SYNTHESIS OF CYCLIC AZOBENZENE ANALOGUES FOR INCORPORATION INTO OLIGONUCLEOTIDES, PEPTIDES AND POLYMERS

(A) In recent years, considerable amount of effort has contributed towards enhancing our understanding of the new photoswitch, cyclic azobenzene, particularly from the theoretical point of view. However, the challenging part with this system was poor efficiency of its synthesis from 2,2’- dinitrodibenzyl and lack of effective methods for further modification which would be useful to incorporate this system into biomolecules as a photoswitch. We report the synthesis of cyclic azobenzene and analogues from 2,2’-dinitrodibenzyl, which would allow for further incorporation of this cyclic azobenzene into biomolecules. Reaction of 2,2’-dinitrodibenzyl with zinc metal powder in the presence of triethylammonium formate buffer (pH-9.5) gave a cyclic azoxybenzene, 11,12-dihydrodibenzo[c,g][1,2]diazocine-5-oxide. The latter compound was converted into cyclic azobenzene analogues (bromo-, chloro-, cyano-, and carboxyl) through subsequent transformations. The carboxylic acid analogue was reacted with D-threoninol to give the corresponding amide, which readily undergoes photo-isomerization upon illumination with light. Upon illumination with light at 400 nm, approximately 70% of cis- isomer of amide was isomerized to trans- isomer. It was observed that cis- to trans- isomerization reached the maximum steady state of light transmission after approximately 40 min, whereas the trans- to cis- isomerization approximately acquired in 2 h to regain full recovery of light transmission. Cyclic azobenzene phosphoramidite was synthesized from DMT-protected D-threoninol linked cyclic azobenzene. (B) In recent years, there has been considerable interest invested towards the synthesis of azobenzene analogues for incorporation into proteins. Among the many azobenzene analogues, the synthesis of bi-functional cyclic azobenzene analogues for the incorporation into proteins is relatively new. In this thesis, we report the synthesis of a cyclic azobenzene biscarboxylic acid from 4-(bromomethyl)benzonitrile. (C) Azobenzene has been widely used in the field of polymer science to study the surface morphology and surface properties of polymers. In this thesis, we report the incorporation of cyclic azobenzene into a commercial polymer 2- (hydroxyethyl)methacrylate. Samples collected after 24 h from the reaction solution showed approximately 9% of incorporation of cyclic azobenzene into polymer compared to samples collected after 10 h, which showed approximately 6% incorporation.

Permissivity of human HeLa cells to bovine adenovirus type 2 (BaV2 infection

Infection of hUlnan cells by bovine adenovirlls type 2 (BAV2) is abortive. To obtain a better understanding of this pllenomel1011, and in particular to identify Wllich steps in the viral replicative cycles are altered dllring this virlls-host cells interaction, we have llndertaken a detailed study of BAV2 infections of the nonpennissive hUlnan IIeLa cells. Using autoradiography and 3H-thymidine-labeled vvhole virus particles for infection of HeLa cells, vve determined that viral attachluent appears normal. Furthermore, Southern analysis revealed that internalization and transport to the nuclells occurs in BAV2 infected HeLa cells. To investigate viral DNi\ synthesis, infectivity assays involving hydroxyllrea, a viral DN-A synthesis inhibitor, were carried out. The results revealed that Bft:LV2 DNA synthesis does not occur in HeLa cells. Fllrtller investigations into viral early gene expression by northern blotting analyses indicated that HeLa cells fail to support expression of EIA. This suggested that abortive infection by BAV2 could be attributed to faiiure of EIA to express. To test the possibility that the failure to express ElA was due to the inability of the host cell to recognize the E lA prOlTIoter, ,ve carried out transient expression transfection experiments using plaslnids \vith the bacterial lacZ linder the control of either BAV2 or i\d5 EIA promoter. X-gal histochelIlical assays sho\ved expression of lacZ from the Ad5 ElA prOlnoter but no expression of lacZ [rOln the BAV2 EIA prOlTIoter. This further suggests that the abortive infection b:y BAV2 could be attributed to failure of EIA to express dlle to a nonfllnctional prOlTIoter in hlunan cells. Thus we speClllated that abortive infection of HeLa cells by adenoviruses may be averted by providing EtA functions in trans. To demonstrate this, we coinfected HeLa cells with Ad5 and BAV2, reasoning that Ad5 could cOlnpensate for EIA deficiency in BAV2. OUf results showed that BAV2 DNA synthesis was indeed Sllpported in HeLa cells coinfected with Ad5dlE3 as revealed by Southern analysis. In contrast, coinfection of HeLa cells \vith BAV2 and Ad5dlElE3 mutallt did not support BLt\V2 DNA synthesis. Interestingly, BAV2 failed to replicate in 293 cells which are constitlltively expressing the El genes. This could ilnply that El is necessary but not sufficient to avert the failllre ofBAV2 to undergo productive infection ofhulnan cells.

Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.

(A) Photoregulation of DNA Functions by Cyclic Azobenzene-tethered Oligonucleotides (B) Site-specific Fluorescent Labeling of DNA using Inverse Electron Demand Diels-Alder Reaction between trans-Cyclooctene Derivatives and BODIPY-Tetrazine Adducts

(A) Most azobenzene-based photoswitches require UV light for photoisomerization, which limit their applications in biological systems due to possible photodamage. Cyclic azobenzene derivatives, on the other hand, can undergo cis-trans isomerization when exposed to visible light. A shortened synthetic scheme was developed for the preparation of a building block containing cyclic azobenzene and D-threoninol (cAB-Thr). trans-Cyclic azobenzene was found to thermally isomerize back to the cis-form in a temperature-dependent manner. cAB-Thr was transformed into the corresponding phosphoramidite and subsequently incorporated into oligonucleotides by solid phase synthesis. Melting temperature measurement suggested that incorporation of cis-cAB into oligonucleotides destabilizes DNA duplexes, these findings corroborate with circular dichroism measurement. Finally, Fluorescent Energy Resonance Transfer experiments indicated that trans-cAB can be accommodated in DNA duplexes. (B) Inverse Electron Demand Diels-Alder reactions (IEDDA) between trans-olefins and tetrazines provide a powerful alternative to existing ligation chemistries due to its fast reaction rate, bioorthogonality and mutual orthogonality with other click reactions. In this project, an attempt was pursued to synthesize trans-cyclooctene building blocks for oligonucleotide labeling by reacting with BODIPY-tetrazine. Rel-(1R-4E-pR)-cyclooct-4-enol and rel-(1R,8S,9S,4E)-Bicyclo[6.1.0]non-4-ene-9-ylmethanol were synthesized and then transformed into the corresponding propargyl ether. Subsequent Sonogashira reactions between these propargylated compounds with DMT-protected 5-iododeoxyuridine failed to give the desired products. Finally a methodology was pursued for the synthesis of BODIPY-tetrazine conjugates that will be used in future IEDDA reactions with trans-cyclooctene modified oligonucleotides.

Analyses, persistence an degradation of the synthetic pyrethroid insecticides permethrin and fenvalerate

Studies on persistence and degradation of the synthetic pyrethroid insecticides, permethrin and fenvalerate, were carried out under natural environmental conditions of the Niagara Peninsula. Permethrin and fenvalerate were treated on apple foliage atrat~s of 0.21 kg(AI)!ha and 0.14 kg(AI)/ha, respectively. The initial cis- and trans-permethrin spray deposits were found to be 13.5 ppm and 19.2 ppm, respectively and 38.0 ppm was observed for the fenvalerate treated sample. Twenty-three days and 84 days after spray application, permethrin residues were 4.0 ppm and 2.7 ppm for the cis-isomer, whereas they were 7.9 ppm and 4.7 ppm for the trans-isomer, respectively. Residues of fenvalerate 23 days and 84 days after spray application were 13.4 ppm and 8.0 ppm, respectively. The values of observed half-life of cis-permethrin, trans-permethrin and fenvalerate were found to be 42 days, 46 days and 51 days, respectively. Studies were extended to quantitatively determine some of the major degradation compounds of permethrin and fenvalerate, which were expected to be produced as results of ester cleavage of the parent compounds. A permethrin treated sample, 84 days after initial spray application, showed 0.25 and 0.8 ppm of cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (C12CA (18), respectively. These two acids were not found as free acids, but found as conjugated compounds. The other expected degradation compounds, 3-phenoxybenzyl alcohol (PBalc (~)),3-phenoxybenz.aldehyde (PBald (38)) and 2- (4-chlorophenyl) isovaleric acid (CPIA (31)) were not detected by the methods employed in this study. The results indicate that these degradation compounds were not present, or, if they were present, their concentrations were too low to detect by the methods used.

