O uso da cocaína e do crack tornou-se um problema de saúde pública importante no Brasil por conta de prejuízos significativos do ponto de vista médico, psicológico e social que ele acarreta. Estudos de gêmeos e, em famílias, sugerem que a dependência de cocaína é uma doença complexa, com participação importante de fatores genéticos. Os estudos genéticos sobre usuários de cocaína são poucos e padecem de problemas metodológicos, tais como, amostras pequenas, com alto grau de miscigenação populacional e um número limitado de marcadores genéticos pesquisados. Além disto, há pouco sendo feito no sentido de verificar como os genes já associados à dependência de cocaína interagem entre si, ou seja, de investigações sobre a epistasia genética. Com o intuito de aprofundar a investigação dos aspectos biológicos da dependência de cocaína, nós estudamos, através de um estudo casocontrole, uma amostra de inicial de 746 pacientes dependentes de crack/cocaína hospitalizados em clínicas especializadas para o tratamento de dependência química na cidade de São Paulo, que foram comparados a 891 controles normais, sem história prévia de abuso ilegal de substâncias. Os objetivos desta tese foram: 1) verificar a associação de três polimorfismos (rs1803274, rs4263329, rs4680662) para o gene da butirilcolinesterase (BCHE), uma enzima envolvida na metabolização da cocaína no desenho do tipo gene candidato; 2) testar a hipótese de interação entre o marcador funcional Val158Met do gene para a enzima catecol-O-metiltransferase (COMT) e os marcadores do tipo VNTR das regiões 3´UTR e Intron8 do gene do transportador da dopamina (DAT1) e; 3) numa análise de caráter exploratório, verificar a interação gene-gene de 40 polimorfismos em 12 genes com plausibilidade biológica para a dependência da cocaína. A análise estatística fez uso de modelos de regressão logística para a interação de marcadores nos dois genes, COMT e DAT1 e, do programa Multifactor Dimensionality Reduction (MDR) para a análise multivariada. A análise envolvendo os marcadores para o gene BCHE não se mostraram associados ao fenótipo da dependência de cocaína porém, encontrou-se uma associação do marcador funcional rs1803274 (p=0,001; OR=5,83; IC95%=2,10 - 16,16) nos usuários exclusivos de crack, a forma cheirada da cocaína quando comparados aos grupos de uso exclusivo da cocaína na forma cheirada ou, de uso das duas formas de administração. Os marcadores do tipo VNTR da DAT1 não interagiram em um modelo de regressão logística com o marcador Val158Met da COMT. Finalmente, os modelos construídos pelo programa MDR não forneceram interações gene-gene que tivessem uma previsibilidade além do acaso. Dentro de uma perspectiva genética, os estudos futuros para a dependência de cocaína devem aprimorar a caracterização fenotípica, por meio de subgrupos divididos por sintomas clínicos e pelo uso de fenótipos intermediários, fazer um rastreio minucioso dos marcadores ao longo dos genes de interesse e, usar de métodos analíticos para as interações gene-gene e gene-ambiente / The use of crack/cocaine has become a major public health problem in Brazil due to its manifold problems in the medical, psychological and social realms. Twin and family studies have documented the role played by genetic factors and environment in cocaine addiction. Genetic association studies in cocaine addiction are few and have methodological problems: small sample size, population stratification and a paucity of genetic markers have been studied so far. There is also a lack of knowledge on how the genes already shown to be associated with cocaine addiction interact, that is, genetic epistasis. In order to advance the knowledge of biological factors in cocaine addiction we investigated, by means of a case-control study, 746 patients with crack/cocaine dependence admitted to specialized clinics for the treatment of drug addiction in the city of São Paulo. They were compared to 891 control subjects with no previous history of illegal drug abuse. The objectives of this thesis were: 1) investigate the association of three SNPs (rs1803274, rs4263329, rs4680662) in the butirilcholinesterase gene (BCHE) that encodes an enzyme involved in cocaine metabolism; 2) test the hypothesis of an interaction between the functional marker Val158Met of the catechol-o-metiltransferase enzyme (COMT) gene and two VNTRs markers, 3´UTR and Intron8, of the dopamine transporter gene (DAT1) and, 3) in an exploratory analysis, investigate the gene-gene interaction of 40 polymorphisms in 12 genes with a biological plausibility for cocaine addiction. Logistic regression was used to assess COMT*DAT1 gene-gene interaction and, the Multifactor Dimensionality Reduction (MDR) program was used for the other multivariate analysis. Genetic variants for the BCHE gene were not associated with cocaine addiction but, an association was found between the functional marker rs1803274 (p=0,001; OR=5,83; IC95%=2,10 - 16,16) and crack users compared to those that snorted cocaine or used both forms of administration. The DAT1 VNTRs did not interact with the COMT Val158Met marker. Finally, the models generated by the MDR program did not provided any predictive gene-gene interaction better than chance. Future studies investigating genetic risk factors for cocaine dependence should improve phenotype characterization (clinically derived subgroups and use of endophenotypes) and should also make a thorough scan of the genetic markers along the genes of interest including gene-gene and gene-environment analysis

