With the increasing demand for assisted reproduction, more knowledge and understanding towards health requirements of oocytes and their inner workings are required. With current IVF success rates of approximately 40%, oocyte and embryo culture conditions in vitro can be improved by first understanding the finer details of oocyte function. As such, there is a need to better understand the mechanisms through which oocytes can acquire certain nutrients. This thesis focuses on the amino acid serine, which has been shown to improve outcome in developing embryos and also plays a variety of roles in the body that may carry over to oocyte health as well. Using radiolabeled [3H] serine, we measured uptake of serine as a function of time throughout growth and meiotic maturation in mouse oocytes. Serine transport appeared in oocytes during growth and became absent in mature eggs. With a competition assay using substrates diagnostic for several different amino acid transporter systems and culture with and without sodium in the external medium, I identified Na+-dependent SNAT7 of the System A/N (SLC38) family to be the most likely transporter in oocytes. Quantitative RT-PCR was consistent with this result. Transporter activity is also not activated by progression of meiotic maturation, as indicated by unperturbed transport when dbcAMP was provided to maintain meiotic arrest. However, a biological regulator of arrest, NPPC, resulted in enhanced transport activity in vitro. This may be due to signalling mechanisms of the NPPC pathway affecting regulation of serine uptake, which presents a direction for future research.

The Group III metabotropic glutamate receptors (mGluR) are located presynaptically on axon terminals and act as autoreceptors and heteroreceptors by inhibiting neurotransmitter release. Much has been learned about these receptors through exogenous application of L-serine-O-phosphate (L-SOP), an endogenous amino acid derivative and known activator of the Group III mGluRs. We hypothesized that L-SOP is the endogenous co-agonist at the high affinity Group III mGluR, mGluR4. We found the EC50 of L-SOP at mGluR4 was 0.5 μM, and determined that the concentration of L-SOP in whole brain was approximately 5 μM. An immunocytochemical survey revealed that cells containing the enzymatic machinery necessary for L-SOP synthesis and metabolism were observed in two brain regions known to express mGluR4, namely, cerebellum and hippocampus. In the cerebellum, the L-SOP synthetic and metabolic enzymes were found in Bergmann glia and Purkinje cells, two cells which form a tripartite synapse with parallel fiber axon terminals where the mGluR4 subtype is exclusively expressed at high levels. In the hippocampus, the L-SOP metabolic enzyme was detected in young neurons emanating from the neurogenic subventricular zone. Attempts to raise endogenous levels of L-SOP by crippling the L-SOP metabolizing enzyme (phosphoserine phosphatase), over-expressing the L-SOP synthesizing enzyme (phosphoserine aminotransferase), or through dietary protein restriction, to study the effects on neurotransmission and neurodevelopment in the central nervous system (CNS) were unsuccessful, suggesting that the production of L-SOP remains stable despite manipulation of the synthetic and metabolic enzymes. Finally, the ability of L-SOP to modulate glutamate release from presynaptic terminals was examined in cerebellar synaptosomes. Co-incident activation of presynaptic mGluR4 and presynaptic GABAA receptors facilitated glutamate release, suggesting that simultaneous activation of parallel fibers and Bergmann glia may serve to enhance synaptic transmission. This observation expands the traditional view of Group III mGluRs acting solely as inhibitory autoreceptors. Taken together, these results provide compelling evidence to support the hypothesis that L-SOP is the endogenous agonist at mGluR4, and possibly other Group III mGluRs.

