A fast atom bombardment mass spectrometry study of tris (3, 6-dioxaheptyl) amine-alkali metal halide complexes and hydrogen bonded complexes

The objective of this thesis was to demonstrate the potential of fast atom bombardment mass spectrometry (FABMS) as a probe of condensed phase systems and its possible uses for the study of hydrogen bonding. FABMS was used to study three different systems. The first study was aimed at investigating the selectivity of the ligand tris(3,6-dioxaheptyl) amine (tdoha) for the alkali metal cations. FABMS results correlated well with infrared and nmr data. Systems where a crown ether competed with tdoha for a given alkali metal cation were also investigated by fast atom bombardment. The results were found to correlate with the cation affinity of tdoha and the ability of the crown ether to bind the cation. In the second and third studies, H-bonded systems were investigated. The imidazole-electron donor complexes were investigated and FABMS results showed the expected H-bond strength of the respective complexes. The effects of concentration, liquid matrix, water content, deuterium exchange, and pre-ionization of the complex were also investigated. In the third system investigated, the abundance of the diphenyl sulfone-ammonium salt complexes (presumably H-bonded) in the FABMS spectrum were found to correlate with qualitative considerations such as steric hindrance and strength of ion pairs.

A study of thermal decompositions of an allylic hydroperoxide /|nby Thomas A. McCarrick. -- 260 St. Catharines [Ont.] : Dept. of Chemistry, Brock University,

The thermal decomposition of 2,3-di~ethy l - J-hydr operox y- 1 - butene , p r epared f rol") singl e t oxygen, has been studied i n three solvents over the tempe r a ture r ange from 1500e to l o00e and t!1e i 111 t ial ~oncentrfttl nn r Ange from O. 01 M to 0.2 M. Analys i s of the kine tic data ind ica te s i nduced homolysis as the n ost probRble mode of d e composition, g iving rise to a 3/2 f S order dependence upon hy d.roperoxide concent :r8.tl on . Experimental activation e nergies for the decomposition were f ound to be between 29.5 kcsl./raole and 30.0 k cal./mole .• \,iith log A factors between 11 . 3 and 12.3. Product studies were conducted in R variety of solvents a s well as in the pr esence of a variety of free r adical initiators . Investigation of the kinetic ch a in length indicated a chain length of about fifty. A degenerat i ve chain branching mechanism 1s proposed which predicts the multi t ude of products which Rre observed e xperimentally as well as giving activation energies and log A factors si~il a r to those found experimentally .

Synthesis of chiral homoallylic alcohols and phthalans through the asymmetric allylation of carbonyl compounds /

The implementation of chiral centres within biologically active compounds has been a perplexing yet motivational force in chemistry. This work presents the attempted formation of a concurrent or sequential tandem catalyzed methodology of enantioselective nucleophilic addition and electrophilic cyclization. The 2'- arylalkynyl- aldehyde, ketone, and imine substrates used within were adeptly chosen with a dually activated structure; 1) for nucleophilic addition to the electrophilic substituents; and 2) for carbophilic activation of the alkyne substituent to undergo cyclization. To accomplish the nucleophilic addition, two distinct allylation methodologies were pursued: (/?)-BINOL catalyzed-allylboration and (5)- BINAP-AgF catalyzed-allylsilylation. BINAP catalyzed enantioselective allylation of 2'-arylalkynyl-aldehydes, to form chiral homoallylic alcohols, was successful. Homoallylic alcohols were isolated with high enantio-purity (>80%), which then underwent sequential cyclization to form chiral allylic phthalans, in moderate yields. An application of this methodology towards the construction of biologically active compounds was included with the partial synthesis of the natural product and H. pylori inhibitor, (+)-Spirolaxine methyl ether.

