Carreira Alkynylations with Paraformaldehyde. A Mild and Convenient Protocol for the Hydroxymethylation of Complex Base-SensitiveTerminal Acetylenes via Alkynylzinc Triflates

A new synthetic protocol for the hydroxymethylation of terminal acetylenes is described that involves stoichiometric Carreira alkynylation with solid paraformaldehyde (HO[CH₂O]_nH) in PhMe at 60 ^oC. Significantly, the method can be successfully applied on acetylenes that possess base-sensitive ester functionality and heterocyclic rings that readily undergo metallation. While N-methylephedrine (NME) is generally the best Zn(OTf)₂-coordinating ligand for promoting hydroxymethylation, TMEDA can serve as a replacement.

Asymmetric Total Synthesis of (+)-Inthomycin C Via O-Directed Free Radical Alkyne Hydrostannation with Ph3SnH and Catalytic Et3B: Reinstatement of the Zeeck-Taylor (3R)-Structure for (+)-Inthomycin C

A new pathway to (+)-inthomycin C is reported that exploits an O-directed free radical hydrostannation reaction on (−)-12 and a Stille cross-coupling as key steps. Significantly, the latter process was effected on 19 where a gauche-pentane repulsive interaction could interfere. Our stereochemical studies on the alkynol (−)-12 and the enyne (+)-7 confirm that Ryu and Hatakeyama’s (3S)-stereochemical revision of (+)-inthomycin C is invalid and that Zeeck and Taylor’s original (3R)-stereostructure for (+)-inthomycin C is correct.

Synthesis of the C(7)-C(22)-Sector of (+)-Acutiphycin Via O-Directed Double Free Radical Alkyne Hydrostannation with Ph3SnH/Et3B, Double I-Sn Exchange and Double Stille Coupling

Abstract Image

Herein a new double O-directed free radical hydrostannation reaction is reported on the structurally complex dialkyldiyne 11. Through our use of a conformation-restraining acetal to help prevent stereocenter-compromising 1,5-H-atom abstraction reactions by vinyl radical intermediates, the two vinylstannanes of 10 were concurrently constructed with high stereocontrol using Ph3SnH/Et3B/O2. Distannane 10 was thereafter elaborated into the bis-vinyl iodide 9 via O-silylation and double I–Sn exchange; double Stille coupling of 9, O-desilylation, and oxidation thereafter furnished 8.

Domino 1,3-Dipolar Cycloadditions of N-Alkyl-α-Amino Esters with Paraformaldehyde: A Direct Access to α-Hydroxymethyl α-Amino Acids

N-Alkyl-α-amino esters undergo a domino reaction, based on the iminium cation generation, with paraformaldehyde, followed by a 1,3-dipolar cycloaddition of the stabilized azometh­ine ylide with another equivalent of formaldehyde. The resulting products are oxazolidines, which can be transformed after hydrolysis into α-hydroxymethyl α-amino acid or its derivatives. The diastereoselective 1,3-dipolar cycloaddition was performed using sarcosine (–)-menthyl or (–)-8-phenylmenthyl esters affording the cyclic product with moderate enantiomeric ratio.

Click Chemistry Inspired Synthesis of Novel Ferrocenyl-Substituted Amino Acids or Peptides

This work reports on the synthesis of a wide range of ferrocenyl-substituted amino acids and peptides in excellent yield. Conjugation is established via copper-catalyzed 1,3-dipolar cycloaddition. Two complementary strategies were employed for conjugation, one involving cycloaddition of amino acid derived azides with ethynyl ferrocene 1 and the other involves cycloaddition between amino acid derived alkynes with ferrocene-derived azides 2 and 3. Labeling of amino acids at multiple sites with ferrocene is discussed. A new route to 1,1'-unsymmetrically substituted ferrocene conjugates is reported. A novel ferrocenophane 19 is accessed via bimolecular condensation of amino acid derived bis-alkyne 9b with the azide 2. The electrochemical behavior of some selected ferrocene conjugates has been studied by cyclic voltammetry.