Husserl's approach to transcendental intersubjectivity

It is our intention in the course of the development of this thesis to give an account of how intersubjectivity is "eidetically" constituted by means of the application of the phenomenological reduction to our experience in the context of the thought of Edmund Husserl; contrasted with various representative thinkers in what H. Spiegelberg refers to as "the wider scene" of phenomenology. That is to say, we intend to show those structures of both consciousness and the relation which man has to the world which present themselves as the generic conditions for the possibility of overcoming our "radical sol itude" in order that we may gain access to the mental 1 ife of an Other as other human subject. It is clear that in order for us to give expression to these accounts in a coherent manner, along with their relative merits, it will be necessary to develop the common features of any phenomenological theory of consdousness whatever. Therefore, our preliminary inquiry, subordinate to the larger theme, shall be into some of the epistemological results of the application of the phenomenological method used to develop a transcendental theory of consciousness. Inherent in this will be the deliniation of the exigency for making this an lIintentional ll theory. We will then be able to see how itis possible to overcome transcendentally the Other as an object merely given among other merely given objects, and further, how this other is constituted specifically as other ego. The problem of transcendental intersubjectivity and its constitution in experience can be viewed as one of the most compelling, if not the most polemical of issues in phenomenology. To be sure, right from the beginning we are forced to ask a number of questions regarding Husserl's responses to the problem within the context of the methodological genesis of the Cartesian Meditations, and The Crisis of European Sciences and Transcendental Phenomenology. This we do in order to set the stage for amplification. First, we ask, has Husserl lived up to his goal, in this connexion, of an apodictic result? We recall that in his Logos article of 1911 he adminished that previous philosophy does not have at its disposal a merely incomplete and, in particular instances, imperfect doctrinal system; it simply has none whatever. Each and every question is herein controverted, each position is a matter of individual conviction, of the interpretation given byaschool, of a "point of view". 1. Moreover in the same article he writes that his goal is a philosophical system of doctrine that, after the gigantic preparatory work. of generations, really be- . gins from the ground up with a foundation free from doubt and rises up like any skilful construction, wherein stone is set upon store, each as solid as the other, in accord with directive insights. 2. Reflecting upon the fact that he foresaw "preparatory work of generations", we perhaps should not expect that he would claim that his was the last word on the matter of intersubjectivity. Indeed, with 2. 'Edmund Husserl, lIPhilosophy as a Rigorous Science" in Phenomenology and theCrisis6fPhilosophy, trans". with an introduction by Quentin Lauer (New York.: Harper & Row, 1965) pp. 74 .. 5. 2Ibid . pp. 75 .. 6. 3. the relatively small amount of published material by Husserl on the subject we can assume that he himself was not entirely satisfied with his solution. The second question we have is that if the transcendental reduction is to yield the generic and apodictic structures of the relationship of consciousness to its various possible objects, how far can we extend this particular constitutive synthetic function to intersubjectivity where the objects must of necessity always remain delitescent? To be sure, the type of 'object' here to be considered is unlike any other which might appear in the perceptual field. What kind of indubitable evidence will convince us that the characteristic which we label "alter-ego" and which we attribute to an object which appears to resemble another body which we have never, and can never see the whole of (namely, our own bodies), is nothing more than a cleverly contrived automaton? What;s the nature of this peculiar intentional function which enables us to say "you think just as I do"? If phenomenology is to take such great pains to reduce the takenfor- granted, lived, everyday world to an immanent world of pure presentation, we must ask the mode of presentation for transcendent sub .. jectivities. And in the end, we must ask if Husserl's argument is not reducible to a case (however special) of reasoning by analogy, and if so, tf this type of reasoning is not so removed from that from whtch the analogy is made that it would render all transcendental intersubjective understandtng impos'sible? 