1,1-enediamines and β-substituted enamines in heterocyclic synthesis

The work in this thesis mainly deals with l,l-enediamines and ~ -substituted enamines (push-pull olefines) and their reactions, leading to the formation of a number of heterocycles. Various ~-substituted enamines were prepared by a 'one pot synthesis' in which a l,l-enediamine presumably acts as an intermediate. These enamines, various substituted crotonamides and propenamides, were made by using two different orthoesters, various secondary and primary amines and cyanoacetamide. Their structures, mechanism of formation and geometry are discussed. A synthetic route to various unsymmetrically substituted pyridines was examined. Two substituted pyridinones were obtained by using two different ~-substituted enamines and cyanoacetamide. In one case a dihydropyridine was isolated. This dihydropyridine, on heating in acidic conditions, gave a pyridinone, which confirmed this dihydropyridine as an intermediate in this pyridine synthesis. A new synthetic method was used to make highly substituted pyridinones, which involved the reaction of l,l-enediamines with the ~-substituted enamines. A one pot synthesis and an interrupted one pot synthesis were used to make these pyridinones. Two different orthoesters and three different secondary amines were used. Serendipitous formation of a pyrimidinone was observed when pyrrolidine was used as the secondary amine and triethyl orthopropionate was used as the orthoester. In all cases cyanoacetamide was used as the carbon acid. This pyridine synthesis was designed with aI, l-enediamine as the Michael donor and the ~ -substituted enamines as Michael acceptors. Substituted ureas were obtained in two cases, which was a surprise. Some pyrimidines were made by reacting two substituted enamines with two different amidines. When benzamidine was used, the expected pyrimidines were obtained. But, when 2-benzyl-2-thiopseudourea (which is also an amidine) was used, of the two expected pyrimidines, only one was obtained. In the other case, an additional substitution reaction took place in which the S-benzyl group was lost. An approach to quinazolone and benzothiadiazine synthesis is discussed. Two compounds were made from 1, I-dimorpholinoethene

An aryne route to aporphine alkaloids and related topics / |nIjaz Ahmad Chanda. -- 260 St. Catharines, Ont. : [s. n.],

This research was directed towards the investigation and development of an aryne route to the syntheses of aporphi ne and dibenzopyrrocolinium (dibenzoindolizinium) alkaloids and to the stability of the latter under the conditions used for aryne formation. The work c an be divided into three main sections . i) - Synthesis of Glaucine 6-Bromo-3,4-dimethoxyphenylacetic acid, prepared by the action of bromine i n acetic acid on3,4-dimethoxyphenylacetic a cid, was converted into its acid chloride by t he action of thionyl chloride. This on treatment with 3,4- dimethoxyphenylethylamine pr ovided N-(3, 4-dimethoxyphenylethyl)- 2-(2-bromo-4,S-dimethoxyphenyl)-acetamide which on dehydration with phosphoryl chloride (Bischler Napieralski reaction) in dry benzene afforded l -(2-bromo-4,S-dimethoxybenzyl)- 3,4-dihydro-6,7-dimethoxyisoquinoline, isolated as hydrochl oride. A new method o f destroying the excess of phosphoryl chloride was developed which proved to be quite useful. Methylation of the dihydroisoquinoline'with methyl iodide in methanol , and subsequent reduction with sodium borohydride provided (±)-6-bromolaudanosine. Act ion of potassamide or sodamide in anhydrous liquid ammonia on (±)-6-bromolaudanosine yielded the corresponding amino derivative along with other products. Diazotization and ring closure of (±)-6-aminolaudanosine then a f forded (±)-glaucine which was isolated as methiodide. ii) - Intramolecular Capture of Aryne During Glaucine Synthesis, and Subsequent Reactions . This section deals with the by-products formed under the conditions of the aryne stage of t he glaucine synthesis. The crude product, obtained in the reaction of potassamide or sodamide in liquid ammonia on (±)-6-bromolaudanosine, was s eparated by chromatography, Three products were separated and identified. a ) - 5,6-Dimethoxy-2-( 3,4-dimethoxy-6-ethylphenyl)-lmethylindole. Two mechanisms are proposed for the formation of this interesting product. This compound also was prepared by the action of potassamide in l,iquid ammonia on 5,6 ,l2,l2atetrahydro- 2,3,9,lO-tetramethoxy-7-methyldibenz[b,g]indolizinium i odide . b) - 5,6-Dimethoxy-2-(3,4-dimethoxy-6-vinylphenyl)-lmethylindoline. Its formation represented a new method of Hofmann degradation . Further confirmation of structure was done by performing the normal Hofmann reaction on 5, 6,12,12a-tetrahydro -2/3,9,lO-tetramethoxy ~7-methyldibe nz[ b,g]indolizinium iodide. The indoline prepared i n this way was identical in all respects with that prepared above . c) - 1- (2-amino-4,5-dimethoxybenzyl ) -l,2,3,4-tetrahydro-2- methyl-6,7-dimethoxyisoquinoline, was converted t o glaucine as stated in section 1 . iii) - Attempt:,ed Sxnthesis of Liriodenine Piperonal was converted into 3,4-methylenedioxyinitrostyrene which on reduction with lithium aluminium hydride provided 3,4-methylenedioxyphenylethylamine. The method of extraction after the reduction was improved t o some extent. The amine on condensation with m-chlorophenylacetyl chloride, prepared by the action of oxalyl chloride on 3,4-methylenedioxyphenylacetic acid, provided N-[ ~ -(3,4-methylenedioxyphenyl)- e thyl)-3-chlorophenylacetamide. This on dehydration with phosphoryl chloride in dry benzene followed by air oxidation afforded l-(3-chlorobenzoyl)-6,7-methylenedioxyi soquinoline. This compound on r eaction with potassamide in liquid ammonia afforded a crude product from which. one product was separated by chromatography i n a pure condition . This yellow compound analysed as,c17Hl ON2021 and was t he main product i n the reaction ; a t entative structure is proposed. A second compound, not obtained in pure condition, was submitted to Pschorr reaction in the hope of obtaining liriodenine, but without success.