The Group III metabotropic glutamate receptors (mGluR) are located presynaptically on axon terminals and act as autoreceptors and heteroreceptors by inhibiting neurotransmitter release. Much has been learned about these receptors through exogenous application of L-serine-O-phosphate (L-SOP), an endogenous amino acid derivative and known activator of the Group III mGluRs. We hypothesized that L-SOP is the endogenous co-agonist at the high affinity Group III mGluR, mGluR4. We found the EC50 of L-SOP at mGluR4 was 0.5 μM, and determined that the concentration of L-SOP in whole brain was approximately 5 μM. An immunocytochemical survey revealed that cells containing the enzymatic machinery necessary for L-SOP synthesis and metabolism were observed in two brain regions known to express mGluR4, namely, cerebellum and hippocampus. In the cerebellum, the L-SOP synthetic and metabolic enzymes were found in Bergmann glia and Purkinje cells, two cells which form a tripartite synapse with parallel fiber axon terminals where the mGluR4 subtype is exclusively expressed at high levels. In the hippocampus, the L-SOP metabolic enzyme was detected in young neurons emanating from the neurogenic subventricular zone. Attempts to raise endogenous levels of L-SOP by crippling the L-SOP metabolizing enzyme (phosphoserine phosphatase), over-expressing the L-SOP synthesizing enzyme (phosphoserine aminotransferase), or through dietary protein restriction, to study the effects on neurotransmission and neurodevelopment in the central nervous system (CNS) were unsuccessful, suggesting that the production of L-SOP remains stable despite manipulation of the synthetic and metabolic enzymes. Finally, the ability of L-SOP to modulate glutamate release from presynaptic terminals was examined in cerebellar synaptosomes. Co-incident activation of presynaptic mGluR4 and presynaptic GABAA receptors facilitated glutamate release, suggesting that simultaneous activation of parallel fibers and Bergmann glia may serve to enhance synaptic transmission. This observation expands the traditional view of Group III mGluRs acting solely as inhibitory autoreceptors. Taken together, these results provide compelling evidence to support the hypothesis that L-SOP is the endogenous agonist at mGluR4, and possibly other Group III mGluRs.

Sudden changes in nutrient resources are common in the natural environment. Cells are able to adapt and propagate under changing environmental conditions by making adjustments in their cellular processes. These cellular adaptations involve genome-wide transcriptional reprogramming that results in the induction or repression of metabolic pathways. Specific enzymes are then synthesised and activated to maximise the use of the newly available nutrient sources. L-serine is one of the twenty proteinogenic amino acids, and can be synthesised in yeast by the glycolytic and gluconeogenic pathways when growing on fermentable or non-fermentable carbon sources or taken up from the environment when available. L-serine is metabolically linked to glycine and is a predominant donor of one-carbon units in one-carbon metabolism. L-serine is also a source of pyruvate and ammonia and contributes to other cellular processes including the biosynthesis of cysteine and phospholipids. Previous work has shown that yeast cells exhibit transcriptional induction of the one-carbon pathway and the genes involved in the synthesis of purine and methionine after the addition of 10 mM glycine. Here it is shown that addition of 10 mM L-serine did not, however, elicit the same transcriptional response. This is primarily due to differences in the uptake of glycine and L-serine in yeast. High concentrations of extracellular L-serine were required for yeast to show an increase in intracellular L-serine concentration of the magnitude required to trigger a noticeable cellular response. Despite L-serine and glycine being interconvertable via the SHMT isozymes and being a one-carbon donor, the genome-wide transcriptional response exhibited by cells in response to L-serine addition was markedly different to that seen for glycine. The predominant response to an increase in intracellular L-serine was the induction of the general amino acid control system and the CHA1 gene encoding the serine (threonine) dehydratase. Unlike glycine, addition of L-serine triggered only minor induction of the one-carbon pathway. A large portion of intracellular L-serine was converted to pyruvate and ammonia in the mitochondrion as the result of induction of CHA1. The high intracellular concentration of L-serine stimulated the cell to increase the production of oxaloacetate and to increase the biosynthesis of L-aspartate. Transient increases in the intracellular L-glutamate and L-glutamine were also observed after the addition of L-serine. The work presented in this study shows that large increase in the intracellular concentration of amino acid is required to trigger a significant transcriptional response. Yeast cells exhibit different transcriptional and metabolic responses to the addition of L-serine and glycine even though these two amino acids are closely metabolically linked. Addition of L-serine provokes the GAAC response, expression of the CHA1 gene and stimulates the biosynthesis of L-aspartate in yeast whereas addition of glycine induces the one-carbon pathway which leads to the biosynthesis of the purine nucleotides.