Corroles : synthesis, functionalization and application as chemosensors

O trabalho de investigação apresentado nesta dissertação foi desenvolvido tendo como objectivo a síntese e funcionalização de meso-triarilcorróis para utilização como quimiossensores. Este trabalho encontra-se apresentado ao longo de cinco capítulos. No primeiro capítulo são apresentadas as características gerais, as metodologias de síntese e de funcionalização de macrociclos de tipo corrólico, e descrevemse algumas aplicações em que têm sido utilizados. São ainda abordadas algumas das propriedades e características dos quimiossensores e os mecanismos de deteção de diversos analítos. No segundo capítulo, após uma pequena introdução às reações de Wittig e de Diels-Alder, escolhidas para a funcionalização do macrociclo corrólico, descreve-se o estudo efectuado para a obtenção do complexo de gálio(III) do 3- vinil-5,10,15-tris(pentafluorofenil)corrol e o seu comportamento como dieno, em reações de Diels-Alder na presença dos dienófilos 1,4-benzoquinona e 1,4- naftoquinona. Desses estudos resultaram dois aductos cuja habilidade sensorial, bem como a dos seus precursores, foi estudada, em solução, na presença de aniões esféricos (F-, Br-, Cl-), lineares (CN-) e volumosos (CH3COO-, H2PO4 -). Dos macrociclos estudados verificou-se que o corrol base-livre 5,10,15-tris(pentafluorofenil)corrol apresenta uma elevada sensibilidade para o anião fluoreto (F-), e que a coordenação do núcleo corrólico com gálio(III) diminui a afinidade para este anião. Em geral, todos os compostos mostraram afinidade para o anião cianeto (CN-) mesmo quando em suportes poliméricos. O gel de poliacrilamida revelou-se muito promissor na determinação de CN- em amostras de água. No terceiro capítulo é avaliada a reatividade do complexo de gálio(III) do 3- vinil-5,10,15-tris(pentafluorofenil)corrol ainda como dieno mas agora na presença de um dienófilo linear, o acetilenodicarboxilato de dimetilo. Desse estudo resultaram dois novos derivados corrólicos. A habilidade sensorial dos mesmos perante os aniões fluoreto, cianeto, acetato, e fosfato foi avaliada por espectroscopia de absorção e emissão tendo um dos aductos mostrado ser colorimétrico para o anião cianeto. No quarto capítulo descreve-se a síntese e caracterização de dois conjugados do tipo corrol-cumarina, resultantes de reações de Hetero-Diels-Alder entre o 3-vinil-5,10,15-tris(pentafluorofenil)corrolatogálio(III)(piridina) e orto-quinonasmetídeos gerados in situ a partir de reacções de Knoevenagel entre cumarinas e paraformaldeído. Realizaram-se estudos de afinidade sensorial para aniões e catiões com estes macrociclos, bem como com conjugados porfirinacumarina análogos. A inserção de uma unidade cumarina conferiu uma excepcional solubilidade tendo os novos derivados apresentado solubilidade em etanol. No quinto e último capítulo desta dissertação é avaliada a capacidade sensorial do 5,10,15-tris(pentafluorofenil)corrol e da sua espécie monoaniónica, para os catiões metálicos Na+, Ca2+, Cu2+, Cd2+, Pb2+, Hg2+, Ag+, Al3+, Zn2+, Ni2+, Cr3+, Ga3+, Fe3+ em tolueno e acetonitrilo. Os macrociclos corrólicos mostraram ser selectivos e colorimétricos para o catião Hg2+. Neste trabalho descreve-se ainda a síntese do derivado -iminocorrol, que após funcionalização com o 3-isocianatopropiltrimetoxisilano originou um derivado do tipo alcoxisilano, que foi, posteriormente, ancorado a nanopartículas comerciais de sílica. As novas nanopartículas ancoradas com o alcoxisilano corrol foram estudadas na presença de Cu2+, Hg2+ e Ag+. Na presença do catião Ag+ assistiu-se a uma mudança de cor, de verde para amarelo.