Chelated fluoroboron cations : synthesis and multinuclear nmr studies

The preparation of chelated difluoroboron cations (DD)BF2+, where DD is a saturated polydentate tertiary-amine or polydentate aromatic ligand, has been systematically studied by using multinuclear solution and solid state nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Three new methods of synthesis of (DD)BF2+ cations are reported, and compared with the previous method of reacting a chelating donor with Et20.BF3. The methods most effective for aromatic donors such as 1,1O-phenanthroline are ineffective for saturated polydentate tertiary-amines like N,N,N' ,Nil ,Nil-pentamethyldiethylenetriamine. Polydentate tertiary-amine donors that form 5-membered rings upon bidentate chelation were found to chelate effectively when the BF2 source contained two leaving groups (a heavy halide and a Lewis base such as pyridine =pyr or isoxazole =ISOX), i.e., pyr.BF2X (X = CI or Br), ISOX.BF2X and (pyr)2BF2+. Those that would form 6membered rings upon chelation do not chelate by any of the four methods. Polydentate aromatic ligands chelate effectively when the BF2 source contained a weak Lewis base, e.g., ISOX.BF3, ISOX.BF2X and Et20.BF3. Bidentate chelation by polydentate tertiaryamine and aromatic donors leads to nmr parameters that are significantly different then their (D)2BF2+ relatives (D =monod~ntate t-amines or pyridines). The chelated haloboron cations (DD)BFCI+, and (DD)BFBr+ were generated from D.BFX2 adducts for all ligands that form BF2+ cations above. In addition, the (DD)BCI2+ and (DD)BBr2+ cations were formed from D.BX3 adducts by the chelating aromatic ligands, except for the aromatic ligand 1,8-bis(dimethylamino)naphthalene, which formed only the (DD)BF2+ cation, apparently due to its extreme steric hindrance. Chelation by a donor is a two-step reaction. For polydentate tertiary-amine ligands, the two rates appear to be very dependent on the two possible leaving groups on the central boron atom. The order of increasing ease of displacement for the donors was: pyr < Cl < Br < ISOX. The rate of chelation by polydentate aromatic ligands appears to be dependent on the displacement of the first ligand from the boron. The order of increasing ease of displacement for the donors was: pyr < CI < ISOX ~ Br < Et20.

The 21-dehydroxylation of corticosteroids: evidence for an enol intermediate and the hydroxylation of steroids by fungal cyto-chrome P450: evidence for a stepwise mechanism

Two enzyme mechanisms were examined: the 21-dehydroxylation of corticosteroids by the anaerobe Eubacterium l en tum, and the hydroxylation of steroids by fungal cytochrome P450. Deuterium labelling techniques were used to study the enzymic dehydroxylation. Corticosteroids doubly labelled (2H) at the C-21 position were incubated with a culture of Eubacterium lentum. It was found that t he enzymic dehydroxylation proceeded with the loss of one 2H f rom C-21 per molecule of substrate. The kinetic isotope ef fect f or the reaction was found to be k~kD = 2. 28. These results suggest that enzyme/substr ate binding in this case may proceed via t he enol form of the substrate. Also , it appears that this binding is, at least in part, the rate determining step of t he reaction. The hydroxylation of steroids by fungal cytochrome P450 was examined by means of a product study. Steroids with a double bond at the A8 (9), ~( lO ), or ~ (ll) position were synthesized. These steroids were then incubated with fungal strains known to use a cytochrome P450 monooxygenase to hydroxylate at positions allylic to these doubl e bonds. The products formed in these incubations indicated that the double bonds had migrated during allylic hydroxylat ion. This suggests that a carbon centred radical or ion may be an intermediate i n the cytochrome P450 cat alytic cycle.