L-serine, a non-essential amino acid, plays a role in the biosynthesis of the amino acids, proteins, purine and pyrimidine nucleotides. It is important for the proper functioning of the nervous system. It has been considered in the treatment of patients with schizophrenia, depression, chronic fatigue syndrome and psychomotor retardation, and of the seizures encountered in patients with rare inborn errors of L-serine biosynthesis. However, there are no reports in the literature of the direct cardiovascular effects of L-serine. Using normotensive Sprague-Dawley rats, Sprague-Dawley rats rendered hypertensive by chronic treatment with the nitric oxide (NO) synthase inhibitior NG nitro L-arginine methyl ester (L-NAME) and spontaneously hypertensive rats (SHR), the present study examined the in vitro and in vivo effects of L-serine. In vitro studies focused on L-serine induced changes in phenylephrine constricted third order branches of rat mesenteric arterioles while the in vivo studies examined the effects of intravenous infusion of L-serine on mean arterial pressure (MAP) and heart rate (HR) in intact anaesthetized rats. L-serine (10 to 200 µmol/L) evoked concentration-dependent vasodilatation in phenylephrine constricted endothelium-intact, but not in endothelium-denuded, rat mesenteric arterioles. The vasodilator responses to L-serine were absent in the combined presence of apamin, a calcium activated small conductance potassium (SKCa) channel inhibitor, and TRAM-34, a calcium activated intermediate conductance potassium (IKCa) channel inhibitor, or ouabain, a sodium pump inhibitor and barium (Ba2+), an inward rectifying potassium (Kir) channel inhibitor, or when the vessels were depolarized by potassium chloride. The maximal vasodilatation response (Emax) to L-serine was higher in vessels from L-NAME treated rats (40%) than from control rats (20%). In anesthetized rats, L-serine evoked a rapid, reversible, dose-dependent fall in MAP (without a significant change in HR), which was more pronounced in L-NAME treated rats (> 60 mmHg) than in normotensive control rats (25 mmHg). The fall in MAP was inhibited (p<0.01) by apamin plus charybdotoxin pretreatment. Charybdotoxin was used in place of Tram-34 in in vivo studies since Tram-34 is not soluble in water or saline. In age matched Sprague-Dawley, Wistar-Kyoto (WKY) and SHR strains, D-serine had the same effects on MAP and HR as L-serine; however, L-serine evoked a greater maximal fall in MAP in all strains, and the effect was more pronounced in hypertensive rats. In contrast, the infusion of glycine, a metabolite of L-serine led to a dose-dependent fall in MAP in normotensive rats but a dose-dependent increase in MAP in both SHR and L-NAME treated hypertensive WKY rats. Both the depressor and pressor responses to glycine were abolished by pretreatment with the N-methyl D-aspartate receptor antagonist, MK-801. Regional hemodynamic studies performed using the fluorescent tagged microsphere distribution technique revealed that the fall in MAP and profound decrease in total peripheral resistance (TPR) evoked by acute L-serine infusion is due to increased blood flow in the splanchnic region and more particularly in the small intestinal vascular beds. This effect is blocked by the combined treatment with the KCa channel inhibitors, apamin plus charybdotoxin. Although resting MAP and TPR are higher, and cardiac output (CO) is lower both in SHR and in WKY rats rendered hypertensive by L-NAME treatment compared to normotensive WKY rats, L-serine infusion leads to a rapid fall in TPR and MAP, and an increase in CO in all models. This effect was more profound in the hypertensive rats. These findings suggest that L-serine could be helpful in overcoming splanchnic organ failure observed in patients with cardiopulmonary bypass. In addition, L-serine, either alone or in combination with other antihypertensive medications, could be considered in the management of endothelial dysfunctional states with reduced NO bioavailability such as hypertension and diabetes.