Padrão de distribuição e caracterização morfológica de fibras serotonérgicas nos núcleos da linha Média/ intralaminares do tálamo do mocó (Kerodon rupestris)

The midline/intralaminar nuclei form a remarkable group of nuclei of the medial and dorsal thalamus. The midline nuclei, in rats, comprises the paratenial nuclei (PT), paraventricular (PV), intermediodorsal (IMD), reuniens (Re) and rhomboid (Rh). The intralaminar nuclei comprises the central medial (CM), paracentral (PC), central lateral (CL) and parafascicular (PF). Such nuclei have dense serotonergic innervation originating from the brainstem, especially from the so-called ascending activation system. These nuclei, in turn, send projections to various cortical and subcortical areas, specifically to limbic areas, which suggests the important role of this neurotransmitter in the limbic circuitry. The aim of this study was to characterize the distribution pattern and morphology of serotonin fibers in the nuclei of the midline and intralaminar thalamic of rocky cavy (Kerodon rupestris), a tipical rodent from brazilizan northeast. To reach this aim we used four rock cavies adults. Following the transcardially perfusion with paraformaldehyde and brain microtomy steps was performed immunohistochemistry for serotonin (5-HT), Nissl technique and subsequent achievement and image analysis to characterize the cytoarchitecture of these nuclei and the serotonergic fibers visualized. An analysis was made of Relative Optical Density (ROD) to semi-quantify the concentration of serotonin fibers in the areas of interest. Thus, we observed a cytoarchitectonic arrangement of these nuclei similar to that found in rats. In case of fibers distribution, those immunoreactive to 5-HT were presented in a higher concentration according as ROD in the midline nuclei relative to intralaminar; Re being the core which has a higher pixel value followed by the PV , Rh, IMD and PT. In intralaminar CL showed higher pixels, followed by nuclei CM, PC and PF. The serotonergic fibers were classified as number of varicosities and axon diameter, therefore find three types of fibers distributed through this nuclear complex: fibers rugous, granular and semi-granular. In PV fibers predominated rugous; in PT fibers predominated granular; IMD, CL and PF fibers were represented by semi-granular and Re, Rh, PC and CM fibers showed granular and semi-granular. Morphological characterization of serotonergic fibers and differences in density between the nuclei may suggest different patterns of synaptic organization of this neurotransmitter beyond confirming his large repertoire functional

Microwave-induced fast decalcification of rat bone for electron microscopic analysis: An ultrastructural and cytochemical study

Bone decalcification is a time-consuming process. It takes weeks and preservation of the tissue structure depends on the quality and velocity of the demineralization process. In the present study, a decalcification methodology was adapted using microwaving to accelerate the decalcification of rat bone for electron microscopic analysis. The ultrastructure of the bone decalcified by microwave energy was observed. Wistar rats were perfused with paraformaldehyde and maxillary segments were removed and fixed in glutaraldehyde. Half of specimens were decalcified by conventional treatment with immersion in Warshawsky solution at 4oC during 45 days, and the other half of specimens were placed into the beaker with 20 mL of the Warshawsky solution in ice bath and thereafter submitted to irradiation in a domestic microwave oven (700 maximum power) during 20 s/350 W/±37°C. In the first day, the specimens were irradiated 9 times and stored at 40°C overnight. In the second day, the specimens were irradiated 20 times changing the solution and the ice after each bath. After decalcification, some specimens were postfixed in osmium tetroxide and others in osmium tetroxide and potassium pyroantimonate. The specimens were observed under transmission electron microscopy. The results showed an increase in the decalcification rate in the specimens activated by microwaving and a reduction of total experiment time from 45 days in the conventional method to 48 hours in the microwave-aided method.