The preparation and reactions of some 1, 2-dipolar species /|nI. D. Brindle. -- 260 St. Catharines, Ont. : [s. n.],

This research was directed mainly towards the investigation of the reactions of allylic amineimides. The work can be divided into two main sections. Section 1 of the thesis deals mainly with thermolysis studies of amineimides. Sections 1a and 1b represent a comprehensive survey of amineimide literature up to 1971. N-A1ly1-N,N-dirnethylarnine-benzirnide was prepared and rearranged at 1400 to l-allyl-1-benzoyl-2,2-dimethylhydrazine. A tentative mechanism involving an initial migration to the carbonyl oxygen was disproved by incorporating the amineimide system into a five-membered ring. N,N~Dimethyl-N-propargylamine-benzimidedid not rearrange on heating; but the hydrobromide, on heating, disproportionated to give 1-benzoyl~2,2,2-trimethylhydraziniumbromide and I-benzoyl-2,2~ dimethylhydrazine. l-Ally'l--l, I-dimethyl-2-benzoy-lhydrazinium bromide and 1~benzoy-1-2,2, 2-trimethy-lhydrazinium iodide both disproportionated to give l~benzoyl-2,2-dimethylhydrazine. Section 1 concludes with a discussion of the mechanisms of ally'lic migrations in amineimides proposed by J. E. Baldwin. Section 2 deals with the formation of five-membered heterocyclic compounds from amineimides by bromination. 1,1-Dimethyl-2benzoyl- 4-bromopyrazolidinium bromide was formed from N-allyl-N,Ndime thy-lamtne-benzimide , 1,1-dimethyl-2-benzoyl-4-bromopyrazol-3enium bromide from N,N~dimethyl-N-propargylamine~benzimidevia the unusual acetylenic "bromonium" ion. Hydrogenolysis of both heterocyclic compounds gave the same product. The preparation was extended by forming 2,2-dimethyl-4-bromoisoxazolinium bromide from N-allylN, N-dimethylamine-N-oxide. Sections 3 and 4 cover a number of unsuccessful attempts to synthesise other amineimides and l,2-dipolar species.

Modifications to the Suzuki reaction and mechanistic insights on the NBS mediated cleavage of benzylidene acetals /

One of the most challenging tasks for a synthetic organic chemist today, is the development of chemo, regio, and stereoselective methodologies toward the total synthesis of macromolecules. r . The objective of my thesis was to develop methodologies towards this end. The first part of my project was to develop highly functionalized chirons from D-glucose, a cheap, chiral starting material, to be utilized in this capacity. The second part of the project dealt with modifying the carbon-carbon bond forming Suzuki reaction, which is utilized quite often as a means of combining molecular sub units in total synthesis applications. As previously stated the first area of the project was to develop high value chirons from D-glucose, but the mechanism of their formation was also investigated. The free radical initiated oxidative fragmentation of benzylidene acetals was investigated through the use of several test-case substrates in order to unravel the possible mechanistic pathways. This was performed by reacting the different acetals with N-bromosuccinimide and benzoyl peroxide in chlorobenzene at 70^C in all cases. Of the three mechanistic pathways discussed in the literature, it was determined, from the various reaction products obtained, that the fragmentation of the initial benzylic radical does not occur spontaneously but rather, oxidation proceeds to give the benzyl bromide, which then fragments via a polar pathway. It was also discovered that the regioselectivity of the fragmentation step could be altered through incorporation of an allylic system into the benzylidene acetal. This allows for the acquisition of a new set of densely functionalized. chiral, valuable synthetic intermediates in only a few steps and in high yields from a-Dglucose. The second part of the project was the utilization of the phosphonium salt room temperature ionic liquid tetradecyltrihexylphosphonium chloride (THPC) as an efficient reusable medium for the palladium catalyzed Suzuki cross-coupling reaction of aryl halides, including aryl chlorides, under mild conditions. The cross-coupling reactions were found to proceed in THPC containing small amounts of water and toluene using potassium phosphate and 1% Pd2(dba)3. Variously substituted iodobenzenes, including electron rich derivatives, reacted efficiently in THPC with a variety of arylboronic acids and afforded complete conversion within 1 hour at 50 ^C. The corresponding aryl bromides also reacted under these conditions with the addition of a catalytic amount of triphenylphosphine that allowed for complete conversion and high isolated yields. The reactions involving aryl chlorides were considerably slower, although the addition of triphenylphosphine and heating at 70 ^C allowed high conversion of electron deficient derivatives. Addition of water and hexane to the reaction products results in a triphasic system in which the top hexane phase contained the biaryl products, the palladium catalyst remained fully dissolved in the central THPC layer, while the inorganic salts were extracted into the lower aqueous phase. The catalyst was then recycled by removing the top and bottom layers and adding the reagents to the ionic liquid which was heated again at 50 ^C; resulting in complete turnover of iodobenzene. Repetition of this procedure gave the biphenyl product in 82-97% yield (repeated five times) for both the initial and recycled reaction sequences.