L-serine, a non-essential amino acid, plays a role in the biosynthesis of the amino acids, proteins, purine and pyrimidine nucleotides. It is important for the proper functioning of the nervous system. It has been considered in the treatment of patients with schizophrenia, depression, chronic fatigue syndrome and psychomotor retardation, and of the seizures encountered in patients with rare inborn errors of L-serine biosynthesis. However, there are no reports in the literature of the direct cardiovascular effects of L-serine. Using normotensive Sprague-Dawley rats, Sprague-Dawley rats rendered hypertensive by chronic treatment with the nitric oxide (NO) synthase inhibitior NG nitro L-arginine methyl ester (L-NAME) and spontaneously hypertensive rats (SHR), the present study examined the in vitro and in vivo effects of L-serine. In vitro studies focused on L-serine induced changes in phenylephrine constricted third order branches of rat mesenteric arterioles while the in vivo studies examined the effects of intravenous infusion of L-serine on mean arterial pressure (MAP) and heart rate (HR) in intact anaesthetized rats. L-serine (10 to 200 µmol/L) evoked concentration-dependent vasodilatation in phenylephrine constricted endothelium-intact, but not in endothelium-denuded, rat mesenteric arterioles. The vasodilator responses to L-serine were absent in the combined presence of apamin, a calcium activated small conductance potassium (SKCa) channel inhibitor, and TRAM-34, a calcium activated intermediate conductance potassium (IKCa) channel inhibitor, or ouabain, a sodium pump inhibitor and barium (Ba2+), an inward rectifying potassium (Kir) channel inhibitor, or when the vessels were depolarized by potassium chloride. The maximal vasodilatation response (Emax) to L-serine was higher in vessels from L-NAME treated rats (40%) than from control rats (20%). In anesthetized rats, L-serine evoked a rapid, reversible, dose-dependent fall in MAP (without a significant change in HR), which was more pronounced in L-NAME treated rats (> 60 mmHg) than in normotensive control rats (25 mmHg). The fall in MAP was inhibited (p<0.01) by apamin plus charybdotoxin pretreatment. Charybdotoxin was used in place of Tram-34 in in vivo studies since Tram-34 is not soluble in water or saline. In age matched Sprague-Dawley, Wistar-Kyoto (WKY) and SHR strains, D-serine had the same effects on MAP and HR as L-serine; however, L-serine evoked a greater maximal fall in MAP in all strains, and the effect was more pronounced in hypertensive rats. In contrast, the infusion of glycine, a metabolite of L-serine led to a dose-dependent fall in MAP in normotensive rats but a dose-dependent increase in MAP in both SHR and L-NAME treated hypertensive WKY rats. Both the depressor and pressor responses to glycine were abolished by pretreatment with the N-methyl D-aspartate receptor antagonist, MK-801. Regional hemodynamic studies performed using the fluorescent tagged microsphere distribution technique revealed that the fall in MAP and profound decrease in total peripheral resistance (TPR) evoked by acute L-serine infusion is due to increased blood flow in the splanchnic region and more particularly in the small intestinal vascular beds. This effect is blocked by the combined treatment with the KCa channel inhibitors, apamin plus charybdotoxin. Although resting MAP and TPR are higher, and cardiac output (CO) is lower both in SHR and in WKY rats rendered hypertensive by L-NAME treatment compared to normotensive WKY rats, L-serine infusion leads to a rapid fall in TPR and MAP, and an increase in CO in all models. This effect was more profound in the hypertensive rats. These findings suggest that L-serine could be helpful in overcoming splanchnic organ failure observed in patients with cardiopulmonary bypass. In addition, L-serine, either alone or in combination with other antihypertensive medications, could be considered in the management of endothelial dysfunctional states with reduced NO bioavailability such as hypertension and diabetes.

