calibration curves demonstrated good linearity in the concentration ranges 1.3-600 ng/mL for myosmine and 4.9-900 ng/mL for anabasine, respectively. The proposed method has been used to analyze successfully trace alkaloids in cigarette samples.

Analyses of tobacco alkaloids by cation-selective exhaustive injection sweeping microemulsion electrokinetic chromatography.

In this study, an on-line concentration method which coupled cation-selective exhaustive injection (CSEI) sweeping technology with microemulsion electrokinetic chromatography (MEEKC) was used to detect and analyze several tobacco alkaloids (nornicotine, anabasine, anatabine, nicotine, myosmine and cotinine) that are commonly found in various tobacco products. First, the effects of microemulsion compositions (oil, cosurfactant and solution pH) were examined in order to optimize the alkaloid separations in conventional MEEKC. The pH value and the injection length of basic plug were found to be the predominant influences on the alkaloid stacking. This optimal CSEI sweeping MEEKC method provided approximately 180- to 540-fold increase in detection sensitivity in terms of peak height without any loss in separation efficiency when compared to normal MEEKC separation. Furthermore, this proposed CSEI sweeping MEEKC method was applied successfully for the detection of the minor alkaloids nornicotine, anabasine and anatabine in tobacco products.

Synthesis, insecticidal activity, and QSAR of novel nitromethylene neonicotinoids with tetrahydropyridine fixed cis configuration and exo-ring ether modification.

To keep the nitro group in the cis position, a series of nitromethylene neonicotinoids containing a tetrahydropyridine ring with exo-ring ether modifications were designed and synthesized. All of the compounds were characterized and confirmed by 1H NMR, high-resolution mass spectroscopy, elemental analysis, and IR. The bioassay tests showed that some of them exhibited good insecticidal activities against pea aphids. On the basis of 10 nitromethylene derivatives, the quantitative structure-bioactivity relationship (QSAR) was analyzed and established. The results suggested that AlogP98 and Dipole_Mopac might be the important parameters related with biological activities.

Insecticidal and neuroblocking potencies of variants of the imidazolidine moiety of imidacloprid-related neonicotinoids and the relationship to partition coefficient and charge density on the pharmacophore.

The pharmacophore of the neonicotinoid insecticide imidacloprid, nitroiminoimidazolidine, was modified to heterocycles such as thiazolidine, pyrrolidine, dihydroimidazole, dihydrothiazole, and pyridone conjugated to nitroimine (=NNO2) or nitromethylene (=CHNO2). Their 6-chloro-3-pyridylmethyl or 5-chloro-3-thiazolylmethyl derivatives were examined for insecticidal activity against the American cockroach by injection and for neuroblocking activity using the cockroach ganglion. Most of the compounds having the neonicotinoidal pharmacophore exhibited insecticidal activity at the nanomolar level, which was enhanced in the presence of synergists, and high neuroblocking activity at the micromolar level. Quantitative analysis for the compounds showed that the neuroblocking potency is proportional both to the Mulliken charge on the nitro oxygen atom and to the partition coefficient log P value. The equation for the insecticidal versus neuroblocking potencies indicated that both potencies are related proportionally with each other when the other factors are the same.

Influence of agonists and antagonists on the segmental motion of residues near the agonist binding pocket of the acetylcholine-binding protein.

Using the Lymnaea acetylcholine-binding protein as a surrogate of the extracellular domain of the nicotinic receptor, we combined site-directed labeling with fluorescence spectroscopy to assess possible linkages between ligand binding and conformational dynamics. Specifically, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl methanethiosulfonate was conjugated to a free cysteine on loop C and to five substituted cysteines at strategic locations in the subunit sequence, and the backbone flexibility around each site of conjugation was measured with time-resolved fluorescence anisotropy. The sites examined were in loop C (Cys-188 using a C187S mutant), in the beta9 strand (T177C), in the beta10 strand (D194C), in the beta8-beta9 loop (N158C and Y164C), and in the beta7 strand (K139C). Conjugated fluorophores at these locations show distinctive anisotropy decay patterns indicating different degrees of segmental fluctuations near the agonist binding pocket. Ligand occupation and decay of anisotropy were assessed for one agonist (epibatidine) and two antagonists (alpha-bungarotoxin and d-tubocurarine). The Y164C and Cys-188 conjugates were also investigated with additional agonists (nicotine and carbamylcholine), partial agonists (lobeline and 4-hydroxy,2-methoxy-benzylidene anabaseine), and an antagonist (methyllycaconitine). With the exception of the T177C conjugate, both agonists and antagonists perturbed the backbone flexibility of each site; however, agonist-selective changes were only observed at Y164C in loop F where the agonists and partial agonists increased the range and/or rate of the fast anisotropy decay processes. The results reveal that agonists and antagonists produced distinctive changes in the flexibility of a portion of loop F.

Relative toxicities and neuromuscular nicotinic receptor agonistic potencies of anabasine enantiomers and anabaseine.

Anabasine occurring in wild tree tobacco (Nicotiana glauca) and anabaseine occurring in certain animal venoms are nicotinic receptor agonist toxins. Anabasine lacks the imine double bond of anabaseine; the two possible enantiomers of anabasine occur in N. glauca. A comparision of the relative potencies of S- and R-anabasine has not been previously reported. We separated the enantiomers of anabasine by reaction of the racemic N. glauca natural product with 9-fluorenylmethoxycarbonyl-L-alanine (Fmoc-L-Ala-OH) to give diastereomers, which were separated by preparative reversed phase HPLC. The S- and R-anabasine enantiomer fractions were then obtained by Edman degradation. A mouse bioassay was used to determine the relative lethalities of S- and R-enriched anabasine enantiomers. The intravenous LD50 of the (+)-R-anabasine rich fraction was 11 +/- 1.0 mg/kg and that of the (-)-S-anabasine-rich fraction was 16 +/- 1.0 mg/kg. The LD50 of anabaseine was 0.58 +/- 0.05 mg/kg. Anabaseine was significantly more toxic in the mouse bioassay than S-anabasine (27-fold) and R-anabasine (18-fold). The relative agonistic potencies of these three alkaloids on human fetal nicotinic neuromuscular receptors were of the same rank order: anabaseine>>R-anabasine>S-anabasine.