Synthesis of new xanthene derivatives

Xanthene dyes, including fluorescein, are a well-known class of fluorescent dyes, which have widespread applications in natural sciences. The synthesis of xanthene derivatives via acid catalyzed condensation of substituted phenols with phthalic anhydride, to afford the asymmetric derivatives, is well established. The high temperature, harsh reaction conditions and often low yields make this method less convenient. The synthesis of xanthene dyes by direct modification of the fluorophore moiety is a great option to circumvent the above mentioned drawbacks. rnOur new synthetic strategy for the preparation of novel asymmetric xanthene dyes via direct conversion of hydroxyl groups on 3'- and 6'-positions into leaving groups by mesylation is reported. It was discovered that 3',6'-di-mesylated fluorescein underwent a nucleophilic aromatic substitution with sulfur nucleophiles and afforded new asymmetric xanthene sulfides. rnThe impact of substituents possessing an electron-withdrawing character such as chlorines and bromines was investigated with the aim to improve the aromatic substitution on the electron-rich fluorescein structure. It was observed that the incorporation of these groups did not considerably affect the substitution reaction and the yields were comparable with the unsubstituted fluorescein. rnThis strategy provided novel fluorescent probes with the linker suitable to further modifications. The modifications of the linker delivered fluorescein derivatives that could be used as fluorescent labels in peptides, oligonucleotides and for cell imaging. rnThe hydroxyl group on the linker was modified to achieve potent bioconjugate functionality such as azide. The new fluorescent azides were obtained in a 4-step synthesis, namely 2-(6-(2-azidoethylthio)-3-oxo-3H-xanthen-9-yl)benzoic acid with an overall yield of 13%, its 2',7'-dichloro derivative with an overall yield of 10% and its 2',4',5'-tribromo derivative with an overall yield of 1%, respectively. rnAn asymmetric xanthene sulfide with an amino functionality placed on the aliphatic linker, namely 2-(6-((2-aminoethyl)thio)-3-oxo-3H-xanthen-9-yl)benzoic acid, was obtained in a 3-step synthesis with an overall yield of 33%. rnThe impact of the substitution with sulfur nucleophiles on the 6'-position of the xanthene moiety on its fluorescent characteristics was investigated. In comparison with fluorescein new asymmetric xanthene sulfides afforded lower extinction coefficients and fluorescent quantum yields. On the other hand, the substitution with a sulfur nucleophile significantly improved the photostability of xanthene dyes. It was shown that after 10 hours of continuous excitation, the asymmetric sulfur-containing xanthene fluorophores exhibited 58-94% of their initial fluorescent intensities. This observation suggested that the novel dyes were 1-2 orders of magnitude more stable than fluorescein. rnThe azido-modified xanthenes were “clicked” via Cu(I)-catalyzed azide-alkyne cycloaddition with an oligonucleotide, which contained the terminal alkyne residue. rn

Overexpressed Ly-6A.2 mediates cell-cell adhesion by binding a ligand expressed on lymphoid cells.

The Ly-6 locus encodes several cell surface proteins whose functions are unknown. Although it is hypothesized that these proteins may be receptors, there is no direct evidence that they bind a ligand. Herein we present evidence that Ly-6A.2, a Ly-6 protein expressed on T lymphocytes, binds a ligand expressed on normal thymocytes and splenic B and T cells. We find that transgenic thymocytes that overexpress Ly-6A.2 spontaneously aggregate in culture. This homotypic adhesion requires the overexpression of Ly-6A.2 because it is not observed in cultures of nontransgenic thymocytes. The aggregation of Ly-6A.2 transgenic thymocytes is inhibited by phosphatidylinositol-specific phospholipase C (which removes Ly-6A.2 and other glycosylphosphatidylinositol-anchored proteins from the membrane). Some anti-Ly-6A.2 monoclonal antibodies, including nonactivating ones and Fab' fragments, inhibit this aggregation. In contrast, other anti-Ly-6A.2 monoclonal antibodies increase the aggregation of transgenic but not nontransgenic thymocytes. To further examine whether Ly-6A.2 mediates adhesion (versus inducing another adhesion pathway) reaggregation assays were performed with paraformaldehyde-fixed Tg+ thymocytes. Paraformaldehyde-fixed Tg+ thymocytes reaggregate in culture and this aggregation is also blocked by phosphatidyl-inositol-specific phospholipase C and anti-Ly-6A.2 monoclonal antibodies. These results indicate that the homotypic adhesion of cultured Ly-6A.2 transgenic thymocytes is directly mediated by Ly-6A.2 and, more importantly, strongly suggests that Ly-6A.2 binds a ligand that is expressed on thymocytes. Tg+ thymocytes also bind to nontransgenic thymocytes, B cells, and T cells, indicating that normal cells naturally express the Ly-6A.2 ligand.