Tyrosine (4-hydroxyphenylalanine) is one of the naturally occurring 22 amino acids. The importance of tyrosine is due to the presence of its phenolic side chain. In biological systems, the tyrosyl residue in proteins is found to be sulfated, phosphorylated and nitrated. Upon oxidation with dioxygenases, Tyr residue forms dopaquinone which undergoes a series of reactions ultimately leading to the formation of melanin. Tyr is also a precursor to neurotransmitters (catechol amines namely dopamine, epinephrine and norepinephrine) and thyroid harmones T4 and T3. Tyr residue is also found to be cross linked with other amino acid residues in the active site of certain proteins. Tyr-Tyr cross link has also been associated with neurodegenerative diseases. Tyr residue in proteins has been targeted widely for site selective modifications. A series of chemical modifications like acylation, allylation, ene-type reaction, iodination with radiolabeled iodine, formation of Tyr-Tyr cross link with oxidants and aminoalkylation have been carried out on surface exposed Tyr residues in proteins. Apart from these chemical modifications of Tyr on protein surface, a couple of free Tyr-based scaffolds have also been developed for different applications. Similar to tyrosine-based scaffolds, several phenolate-based scaffolds have also been developed for various applications. Several phenolate-based binuclear metal complexes have been developed as mimics of the active site of metalloenzymes. Moreover, by varying the substituent in the phenolate scaffold, the redox properties of metal bound in these systems can be tuned. The thesis consists of five chapters. The first chapter gives general idea about tyrosine-and phenolate-based scaffolds. The first chapter also gives introduction to zinc(II)-containing enzymes metallo-β-lactamases (mβls) and phosphotriesterase (PTE) and their functional mimics. The importance of copper(II)-containing enzyme, catechol oxidase and its mimics has also been discussed. The significance and formation of o-dityrosine (Tyr-Tyr cross link) has also been briefly discussed. In chapters 2 and 3, a couple of phenolate-based ligands and their corresponding zinc(II)- and copper(II)- complexes have been synthesized and have been checked as mimics of zinc(II)-containing enzymes (mβl and PTE) and copper-containing enzyme catechol oxidase, respectively. In chapter 4, a series of tyrosine-based ligands have been designed and their in situ copper(II) complexes have been tested as mimics of catechol oxidase. In chapter 5, the effect of neighboring amino acid in the formation of Tyr-Tyr cross link has been studied. In chapter 2, a couple of zinc(II) complexes have been synthesized and studied as mimic of zinc(II)-containing enzymes mβl and PTE. Metallo-β-lactamases (mβls) are zinc(II)-containing enzymes which exist in both mono- and binuclear forms. Mβls are capable of hydrolyzing β-lactam ring in antibiotics and make them inactive (Scheme 1(A)). To date, an effective inhibitor for this enzyme is not known. Hence, in order to understand the nature of the enzyme a couple of synthetic mimics are known. However, in most of the synthetic mimics both the metal ions are in symmetrical environment. Therefore, we have attempted to design a few unsymmetrical phenolate- based ligands and their zinc(II) complexes. The unsymmetrical phenolate-based ligands HL1 and HL2 have been synthesized by sequential mannich reaction with formaldehyde and two different amines. Complexes 1 and 2 are obtained from ligands HL1 and HL2, respectively (Figure 1). For comparative purpose, the symmetrical ligands HL3 and HL4, and their zinc(II)-complexes 3 and 4 have been synthesized by reported procedures (Figure 1). The efficiency of the complexes 1-4 towards the hydrolysis of oxacillin has been studied. It has been observed that the binuclear zinc(II) complexes with metal-bound water molecule 1 and 4 are able to hydrolyze oxacillin at much faster rates compared to that of mononuclear complexes 2 and 3. However, between 1 and 4, there is no appreciable change in activity, indicating that the slight change in ligand environment has no significant role. PTE is a binuclear zinc(II)-containing enzyme, capable of hydrolyzing toxic organphosphotriesters to less toxic diesters (Scheme 1(B)). As the binuclear active site of mβl is comparable with that of phosphotriesterase (PTE), PTE activity of complexes 1-4 has been studied. Although the binuclear zinc(II)-complexes 1 and 4 are able to hydrolyze PNPDPP (p-nitrophenyl diphenyl phosphate) initially, these