2. HistoticalandEidetic Priority: The Necessity of Abstraction 4. The problem is not a simple one. What is being sought are the conditions for the poss ibili:ty of experi encing other subjects. More precisely, the question of the possibility of intersubjectivity is the question of the essence of intersubjectivity. What we are seeking is the absolute route from one solitude to another. Inherent in this programme is the ultimate discovery of the meaning of community. That this route needs be lIabstract" requires some explanation. It requires little explanation that we agree with Husserl in the aim of fixing the goal of philosophy on apodictic, unquestionable results. This means that we seek a philosophical approach which is, though, not necessarily free from assumptions, one which examines and makes explicit all assumptions in a thorough manner. It would be helpful at this point to distinguish between lIeidetic ll priority, and JlhistoricallJpriority in order to shed some light on the value, in this context, of an abstraction.3 It is true that intersubjectivity is mundanely an accomplished fact, there havi.ng been so many mi.llions of years for humans to beIt eve in the exi s tence of one another I s abili ty to think as they do. But what we seek is not to study how this proceeded historically, but 3Cf• Maurice Natanson;·TheJburne in 'Self, a Stud in Philoso h and Social Role (Santa Cruz, U. of California Press, 1970 . rather the logical, nay, "psychological" conditions under which this is possible at all. It is therefore irrelevant to the exigesis of this monograph whether or not anyone should shrug his shoulders and mumble IIwhy worry about it, it is always already engaged". By way of an explanation of the value of logical priority, we can find an analogy in the case of language. Certainly the language 5. in a spoken or written form predates the formulation of the appropriate grammar. However, this grammar has a logical priority insofar as it lays out the conditions from which that language exhibits coherence. The act of formulating the grammar is a case of abstraction. The abstraction towards the discovery of the conditions for the poss; bi 1 ity of any experiencing whatever, for which intersubjective experience is a definite case, manifests itself as a sort of "grammar". This "grammar" is like the basic grammar of a language in the sense that these "rulesil are the ~ priori conditions for the possibility of that experience. There is, we shall say, an "eidetic priority", or a generic condition which is the logical antecedent to the taken-forgranted object of experience. In the case of intersubjectivity we readily grant that one may mundanely be aware of fellow-men as fellowmen, but in order to discover how that awareness is possible it is necessary to abstract from the mundane, believed-in experience. This process of abstraction is the paramount issue; the first step, in the search for an apodictic basis for social relations. How then is this abstraction to be accomplished? What is the nature of an abstraction which would permit us an Archimedean point, absolutely grounded, from which we may proceed? The answer can be discovered in an examination of Descartes in the light of Husserl's criticism. 3. The Impulse for Scientific Philosophy. The Method to which it Gives Rise. 6. Foremost in our inquiry is the discovery of a method appropriate to the discovery of our grounding point. For the purposes of our investigations, i.e., that of attempting to give a phenomenological view of the problem of intersubjectivity, it would appear to be of cardinal importance to trace the attempt of philosophy predating Husserl, particularly in the philosophy of Descartes, at founding a truly IIscientific ll philosophy. Paramount in this connexion would be the impulse in the Modern period, as the result of more or less recent discoveries in the natural sciences, to found philosophy upon scientific and mathematical principles. This impulse was intended to culminate in an all-encompassing knowledge which might extend to every realm of possible thought, viz., the universal science ot IIMathexis Universalis ll •4 This was a central issue for Descartes, whose conception of a universal science would include all the possible sciences of man. This inclination towards a science upon which all other sciences might be based waS not to be belittled by Husserl, who would appropriate 4This term, according to Jacab Klein, was first used by Barocius, the translator of Proclus into Latin, to designate the highest mathematical discipline. . 