Macrophages and lymphocyte responiveness to mitogen

Purified B-cells fail to proliferate in response to the strong thymus-independent (TI) antigen Lipopolysaccharide (LPS) in the absence of macrophages (Corbel and Melchers, 1983). The fact that macrophages, or factors derived from them are required is supported by the inability of marginal zone B-cells in infants to respond to highly virulent strains of bacteria such as Neisseria meningitidis and Streptococcus pneumoniae (Timens, 1989). This may be due to the lack of CD21 expression on B-cells in infants which could associate with its co-receptor (C3d) on adjacent macrophages. It is not clear whether cell surface contacts and/or soluble products are involved in lymphocyte-macrophage interactions in response to certain antigens. This thesis describes the importance of the macrophage in lymphocyte responses to T-dependent (TD) and TI antigens. The major findings of this thesis were as follows: (1). Macrophages were essential for a full proliferative response to a range of T - and B-cell mitogens and TI-1 and TI-2 antigens, including Concanavalin A, LPS, Pokeweed mitogen (PWM), Dextran sulphate, Phytohaemagglutinin-P (PHA-P) and Poly[I][C]. (2). A ratio of 1 macrophage to 1000 lymphocytes was sufficient for the mitogens to exert their effects. (3). The optimal conditions were established for the activation of an oxidative burst in cells of the monocyte/macrophage lineage as measured by luminometry. The order of ability was OpZ >PMA/lonomycin >f-MLP >Con A >DS >PHA >Poly[I][C] >LPS >PWM. Responses were only substantial and protracted with OpZ and PMA. Peritoneal macrophages were the most responsive cells, whereas splenic and alveolar macrophages were significantly less active and no response could be elicited with Kupffer cells, thus demonstrating heterogeneity between macrophages. (4). Activated macrophages that were then fixed with paraformaldehyde were unable to restore mitogenic responsiveness, even with a ratio of 1 macrophage to 5 lymphocytes. (5). Although highly purified T- and B-cells could respond to mitogen provided live macrophages were present, maximum activation was only observed when all 3 cell types were present. (6). Supernatants from purified macrophage cultures treated with a range of activators were able to partially restore lymphocyte responses to mitogen in macrophage-depleted splenocyte cultures, and purified T - and B-cell cultures. In fact supernatants from macrophages treated with LPS for only 30 minutes could restore responsiveness. Supernatants from OpZ treated macrophages were without effect. (7). Macrophage supernatants could not induce proliferation in the absence of mitogen. They therefore provide a co-mitogenic signal required by lymphocytes in order to respond to mitogen. (8). Macrophage product profiles revealed that LPS and Con A-treated macrophage supernatants showed elevated levels of IL-1β, TNF -α L TB4 and TXB2. These products were therefore good candidates as the co-mitogenic factor. The possible inhibitory factors secreted by OpZ-treated macrophages were PGE2, IL-10 and NO. (9). The removal of cytokines, eicosanoids and TNF-α from LPS-treated macrophage supernatants using Cycloheximide, Dexamethasone and an MMPI respectively, resulted in the inability of these supernatants to restore macrophage-depleted lymphocyte responses to mitogen. (10). rIL-1β and rTNF-α are co-mitogenic factors, as macrophage-depleted lymphocytes incubated with rIL-1β and rTNF-α can respond to mitogen.

Optimizing Western Blots for the Detection of Endogenous α-Synuclein in the Enteric Nervous System

International audience

Reaction of Aromatic Peroxyl Radicals with Alkynes: A Mass Spectrometric and Computational Study Using the Distonic Radical Ion Approach

The reaction of the aromatic distonic peroxyl radical cations N-methyl pyridinium-4-peroxyl (PyrOO center dot+) and 4-(N,N,N-trimethyl ammonium)-phenyl peroxyl (AnOO center dot+), with symmetrical dialkyl alkynes 10?ac was studied in the gas phase by mass spectrometry. PyrOO center dot+ and AnOO center dot+ were produced through reaction of the respective distonic aryl radical cations Pyr center dot+ and An center dot+ with oxygen, O2. For the reaction of Pyr center dot+ with O2 an absolute rate coefficient of k1=7.1X10-12 cm3 molecule-1 s-1 and a collision efficiency of 1.2?% was determined at 298 K. The strongly electrophilic PyrOO center dot+ reacts with 3-hexyne and 4-octyne with absolute rate coefficients of khexyne=1.5X10-10 cm3 molecule-1 s-1 and koctyne=2.8X10-10 cm3 molecule-1 s-1, respectively, at 298 K. The reaction of both PyrOO center dot+ and AnOO center dot+ proceeds by radical addition to the alkyne, whereas propargylic hydrogen abstraction was observed as a very minor pathway only in the reactions involving PyrOO center dot+. A major reaction pathway of the vinyl radicals 11 formed upon PyrOO center dot+ addition to the alkynes involves gamma-fragmentation of the peroxy O?O bond and formation of PyrO center dot+. The PyrO center dot+ is rapidly trapped by intermolecular hydrogen abstraction, presumably from a propargylic methylene group in the alkyne. The reaction of the less electrophilic AnOO center dot+ with alkynes is considerably slower and resulted in formation of AnO center dot+ as the only charged product. These findings suggest that electrophilic aromatic peroxyl radicals act as oxygen atom donors, which can be used to generate alpha-oxo carbenes 13 (or isomeric species) from alkynes in a single step. Besides gamma-fragmentation, a number of competing unimolecular dissociative reactions also occur in vinyl radicals 11. The potential energy diagrams of these reactions were explored with density functional theory and ab initio methods, which enabled identification of the chemical structures of the most important products.