complexes are not able to effect complete hydrolysis. This is due to the inhibition of complexes 1 and 4 by hydrolyzed product, diester. However with mononuclear complexes 2 and 3 no such inhibitions is possible, and are capable of hydrolyzing PNPDPP at comparatively faster rates than 1 and 4. Scheme 1. Function of metallo-β-lactamase and phosphotriesterase. (A) Hydrolysis of β-lactam ring in antibiotics by metallo-β-lactamase. (B) Hydrolysis of organophosphotriesters to diesters by phosphotriesterase. Figure 1. Chemical structures of ligands HL1-HL4 and their corresponding zinc(II)complexes 1-4. In chapter 3, a couple of copper(II) complexes have been synthesized and their catechol oxidase activity has been studied. Catechol oxidase belongs to the class of oxidoreductase and it catalyzes the oxidation of a wide range of o-diphenols to o-quinones through the reduction of molecular oxygen to water (Scheme 2). A four new µ4-oxo-bridged tetranuclear copper(II) complexes (5-8) have been synthesized (Figure 2). The ability of these complexes to catalyze the oxidation of 3,5-DTBC (3,5-Di-tert-butylcatechol) to 3,5-DTBQ (3,5-Di-tert-butylquinone) has been studied. A detailed kinetic study has been carried out which reveals that the complexes with exogenous acetate ligands (5 and 6) are better catechol oxidase mimics compared to complexes with exogenous chloride ligands (7 and 8). This observation is due to the labile nature of acetate compared to chloride, as the displacement of exogenous ligand is essential for the binding of substrate to the catalyst. Based on mass spectral analysis a plausible mechanism has been proposed for the oxidation of 3,5-DTBC by these complexes. Scheme 2. Oxidation of catechol by catechol oxidase. Figure 2. Chemical structures of copper(II) complexes 5-8. In chapter 4, by following the analogy between phenol and tyrosine, a series of binucleating ligands of tyrosine or tyrosyl dipeptides (Figures 3 and 4) have been synthesized by Mannich reaction under mild conditions. The in situ complexation of these fifteen new binucleating ligands (HL5-HL19) with copper(II) chloride has been observed. In situ complexation was followed by UV-visible and mass spectral analysis. These in situ complexes were able to oxidize 3,5-DTBC at slower rate compared to that of the tetranuclear complexes reported in chapter 3. The catecholase activity has also been tested with the addition of base. A slight enhancement in activity of in situ complexes has been observed in the presence of base. Based on mass spectral evidences, a plausible mechanism for the oxidation of catechol by these in situ complexes has been proposed. Figure 3. Binucleating ligands (Mannich bases) of boc-protected tyrosine and tyrosyl dipeptides. Figure 4. Binucleating ligands (Mannich bases) of boc-deprotected tyrosyl dipeptides. In chapter 5 of the thesis, the effect of neighboring amino acid residue in the formation of o,o-dityrosine (Tyr-Tyr cross link) has been studied. o,o’-Dityrosine is a specific marker for oxidative/nitrosative stress. The increase in concentration of dityrosine is associated with several disease states. A detailed study has been carried out in order to find out the effect of neighboring amino acid residues in the rate of formation of dityrosine of several tyrosyl dipeptides. The formation of dityrosine has been carried out with horseradish peroxidase(HRP) and H2O2 (Scheme 3). Except Cys-Tyr, all other tyrosyl dipeptides, form corresponding dityrosine with HRP/ H2O2. With Cys-Tyr, the formation of corresponding disulfide is observed. The appreciably higher rate of dityrosine formation of Phe-Tyr is attributed to the presence of strong hydrophobic environment around the active site of HRP. Among the polar tyrosyl peptides, the positively charged peptides (Arg-Tyr, Lys-Tyr) undergo dityrosine formation at much faster rate compared to that of negatively charged dipepptides (Asp-Tyr, Glu-Tyr). This trend is in accordance with the pKa of neighboring amino acid residues. The positively charged neighboring residues with higher pKa stabilizes ionized tyrosine, hence the rate of dityrosine formation is higher for them. As positively charged neighboring residue enhances the rate of dityrosine formation, the effect of externally added L-Arg has been studied. A coupling of a few biologically relevant tyrosine derivatives has been studied. The derivatives in which one of the ortho-positions of tyrosine is blocked, does not undergo coupling under the experimental conditions employed. Scheme 3. Formation of dityrosine of Ile-Tyr from Ile-Tyr in the presence of H2O2 catalyzed by HRP. (For structural formula and figures pl refer the abstract pdf file)