7. it himself in hopes of establishing, for the very first time, philosophy as a "rigorous science". It bears emphasizing that this in fact was the drive for the hardening of the foundations of philosophy, the link between the philosophical projects of Husserl and those of the philosophers of the modern period. Indeed, Husserl owes Descartes quite a debt for indicating the starting place from which to attempt a radical, presupositionless, and therefore scientific philosophy, in order not to begin philosophy anew, but rather for the first time.5 The aim of philosophy for Husserl is the search for apodictic, radical certitude. However while he attempted to locate in experience the type of necessity which is found in mathematics, he wished this necessity to be a function of our life in the world, as opposed to the definition and postulation of an axiomatic method as might be found in the unexpurgated attempts to found philosophy in Descartes. Beyond the necessity which is involved in experiencing the world, Husserl was searching for the certainty of roots, of the conditi'ons which underl ie experience and render it pOssible. Descartes believed that hi~ MeditatiOns had uncovered an absolute ground for knowledge, one founded upon the ineluctable givenness of thinking which is present even when one doubts thinking. Husserl, in acknowledging this procedure is certainly Cartesian, but moves, despite this debt to Descartes, far beyond Cartesian philosophy i.n his phenomenology (and in many respects, closer to home). 5Cf. Husserl, Philosophy as a Rigorous Science, pp. 74ff. 8 But wherein lies this Cartesian jumping off point by which we may vivify our theme? Descartes, through inner reflection, saw that all of his convictions and beliefs about the world were coloured in one way or another by prejudice: ... at the end I feel constrained to reply that there is nothing in a all that I formerly believed to be true, of which I cannot in some measure doubt, and that not merely through want of thought or through levity, but for reasons which are very powerful and maturely considered; so that henceforth I ought not the less carefully to refrain from giving credence to these opinions than to that which is manifestly false, if I desire to arrive at any certainty (in the sciences). 6 Doubts arise regardless of the nature of belief - one can never completely believe what one believes. Therefore, in order to establish absolutely grounded knowledge, which may serve as the basis fora "universal Science", one must use a method by which one may purge oneself of all doubts and thereby gain some radically indubitable insight into knowledge. Such a method, gescartes found, was that, as indicated above by hi,s own words, of II radical doubt" which "forbids in advance any judgemental use of (previous convictions and) which forbids taking any position with regard to their val idi'ty. ,,7 This is the method of the "sceptical epoche ll , the method of doubting all which had heretofor 6Descartes,Meditations on First Philosophy, first Med., (Libera 1 Arts Press, New York, 1954) trans. by L. LaFl eur. pp. 10. 7Husserl ,CrisiS of Eliroeari SCiences and Trariscendental Phenomenology, (Northwestern U. Press, Evanston, 1 7 ,p. 76. 9. been considered as belonging to the world, including the world itself. What then is left over? Via the process of a thorough and all-inclusive doubting, Descartes discovers that the ego which performs the epoche, or "reduction", is excluded from these things which can be doubted, and, in principle provides something which is beyond doubt. Consequently this ego provides an absolute and apodictic starting point for founding scientific philosophy. By way of this abstention. of bel ief, Desca'rtes managed to reduce the worl d of everyday 1 ife as bel ieved in, to mere 'phenomena', components of the rescogitans:. Thus:, having discovered his Archimedean point, the existence of the ego without question, he proceeds to deduce the 'rest' of the world with the aid of innate ideas and the veracity of God. In both Husserl and Descartes the compelling problem is that of establ ishing a scientific, apodictic phi'losophy based upon presuppos itionless groundwork .. Husserl, in thi.s regard, levels the charge at Descartes that the engagement of his method was not complete, such that hi.S: starting place was not indeed presupositionless, and that the validity of both causality and deductive methods were not called into question i.'n the performance of theepoche. In this way it is easy for an absolute evidence to make sure of the ego as: a first, "absolute, indubitablyexisting tag~end of the worldll , and it is then only a matter of inferring the absolute subs.tance and the other substances which belon.g to the world, along with my own mental substance, using a logically val i d deductive procedure. 8 8Husserl, E.;' Cartesian 'Meditation;, trans. Dorion Cairns (Martinus Nijhoff, The Hague, 1970), p. 24 ff.