Depalladation of Neutral Monoalkyne- and Dialkyne-Inserted Palladacycles and Alkyne Insertion/Depalladation Reactions of Cationic Palladacycles Derived from N,N `,N `'-Triarylguanidines as Facile Routes for Guanidine-Containing Heterocycles/Carbocycles: Synthetic, Structural, and Mechanistic Aspects

Depalladation of the monoalkyne-inserted cyclopalldated guanidines (kappa 2(C,N)Pd(2,6-Me2C5H3N)Br] (I and II) in PhCl under reflux conditions and that of the dialkyne-inserted cyclopalladated guanidine kappa(2)(C,N):eta(2)(C=C)PdBr] (III) in pyridine under reflux conditions afforded a guanidine-containing indole (1), imidaziondole (2), and benzazepine (3) in 80%, 67%, and 76%, yields, respectively. trans-L2PdBr2] species (L = 2,6-Me2C5H3N, C5H5N) were also isolated in the aforementioned reactions in 35%, 42%, and 40% yields. Further , the reaction of the cyclopalladated guanidine kappa(2)(C,N)Pd(mu-Br)](2) (IV) with AgBF4 in a CH2Cl2/MeCN mixture afforded the cationic pincer type cyclopalladated guanidine kappa(3)(C,N,O)Pd(MeCN)]BF4] (4) in 85% yield and this palladacycle upon crystallization in MeCN and the reaction of kappa(2)(C,N)Pd(mu-Br)](2) (V) with AgBf(4) in a CH2Cl2/MeCN mixture afforded the cationic palladacycles {kappa(2)(C,N)Pd(MeCN)(2)]BF4](5 and 6) in 89% and 91% yields, respectively. The separate reactions of 4 with 2 equiv of methyl phenylpropiolate (MPP) or diphenylacetylene (DPA) and the reaction of 5 with 2 equiv of MPP in PhCl at 110 degrees C afforded the guanidine-containing quinazolinium tetrafluoroborate 7 in 25-32% yields. The reaction of 6 with 2 equiv of DPA under otherwise identical conditions afforded the unsymmetrically substituted guanidinium tetrafluoroborate 8, containing a highly substituted naphthalene unit, in 82% yield. Compounds 1-8 were characterized by analytical and spectroscopic techniques, and all compounds except 4 were characterized by single-crystal X-ray diffraction. The Molecular structure of 2 and 3 are nove, as the framework in the former arises due to the formation of two C-N bonds upon depalladation while the butadienyl unit in the latter revealed cis,cis stereochemistry, a-feature unprecedented in alkyne insertion chemistry. Plausible pathways for the formation of heterocycles/carbocycles are proposed. the influence of substitutents on the aryl rings fo the cyclopalladated guanidine moiety and those on alkynes upon the nature of the products in addressed. Heterocycles 1 and 7 revealed the presence of two rotamers in about a 1.00:0.43 ratio in CDCl3 and in about a 1.00:0.14 ratio in CD3OD, respectively, as detected by H-1 NMR spectroscopy while in CD3CN and DMSO-d(6) (1) and CD3CN and CDCl3 (7), these heterocycles revealed the presence of a single rotamer. These spectral features are attributed to the restricted C-N single-bond rotation of the CN3 unit of the guanidine moiety, which possibly arises from steric constraint due to the formation of a N-H center dot center dot center dot Cl hydrogen bond with CDCl3 (1) and N-H center dot center dot center dot O and O-D center dot center dot center dot O hydrogen bonds with CD3OD (7).