Sudden changes in nutrient resources are common in the natural environment. Cells are able to adapt and propagate under changing environmental conditions by making adjustments in their cellular processes. These cellular adaptations involve genome-wide transcriptional reprogramming that results in the induction or repression of metabolic pathways. Specific enzymes are then synthesised and activated to maximise the use of the newly available nutrient sources. L-serine is one of the twenty proteinogenic amino acids, and can be synthesised in yeast by the glycolytic and gluconeogenic pathways when growing on fermentable or non-fermentable carbon sources or taken up from the environment when available. L-serine is metabolically linked to glycine and is a predominant donor of one-carbon units in one-carbon metabolism. L-serine is also a source of pyruvate and ammonia and contributes to other cellular processes including the biosynthesis of cysteine and phospholipids. Previous work has shown that yeast cells exhibit transcriptional induction of the one-carbon pathway and the genes involved in the synthesis of purine and methionine after the addition of 10 mM glycine. Here it is shown that addition of 10 mM L-serine did not, however, elicit the same transcriptional response. This is primarily due to differences in the uptake of glycine and L-serine in yeast. High concentrations of extracellular L-serine were required for yeast to show an increase in intracellular L-serine concentration of the magnitude required to trigger a noticeable cellular response. Despite L-serine and glycine being interconvertable via the SHMT isozymes and being a one-carbon donor, the genome-wide transcriptional response exhibited by cells in response to L-serine addition was markedly different to that seen for glycine. The predominant response to an increase in intracellular L-serine was the induction of the general amino acid control system and the CHA1 gene encoding the serine (threonine) dehydratase. Unlike glycine, addition of L-serine triggered only minor induction of the one-carbon pathway. A large portion of intracellular L-serine was converted to pyruvate and ammonia in the mitochondrion as the result of induction of CHA1. The high intracellular concentration of L-serine stimulated the cell to increase the production of oxaloacetate and to increase the biosynthesis of L-aspartate. Transient increases in the intracellular L-glutamate and L-glutamine were also observed after the addition of L-serine. The work presented in this study shows that large increase in the intracellular concentration of amino acid is required to trigger a significant transcriptional response. Yeast cells exhibit different transcriptional and metabolic responses to the addition of L-serine and glycine even though these two amino acids are closely metabolically linked. Addition of L-serine provokes the GAAC response, expression of the CHA1 gene and stimulates the biosynthesis of L-aspartate in yeast whereas addition of glycine induces the one-carbon pathway which leads to the biosynthesis of the purine nucleotides.

La perte de mémoire est un des premiers symptômes caractéristiques de la maladie d’Alzheimer (MA). Dans les cerveaux des patients, on retrouve des dépôts extracellulaires de plaques amyloïdes ainsi que des agrégats intracellulaires de la protéine tau. Les patients présentent également des déficits du métabolisme cérébral du glucose, une quinzaine d’années avant les premiers défauts cognitifs, suggérant que le métabolisme pourrait contribuer à la physiopathologie de la MA. Pour mieux comprendre les mécanismes qui relient le métabolisme énergétique et l’activité synaptique, nous nous sommes intéressés à la production de L-serine, une molécule dont la synthèse de novo dérive d’un intermédiaire de la glycolyse. La L-serine est le précurseur de la D-sérine, un acide aminé en conformation D présent en grande quantité dans le cerveau. La D-sérine est un co-agoniste des récepteurs au N-méthyl-D-aspartate (NMDA-R) nécessaire à la potentialisation à long terme (LTP) de l’activité synaptique dans l’hippocampe. La voie de biosynthèse de la L-serine est ainsi à l’interface entre métabolisme énergétique et activité synaptique. Afin d’étudier le rôle de cette voie, nous avons mis au point un