Reactions of steroidal epoxides with strong organic bases and an investigation into the syntheses of some labelled pregnane derivatives

Reactions of 5,6- and 4,5-epoxycholestane derivatives with strong bases were investigated. Epoxidation of 3a-acetoxycholest-5-ene also gave a new compound along with the anticipated epoxides. Interconversions of the latter were observed. Some possible mechanisms of its formation and rearrangements have been pIioposed. No reaction was observed with any of the 5,6- and 4,5-steroidal epoxides employed in the present study, using potassium tertiary butoxide under refluxing conditions. n-Butyllithium reacted only with 5,6-epoxycholestanes bearing a ketal moiety at the C3 carbon. Opening of the ketal group was observed with n-butyllithium in the case of a ~-epoxide. The reaction was also investigated in the absence of epoxide functionality. A possible mechanism for the opening of ketal group has been proposed. Lithium diethylamide (LDEA) was found effective in rearranging 5,6- and 4,5-epoxides to their ~orresponding allylic alcohols. These rearrangements presumably proceed via syn-eliminations, however the possibility of a corresponding anti-elimination has not been eliminated. A substituent effect of various functional groups (R = H, OH, OCH2CH20) at C3 has-been observed on product distribution in the LDEApromoted rearrangements of the corresponding epoxides. No reaction of these epoxides was observed with lithium diisopropylamide (LDA) • In the second part of the project, several attempts were made towards the sYRthesis of deoxycorticoste~one~17,2l,2l~d3' a compound desirable for the 2l-dehydroxylation studies of deoxycorticosterone. Several routes were investigated, and some deuterium labelled pregnane derivatives were prepared in this regard. Microbial 21-hydroxylation of progesteronel7,21,21,2l- d4 by ~ niger led to loss of deuterium from C21 of the product. An effort was made to hydroxylate progesterone microbially under neutral condtions.

Synthesis and utilization of guanidine catalysts for applications directed towards the preparation of butenolide and modified amino acid derivatives

Development of guanidine catalysts is explored through direct iminium chloride and amine coupling, alongside a 2-chloro-l,3-dimethyl-IH-imidazol-:-3-ium chloride (DMC) induced thiourea cyclization. Synthesized achiral catalyst N-(5Hdibenzo[ d,t][1,3]diazepin-6(7H)-ylidene)-3,5-bis(trifluoromethyl) aniline proved unsuccessful towards O-acyl migrations, however successfully catalyzed the vinylogous aldol reaction between dicbloro furanone and benzaldehyde. Incorporating chirality into the guanidine catalyst utilizing a (R)-phenylalaninol auxiliary, generating (R)-2-((5Hdibenzo[ d,t] [1,3 ]diazepin-6(7H)-ylidene ) amino )-3 -phenylpropan-l-ol, demonstrated enantioselectivity for a variety of adducts. Highest enantiomeric excess (ee) was afforded between dibromofuranone and p-chlorobenzaldehyde, affording the syn conformation in 96% ee and the anti in 54% ee, with an overall yield of30%. Attempts to increase asymmetric induction were focused on incorporation of axial chirality to the (R)phenylalaninol catalyst using binaphthyl diamine. Incorporation of (S)-binaphthyl exhibited destructive selectivity, whereas incorporation of (R)-binaphthyl demonstrated no effects on enantioselectivity. Current studies are being directed towards identifying the catalytic properties of asymmetric induction with further studies are being aimed towards increasing enantioselectivity by increasing backbone steric bulk.