Investigation of the composition of linear alkylbenzenes with emphasis on the identification and quantitation of some trace compounds using GS/MS system in both electron impact and chemical ionization modes

Linear alkylbenzenes, LAB, formed by the Alel3 or HF catalyzed alkylation of benzene are common raw materials for surfactant manufacture. Normally they are sulphonated using S03 or oleum to give the corresponding linear alkylbenzene sulphonates In >95 % yield. As concern has grown about the environmental impact of surfactants,' questions have been raised about the trace levels of unreacted raw materials, linear alkylbenzenes and minor impurities present in them. With the advent of modem analytical instruments and techniques, namely GCIMS, the opportunity has arisen to identify the exact nature of these impurities and to determine the actual levels of them present in the commercial linear ,alkylbenzenes. The object of the proposed study was to separate, identify and quantify major and minor components (1-10%) in commercial linear alkylbenzenes. The focus of this study was on the structure elucidation and determination of impurities and on the qualitative determination of them in all analyzed linear alkylbenzene samples. A gas chromatography/mass spectrometry, (GCIMS) study was performed o~ five samples from the same manufacturer (different production dates) and then it was followed by the analyses of ten commercial linear alkylbenzenes from four different suppliers. All the major components, namely linear alkylbenzene isomers, followed the same elution pattern with the 2-phenyl isomer eluting last. The individual isomers were identified by interpretation of their electron impact and chemical ionization mass spectra. The percent isomer distribution was found to be different from sample to sample. Average molecular weights were calculated using two methods, GC and GCIMS, and compared with the results reported on the Certificate of Analyses (C.O.A.) provided by the manufacturers of commercial linear alkylbenzenes. The GC results in most cases agreed with the reported values, whereas GC/MS results were significantly lower, between 0.41 and 3.29 amu. The minor components, impurities such as branched alkylbenzenes and dialkyltetralins eluted according to their molecular weights. Their fragmentation patterns were studied using electron impact ionization mode and their molecular weight ions confirmed by a 'soft ionization technique', chemical ionization. The level of impurities present i~ the analyzed commercial linear alkylbenzenes was expressed as the percent of the total sample weight, as well as, in mg/g. The percent of impurities was observed to vary between 4.5 % and 16.8 % with the highest being in sample "I". Quantitation (mg/g) of impurities such as branched alkylbenzenes and dialkyltetralins was done using cis/trans-l,4,6,7-tetramethyltetralin as an internal standard. Samples were analyzed using .GC/MS system operating under full scan and single ion monitoring data acquisition modes. The latter data acquisition mode, which offers higher sensitivity, was used to analyze all samples under investigation for presence of linear dialkyltetralins. Dialkyltetralins were reported quantitatively, whereas branched alkylbenzenes were reported semi-qualitatively. The GC/MS method that was developed during the course of this study allowed identification of some other trace impurities present in commercial LABs. Compounds such as non-linear dialkyltetralins, dialkylindanes, diphenylalkanes and alkylnaphthalenes were identified but their detailed structure elucidation and the quantitation was beyond the scope of this study. However, further investigation of these compounds will be the subject of a future study.

Palladium and platinum complexes of vitamin Bâ

Palladium and platinum complexes of pyridoxamine, pyridoxine and pyridoxal have been prepared. The structures of the complexes PtCI2PM.H20, trans-PdC12 (PN)2 and [PLH+ ]2[PtC16] 2- ,H20 have been determined by use of single crystal x-ray studies. The compounds PdC12PH, trans-PdC12 (PN) 2 , cis-PdCI2 (PN)2 and cis PdC12 (PL)2 were also studied by use of carbon-13 nmr spectroscopy. All the complexes have also been characterised by use of infrared spectral studies. In the complexes, PtCI2PM.H20 and PdC12PM, the ligand pyridoxamine is chela ted to the metal through the aminomethyl nitrogen and the phenolate oxygen atoms whereas in the complexes, trans-PdCI2 (PN)2' cis-PdCI2 (PN)2 and cis-PdC12 (PL)2 the vitamin B6 ligands are coordinated to the metal through the pyridine ring nitrogen. The compounds [PLH+ ]2[PtCI6] 2- .H20 and [PMH2] 2+ [PdCI4] 2- .H20have no direct metal-ligand bonding, In all the complexes, the metal maintains a square planar coordination except in [PLH +] 2[PtCI6] 2- ,H20 where the metal is octahedrally coordinated. PH pyridoxamine [PMH ] 2+ = diprotonated pyridoxamine 2 PN = pyridoxine PL pyridoxal PLH+ protonated pyridoxal

ESI-MS[n] of anticancer pt[iv] organoamido complexes and their interactions with DNA-model compounds /