modèle de délétion conditionnelle de la Phgdh, la première enzyme de la voie de biosynthèse de la L-serine. Nous avons injecté par stéréotaxie des vecteurs adéno-associés permettant l’expression de la Cre recombinase dans l’hippocampe de souris Phgdh(flox/flox), une lignée de souris qui possède des sites LoxP autour des exons 4 et 5 du gène de la Phgdh. Nous avons validé ce modèle en montrant que l’expression de Phgdh ainsi que les taux de D-serine diminuent d’environ 60% dans l’hippocampe des souris injectées. Nous avons ensuite réalisé des enregistrements électrophysiologiques sur tranches et nous avons mis en évidence une diminution de la LTP dans l’hippocampe des souris injectées avec la Cre recombinase. Ces souris présentent également un déficit de mémoire à long terme mis en évidence avec le test de la piscine de Morris. Ces déficits sont restaurés lorsque les souris reçoivent chroniquement un régime enrichi en L-serine. Ces résultats montrent que la biosynthèse de sérine est nécessaire et suffisante pour la plasticité synaptique et la mémoire à long terme.Afin d’étudier le rôle de cette voie dans la MA, nous avons mesuré l’expression de différentes enzymes dans des extraits d’hippocampes de patients atteints de MA et nous avons mis en évidence des changements significatifs dès les stades intermédiaires. Finalement, nous avons étudié un modèle murin de MA, les souris 3xTg, qui présentent des déficits métaboliques, synaptiques et comportementaux. Les déficits de LTP sont restaurés en ajoutant de la L- ou de la D-sérine de façon aigue sur les tranches d’hippocampe. Nous montrons que le déficit de mémoire spatiale à long terme peut être restauré par une supplémentation chronique en D-sérine, suggérant l’importance de cette voie dans le contexte de la MA. / Memory loss is among the first symptoms reported by patients suffering from Alzheimer’s disease (AD). AD is characterized by extracellular amyloid plaques and intracellular aggregations of tau. A decrease of brain glucose metabolism has also been described in the brain of AD patients. Since this decrease appears decades before memory loss, we hypothesize that metabolic deficits could directly contribute to AD physiopathology. To understand the mechanisms linking brain metabolism and synaptic activity, we proposed to study the production of L-serine, a signaling molecule whose de novo synthesis diverts part of the glycolytic flux. L-serine is the precursor of D-serine, a co-agonist of N-methyl-D-aspartate receptors (NMDA-R) that is required to maintain long term potentiation (LTP) of synaptic activity in the hippocampus. Since both L- and D-serine are formed through the activity of the Phosphorylated Pathway that diverts part of the glycolytic flux, any metabolic deficits may impact synaptic activity.We developed a model of conditional Phgdh deletion, the first enzyme of the phosphorylated pathway, by stereotaxically injecting Adeno-Associated Vectors allowing the expression of Cre recombinase in the hippocampus of Phgdh(flox/flox) mice, a mice strain with loxP sites flanking exons 4 and 5 of Phgdh gene. We validated this model showing that Phgdh expression and D-serine level are decreased by 60% in the hippocampus of injected mice. We performed electrophysiological recordings and showed that LTP is significantly reduced in mice injected with Cre recombinase. Those mice also show long term memory deficits in the Morris Water Maze test. Those deficits are restored by chronically feeding Cre injected mice with a diet enriched in L-serine indicating that serine biosynthesis is necessary and sufficient for synaptic plasticity and long term memory.To assess whether this pathway may be involved in AD pathogenesis, we quantified the expression of several enzymes of the serine biosynthesis pathway in human brain samples and found major changes in AD patients even at intermediate stages. To further investigate this hypothesis, we used 3xTg-AD mice, a mouse model for AD showing deficits in brain metabolism, synaptic activity and cognition. LTP deficits in 3xTg mice are restored by acute supplementation of L- or D-serine on hippocampal slices. We show that chronic administration of D-serine restores long term spatial memory. It suggests that serine biosynthesis is an important pathway in AD.