<p><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a></a><a>ABSTRACT</a></p> <p>Phenols are ubiquitous in our surroundings including biological molecules such as L-Dopa metabolites, food components, such as whiskey and liquid smoke, etc. This dissertation describes a new method for detecting phenols, by reaction with Gibbs reagent to form indophenols, followed by mass spectrometric detection. Unlike the standard Gibbs reaction which uses a colorimetric approach, the use of mass spectrometry allows for simultaneous detection of differently substituted phenols. The procedure is demonstrated to work for a large variety of phenols without <i>para</i>‐substitution. With <i>para</i>‐substituted phenols, Gibbs products are still often observed, but the specific product depends on the substituent. For <i>para</i> groups with high electronegativity, such as methoxy or halogens, the reaction proceeds by displacement of the substituent. For groups with lower electronegativity, such as amino or alkyl groups, Gibbs products are observed that retain the substituent, indicating that the reaction occurs at the <i>ortho</i> or <i>meta</i> position. In mixtures of phenols, the relative intensities of the Gibbs products are proportional to the relative concentrations, and concentrations as low as 1 μmol/L can be detected. The method is applied to the qualitative analysis of commercial liquid smoke, and it is found that hickory and mesquite flavors have significantly different phenolic composition.</p> <p>In the course of this study, we used this technique to quantify major phenol derivatives in commercial products such as liquid smoke (catechol, guaiacol and syringol) and whiskey (<i>o</i>-cresol, guaiacol and syringol) as the phenol derivatives are a significant part of the aroma of foodstuffs and alcoholic beverages. For instance, phenolic compounds are partly responsible for the taste, aroma and the smokiness in Liquid Smokes and Scotch whiskies. </p> <p>In the analysis of Liquid Smokes, we have carried out an analysis of phenols in commercial liquid smoke by using the reaction with Gibbs reagent followed by analysis using electrospray ionization mass spectrometry (ESI-MS). This analysis technique allows us to avoid any separation and/or solvent extraction steps before MS analysis. With this analysis, we are able to determine and compare the phenolic compositions of hickory, mesquite, pecan and apple wood flavors of liquid smoke. </p> <p>In the analysis of phenols in whiskey, we describe the detection of the Gibbs products from the phenols in four different commercial Scotch whiskies by using simple ESI-MS. In addition, by addition of an internal standard, 5,6,7,8-tetrahydro-1-napthol (THN), concentrations of the major phenols in the whiskies are readily obtained. With this analysis we are able to determine and compare the composition of phenols in them and their contribution in the taste, smokey, and aroma to the whiskies.</p> <p>Another important class of phenols are found in biological samples, such as L-Dopa and its metabolites, which are neurotransmitters and play important roles in living systems. In this work, we describe the detection of Gibbs products formed from these neurotransmitters after reaction with Gibbs reagent and analysis by using simple ESI‐MS. This technique would be an alternative method for the detection and simultaneous quantification of these neurotransmitters. </p> <p>Finally, in the course of this work, we found that the positive Gibbs tests are obtained for a wide range of <i>para</i>-substituted phenols, and that, in most cases, substitution occurs by displacement of the <i>para</i>-substituent. In addition, there is generally an additional unique second-phenol-addition product, which conveniently can be used from an analytical perspective to distinguish <i>para</i>-substituted phenols from the unsubstituted versions. In addition to using the methodology for phenol analysis, we are examining the mechanism of indophenol formation, particularly with the <i>para</i>-substituted phenols. </p> <p>The importance of peptides to the scientific world is enormous and, therefore, their structures, properties, and reactivity are exceptionally well-characterized by mass spectrometry and electrospray ionization. In the dipeptide work, we have used mass spectrometry to examine the dissociation of dipeptides of phenylalanine (Phe), containing sulfonated tag as a charge carrier (Phe*), proline (Pro) to investigate their gas phase dissociation. The presence of sulfonated tag (SO₃^-) on the Phe amino acid serves as the charge carrier such that the dipeptide backbone has a canonical structure and is not protonated. Phe-Pro dipeptide and their derivatives were synthesized and analyzed by LCQ-Deca mass spectroscopy to get the fragmentation mechanism. To confirm that fragmentation path, we also synthesized dikitopeparazines and oxazolines from all combinations of the dipeptides. All these analyses were confirmed by isotopic labeling experiments and determination and optimization of structures were carried out using theoretical calculation. We have found that the fragmentation of Phe*Pro and ProPhe* dipeptides form sequence specific b₂ ions. In addition, not only is the ‘mobile proton’ involved in the dissociation process, but also is the ‘backbone hydrogen’ is involved in forming b₂ ions. </p> <p> </p>