The general solution behaviour and" the major fragmentation pathways of the anticanceractive PtIV coordination complexes, trans, trans, cis, cis-[PtCIOH{N(pFC6F4) CH2h(pY)2] (1), trans, cis, cis-[Pt(OH)2{N(p-FC6F4)CH2h(Py)2] (2), trans, cis, cis-[Pt(OH)2{N(p-HC6F4)CH2h(Py)2] (3), trans, trans, cis, cis-[PtCIOH{N(pHC6F4) CH2h(Py)2] (4), and trans, trans, cis, cis-[PtOH(OCH3){N(p-HC6F4)CH2h(PY)2] (5) (Py = pyridine) have been deduced by positive-ion tandem-in-time ESI-MS. Overall, the acquired full-scan, positive-ion ESI-MS spectra of 2, 3, and 5 were characterized by the presence of relatively low-intensity [M+Nar and [M+Kt mass spectral peaks, whereas those of 1 and 4 were dominated by extremely intense [M+Hr peaks. Complexes 2 and 3 were also noted to form [2M+Ht and [2M+Nat dilneric cations. The source of Na + and K+ ions is believed to be the sample, the solvent systems used or the transport line carrying the sample solutions into the ES ion source. Further, the fragmentation pathway of all complexes studied was found to be almost identical with concurrent loss of py and H20 molecules, loss of a {N(p-YC6F4)CH2} (Y = F, H) group and/or concomitant release of the latter group and a py ligand being the most conunon. The photochemical degradation behaviour of 1 and 2 was also investigated using either fluorescent or ultraviolet light and some products of that degradation were positively identified. Altogether, light irradiation of solutions of both complexes resulted in cation cationisation, reductive-elimination, ligand-release, ligand-exchange and ligand-addition reactions. Finally, positive- and negative-ion ESI-MSn spectra of 5' -GMP, guanosine, inosine and products of their reactions with 1, 2,3, and 4 were also recorded. On the whole, full-scan ESI-MS spectra of the pure nucleobases revealed the presence of cationic and anionic species that are highly reflective of both their solution ionic composition and their propensity t9 form polymeric clusters. Analyses of mass spectra acquired from their reaction solutions with the aforementioned platinum complexes indicated very slow kinetics. However, all complexes investigated formed, to various degrees, Pt-nucleobase adducts with guanosine and inosine, but not with 5'-GMP. The products included species having coordination numbers of III, IV, V, and VI, among which the first-time· observed, coordinatively saturated, jive-coordinate PtlI-nucleobase complexes were of most interest. The latter complexes are presumably stabilized by 7tback- donation involving the filled d orbitals of the PtII centre and the empty pz· orbital of MeCN. All products, whose peaks appeared inlull-scan ESI-MS spectra, are believed to represent solution species rather than artifacts of gas-phase processes. Finally, negativeion ESI-MSn spectra recorded in reaction solutions of 1 and 4 with guanosine and of the latter complex with inosine revealed the negative-ion-ESI-MS first-time observed, noncovalent, nucleoside-chloride adducts, with the source of chloride anion being complexes 1 and 4 theillselves. In contrast, no such adducts were observed to form with Na25'-GMP or its protonated fonn. Few suggestions are offered for the possible cause(s) behind the absence of such adduct ions.

Enantiodivergent chemoenzymatic synthesis of Balanol and approaches to the synthesis of (+)-Codine

The present thesis reviews the development of a formal enantiodivergent synthesis of the (+)- and (-)-isomers of balanol. This approach commences from a cis-dihydrodiol derived from the enzymatic dihydroxylation of bromobenzene. The stereochemistry of the diol is used to direct the synthesis of two different aziridines, each used in the formal synthesis of one enantiomer of balanol. Also described are several enantioselective approaches to (+ )-codeine. Each strategy begins with the enzymatic dihydroxylation of p-bromoethylbenzene and involves a Mitsunobu inversion and intramolecular Heck reaction as key steps.

Chemoenzymatic synthesis of amaryllidaceae alkaloids and their C-1 analogues : symmetry based approach to total synthesis of thebaine

Described herein is the chemoenzymatic total synthesis of several Amaryllidaceae constituents and their unnatural C-I analogues. A new approach to pancratistatin and related compounds will be discussed along with the completed total synthesis of 7 -deoxypancratistatin and trans-dihydrolycoricidine. Evaluation of all new C-l analogues as cancer cell growth inhibitory agents is described. The enzymatic oxidation of dibromobenzenes by Escherichia coli 1M 109 (pDTG60 1) is presented along with conversion of their metabolites to (-)-conduritol E. Investigation into the steric and functional factors governing the enzymatic dihydroxylation of various benzoates by the same organism is also discussed. The synthetic utility of these metabolites is demonstrated through their conversion to pseudo-sugars, aminocyclitols, and complex bicyclic ring systems. The current work on the total synthesis of some morphine alkaloids is also presented. Highlighted will be the synthesis of several model systems related to the efficient total synthesis of thebaine.