I evaluated the potential of isomers of the low molecular weight phosphomonoester naphthyl phosphate as general diagnostic substrates for differentiating between two families of protein phosphatases: the protein-tyrosine phosphatases [PTPs] and the dual-specificity protein phosphatases [DSPs]. Three PTPs, PTP-1B, Tc-PTPa, and PTP-H1, and three DSPs, Cdc-14, VHR, and IphP, were challenged in vitro with alpha-naphthyl phosphate and beta-naphthyl phosphate. Both the DSPs and PTPs readily hydrolyzed beta-naphthyl phosphate. As expected, the DSPs also hydrolyzed alpha-naphthyl phosphate at rates comparable to beta-naphthyl phosphate and two of the PTPs, PTP-1B and Tc-PTPa, hydrolyzed alpha-naphthyl phosphate at a rate one-tenth that of beta-naphthyl phosphate. However, PTP-H1 hydrolyzed both alpha- and beta- naphthyl phosphate at nearly equal rates. Intriguingly, when challenged with radiolabeled phosphoproteins, PTP-H1 was markedly less stringent, by a factor of 40- to 200- fold, than PTP-1B or Tc-PTPa in its selectivity for [32P]phosphotyrosyl- over [32P]phosphoseryl- proteins in vitro. The DSPs and PTPs listed above also were challenged in vitro with free phosphoserine. Each displayed little or no activity towards free phosphoserine. However, the addition of a hydrophobic "handle" to form N-(cyclohexane carboxyl)-O-phospho-L-serine produced a derivative that was hydrolyzed by IphP at rates comparable to that of the avid substrates p-nitrophenyl phosphate and beta-naphthyl phosphate. VHR also hydrolyzed N-(cyclohexane carboxyl)-O-phospho-L-serine, though at a lower rate than IphP. Cdc14 displayed little activity towards N-(cyclohexane carboxyl)-O-phospho-L-serine. The active site of VHR was mapped and amino acid residues potentially involved in binding N-(cyclohexane carboxyl)-O-phospho-L-serine were identified. The amino acid sequence of VHR was aligned with the amino acid sequences of IphP and Cdc14 to identify the nature of the corresponding residues in IphP and Cdcd14. Low molecular weight phosphomonoesters have proven to be effective in vitro indicators of protein phosphatase activity. They also have shown potential as diagnostic substrates for specific subclasses of protein phosphatases. However, neither alpha- and beta- naphthyl phosphate nor N-(cyclohexane carboxyl)-O-phospho-L-serine proved to be universal discriminatory substrates for the functional subgroups within the family of protein-tyrosine phosphatases. Indeed, the probability of identifying such a substrate would appear to be relatively low. / Master of Science

Neste trabalho foram aprofundados nossos estudos para obtenção da (S)-2-terc-butilamida-4-(3-picolil)piperazina, pela abertura da (S)-2-terc-butilcarboxamida-N-p-tosilaziridina seguida de ciclização, em 78% de rendimento, com o triflato de vinildifenilsulfônio. A aziridina foi preparada por um processo de ciclização, em condições de transferência de fase, partindo-se da L-serina, um aminoácido natural de baixo custo. Esta rota sintética rendeu um material que apresenta a mesma estereoquímica S do fragmento piperazínico usado na síntese do Indinavir, podendo vir a constituir uma via alternativa para a obtenção deste fármaco. / In this work we performed a deeper study for obtaining (S)-2-tert-butylamide-4-(3-picolyl)piperazine by opening (S)-2-tert-butylcarboxamide-N-p-tosylaziridine followed by cyclization, in 78% yield, with diphenylvinylsulfonium trifluoromethanesulfonate. The aziridine were prepared by a cyclization process in phase transfer conditions, starting from L-serine, a low cost amino acid. This synthetic route yielded a material which has the same S piperazinic fragment stereochemistry used in the synthesis of Indinavir, and may constitute an alternative route for obtaining this drug.