Molecular Magnetism: The Design, Synthesis, and Characterization of New Building Blocks for Molecule-based Magnetic Materials

Two classes of building blocks have been prepared and characterized and their coordination chemistry explored working towards the preparation of new molecule-based magnetic materials. In the first project, the amine functionality of 3,3'-diamino-2,2'- bipyridine was exploited for the preparation of a new family of ligands (H2L 1)-(H2L 4). The molecular structures of three ligands have been fully characterized by X-ray crystallography. [molecular structure diagram will not copy here, but is available in full pdf.] The coordination chemistry of these ligands with divalent first row transition metal ions was investigated. For ligand (H2L1), the molecular structures of four coordination complexes with stoichiometries [Zn2(Ll)(OAc)(MeO)]2 (I), [Cu2(L1)(OAc)2 (II), [Li(L1)]3 (III), and [Ni(L1)]3 (IV) were determined by X-ray crystallography. For ligand (H2L2), a Cu(II) complex of stoichiometry [Cu3(L2)(OAc)3MeO] (V) was determined by X-ray crystallography. The magnetic properties of complexes (II), (III), and (V) have been fully elucidated. In project two, synthetic strategies for the preparation of porphyrin molecules bearing triol substituents is presented. Following this approach, three new porphyrin derivatives have been prepared and characterized [Zn(HPTPP-CH2C(CH20H)3)] (VI), [P(TPP)(OCH2C(CH2)H)3)2]+CL- (VII), and [P(OEP)(C6H5)(OCH2C(CH2OH)3)]+Cl- (VIII). Attempts to exchange the labile methoxide bridges of a tetraironIIl single molecule magnet of stoichiometry [Fe4(OMe)6(dpm)6] (Hdpm = dipivaloylmethane) with the triol appended porphyrins will be discussed. [molecular structure diagram will not copy here, but is available in full pdf.]

Action of octopamine and tyramine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10−5 and 10−4 M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10−5 M OA increased synaptically driven contractions by ∼1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

The action of octopamine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10(-5) and 10(-4) M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10(-5) M OA increased synaptically driven contractions by ≈ 1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

Cell-selective modulation of the neuromuscular system in Drosophila

The capacity for all living cells to sense and interact with their environment is a necessity for life. In highly evolved, eukaryotic species, like humans, signalling mechanisms are necessary to regulate the function and survival of all cells in the organism. Synchronizing systemic signalling systems at the cellular, organ and whole-organism level is a formidable task, and for most species requires a large number of signalling molecules and their receptors. One of the major types of signalling molecules used throughout the animal kingdom are modulatory substances (e.x. hormones and peptides). Modulators can act as chemical transmitters, facilitating communication at chemical synapses. There are hundreds of circulating modulators within the mammalian system, but the reason for so many remains a mystery. Recent work with the fruit fly, Drosophila melanogaster demonstrated the capacity for peptides to modulate synaptic transmission in a neuron-specific manner, suggesting that peptides are not simply redundant, but rather may have highly specific roles. Thus, the diversity of peptides may reflect cell-specific functions. The main objective of my doctoral thesis was to examine the extent to which neuromodulator substances and their receptors modulate synaptic transmission at a cell-specific level using D. melanogaster. Using three different modulatory substances, i) octopamine - a biogenic amine released from motor neuron terminals, ii) DPKQDFMRFa - a neuropeptide secreted into circulation, and iii) Proctolin - a pentapeptide released both from motor neuron terminals and into circulation, I was able to investigate not only the capacity of these various substances to work in a cell-selective manner, but also examine the different mechanisms of action and how modulatory substances work in concert to execute systemic functionality . The results support the idea that modulatory substances act in a circuit-selective manner in the central nervous system and in the periphery in order to coordinate and synchronize physiologically and behaviourally relevant outputs. The findings contribute as to why the nervous system encodes so many modulatory substances.