Elucidation of the Retinoid Signalling Pathway Involved in Axon Guidance in Lymnaea stagnalis and Xenopus laevis

The vitamin A metabolite, retinoic acid (RA), is known to play a crucial role in several developmental processes including axial patterning and differentiation. More recently, RA has been implicated in the regenerative process acting through its classical signaling pathway, the nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), to mediate gene transcription. Moreover, RA has been shown to act as a guidance molecule for growth cones of regenerating motorneurons of the pond snail, Lymnaea stagnalis. Our lab has recently shown that RA can induce this morphological response independent of nuclear transcription, however, the role of the retinoid receptors in RA-induced chemoattraction is still unknown. Here, I show that the retinoid receptors, RXR and RAR, may mediate the growth cones response to the metabolically active retinoic acid isomers, all-trans and 9-cis RA, in Lymnaea stagnalis. Data presented here show that both an RXR and RAR antagonist can block growth cone turning in response to application of both isomers. Because no prior investigations have shown growth cone turning of individual vertebrate neurons, I aimed to show that both retinoic acid isomers were capable of inducing growth cone turning of embryonic spinal cord neurons in the frog, Xenopus laevis. For the first time in Xenopus, I showed that both all-trans and 9-cis RA were able to induce significantly more neurite outgrowth from cultured embryonic spinal cord neurons and induce positive growth cone turning of individual growth cones. In addition, I showed that the presence of the RXR antagonist, HX531, blocked 9-cis RA-induced growth cone turning and the RARβ antagonist, LE135, blocked all-trans RA-induced growth cone turning in this species. Evidence provided here shows for the first time, conservation of retinoic acid-induced growth cone turning in a vertebrate model system. In addition, these data show that the receptors involved in this morphological response may be the same in vertebrates and invertebrates.

Investigating the role of retinoic acid in the vertebrate and invertebrate nervous system

Please consult the paper edition of this thesis to read. It is available on the 5th Floor of the Library at Call Number: Z 9999.5 B56 D64 2007

The contribution of skate blade properties to skating speed

The purpose of the study was to investigate the relative contribution of skate blade properties to on-ice skating speed. Thirty-two male ice hockey players (mean age = 19±2.65 yrs.) representing the Ontario Minor Hockey Association (OMHA; Midget AAA and Junior), Canadian Inter University Sport (CIS: Varsity), Ontario hockey league (OHL) and East Coast Hockey League (ECHL), and the playing positions of forwards (n=18) and defense (n=14) were recruited to participate. Skate related equipment worn by the players for the purpose of the research was documented and revealed that 80% of the players wore Bauer skates, Tuuk blade holders and LS2 skate blades. Subjects completed a battery of eight on-ice skating drills used to measure and compare two aspects of skating speed; acceleration [T1(s)] and total time to complete each drill [TT(s)] while skating on three skate blade conditions. The drills represented skills used in the game of hockey, both in isolation (e.g., forward skating, backward skating, stops and starts, and cornering) and in sequence to simulate the combination of skills used in a shift of game play. The three blade conditions consisted of (i) baseline, represented by the blades worn by the player throughout their current season of play; (ii) experimental blades (EB), represented by brand name experimental blades with manufacturers radius of contour and a standardized radius of hollow; and (iii) customized experimental blades (CEB), represented by the same brand name experimental blades sharpened to the players’ preference as identified in the baseline condition. No significant differences were found in acceleration time [T1(s)] or total time to complete [TT(s)] the isolated drills across blade conditions; however significant differences were revealed in both T1(s) and TT(s) measured during the execution of the sequenced drill across blade conditions. A iii Bonferroni post hoc test revealed that players skated significantly faster when skating on the CEB condition compared to the baseline condition (p≤.05). A questionnaire assessing subjects perceived comfort, confidence and effort expended while skating on the experimental blades revealed that players were significantly more comfortable when skating on the CEB versus the EB condition (p≤.05). Outcomes of the study provide evidence to suggest that the experimental skate blades customized with the players preferred blade sharpening characteristics results in faster skating speed in a combination drill representing skills performed in gameplay.